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Chng 2

Cu trc v chc nng ca t bo vi sinh vt


I. Sinh vt nhn s
1. Cc phng php v k thut nghin cu 1.1. Phng php quan st t bo Nh knh hin vi quang hc (knh hin vi thng) vi phng i 1500 - 2000 ln, c bit nh knh hin vi in t thng (TEM) v knh hin vi in t qut (SEM) m khoa hc c th thy c t bo, cu trc siu hin vi ca vi khun vi ng knh khong 1m. - Quan st vi sinh vt trn tiu bn sng: vi sinh vt gia lam v lamella, nhum mc nho thy r mng nhy (capsule), y l phng php hay dng cho nhng vi sinh vt nui cy trn mi trng lng vi knh hin vi thng, quan st c kh nng vn ng ca chng.
Bng 2.1: Mt s phng php nhum mu v nguyn tc s dng
Phg php nhum mu +Nhum n (xanh methylene, carbolfuchsin, tinh th tm, safranin) +Nhum phn ly - Gram - Ziehl - Nielsen Nguyn tc s dng Dung dch ru hoc nc ca cc kim, dng quan st hnh dng vi sinh vt, cch sp xp t bo Vi cc phn ng khc nhau vi thuc nhum c th phn bit c chng Chia cc vi khun thnh 2 nhm ln: Gram dng gi mu tinh th tm, Gram m mt mu khi ty do s nhum mu ph hng safranin Dng phn bit cc loi Mycobacterium v mt s loi Nocardia. Vi khun acid nhum vi carbolfuchsin v x l vi dung dch ru acid, vn gi mu . Vi khun khng acid s mt mu do s nhum mu ph l xanh methylene. Dng pht hin s c mt ca mng nhy, bi v polysaccharide mng nhy khng bt mu thuc nhum bao quanh t bo vi khun nhum mu t S dng pht hin bo t vi khun. khi dng thuc nhum lc malachite vi tiu bn c un nng, thuc nhum s thm nhp vo ni bo t v lm chng nhum mu lc, khi b sung bng safranin s lm phn bao quang bo t nhum mu hng. Dng pht hin tin mao vi khun, s dng thuc lm phng tin mao ri sau nhum bng carbolfuchsin

+Nhum c bit - Nhum m (negative) -Nhum ni (endospore) -Nhum (flagella) tin bo

mao

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- Quan st vi sinh vt trn tiu bn c nh v nhum mu: Phng php nhum Gram v Ziehl - Nielsen cho php nhn bit 2 nhm vi khun Gram dng va Gram m, hnh dng ca bo t, cc vt th n nhp nh ht d tr polyphosphate (ht d nhim sc, ht volutin), cc git m, glycogen Nhng tiu bn c nh ny c quan st bi gic ln X 90 hoc X 100 (bi gic dng du, vt knh c vng en). Tham gia vo c ch nhum mu c cu trc ca thnh t bo v bn cht cc hp cht ca sinh cht khc nhau hai loi vi khun. quan st nhng cu trc siu hin vi ngi ta dng knh hin vi in t TEM v SEM, c th thy c nhng cu to rt nh vi ln vi nanometre. Nhng vi khun Gram+ c mi lin h chng loi pht sinh gn gi nhau, y ngi ta chia thnh hai nhm ph: nhm c hm lng (G + X)% cao hn 50% nh cc Actinomycetales, Corynebacterium, Cellulomonas v nhm c (G + X)% thp hn 50% nh Clostridium, Bacillus, Staphylococcus, Nhng vi khun Gram- c mi quan h chng loi pht sinh cch xa nhau v y ngi ta phn ra rt nhiu nhm ph.
Bng 2.2: Mt s tnh Gram m Tnh cht Phn ng vi ha cht nhum Gram cht khc bit gia vi khun Gram dng v

Gram dng Gram m gi mu tinh th tm, mt mu tm khi ty do t bo c mu ra, nhum mu ph tm hoc ta safranin hay Fuchsin Lp peptidoglucan dy, nhiu lp mng, ch c mt lp c khng c Acid techoic Lp pha ngoi thnh khng c c Lp lipopolysaccharide rt t hoc khng c nhiu, hm lng cao Hm lng lipid v thp (vi khun acid c cao (to thnh lp lp lipid mng lin kt ngoi thnh) lipoprotein vi peptidoglucan) Cu trc gc tin mao hai vng a gc bn vng a gc To c t ch yu l ngoi c ch yu l ni c t t (exotoxins) (endotoxins) Chng chu vi tc nhn kh nng chng chu kh nng chng chu vt l cao thp Mn cm vi lysozyme rt mn cm, d b tan t mn cm (cn phi vi enzyme ny x l ph lp mng ngoi ca peptidoglucan) Mn cm vi Penicillin v cao thp sulfonamide

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Mn cm vi Streptomycine, Chloramphenicol, Tetracycline Kt hp vi thuc nhum kim Mn cm vi cc cht ty anionic Chng chu vi mui Natri Chng chu vi kh hn

thp

cao

cao, cht ch cao cao cao

thp, lng lo thp thp thp

1.2. Phng php tch ly cc thnh phn ca t bo Khi cn nghin cu cc thnh phn ring bit ca t bo, ngi ta phi tch ly cc thnh phn ny nh siu m, enzyme lm tan thnh, cc khng sinh tc ng vo thnh, dng p sut thm thu gy co nguyn sinh, dng sc p c hc, dng siu li tm hoc li tm trong ng gradient Nh cc my o quang ph (Spectrophotometer) chng ta bit r rng v nhanh chng s lng t bo trong dch huyn ph. Nh phng php sc k giy hoc sc k ct chng ta c th thu c cc hp cht ring bit. 2. Hnh dng vi khun v vi khun c Hnh dng vi khun khc nhau gia loi ny v loi khc, i vi nhng vi khun a hnh (polymorphysme) th hnh dng c th khc nhau trong cc giai on sng khc nhau ca chu k sinh trng. Nhng hnh dng chnh ca vi khun: - Cu khun (Coccus): Khi phn chia theo mt phng v dnh nhau ta c song cu khun (Diplococcus), hoc lin cu khun (Streptococcus), phn chia hai phng v dnh vi nhau ta c t cu khun (Tetracoccus), phn chia 3 phng v dnh nhau ta c bt cu khun (Sarcina) hoc phn chia theo nhiu phng ta c t cu khun (Staphylococcus). - Trc khun: bao gm trc khun khng sinh bo t (nh E.coli) v trc khun sinh bo t nh Bacillus, Clostridium vi kch thc khong 2-3 x 1m. - Xon khun: Spirillum, Campylobacter, xon th vi cc vng khc nhau: Spirochaeta, Leptospira vi kch thc 1 x 5-500m. - X khun: gm nhng vi khun thuc b Actinomycetales trong c cc ging quan trng nh Streptomyces, Micromonospora, c kch thc 1-2 x 100-500m.

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Hnh 2.1: Cc nhm hnh thi chnh ca vi khun

- Vi sinh vt hnh sao nh ging Stella v vi sinh vt hnh vung nh ging Haloarcula, mt loi "vi khun" a mn thuc vi sinh vt c.

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3. S cu to t bo vi khun Nh knh hin vi in t khoa hc bit rt r cc t chc di mc t bo ca vi khun nh lp mng nhy, thnh t bo, mng sinh cht, cht nguyn sinh v cc thnh phn quan trng khc.

Hnh 2.2: M hnh v s cu trc t bo vi khun

1. Thnh t bo, 2. Mng sinh cht, 3. Th nhn, 4. Mexosome, 5. Cht d tr, 6. Sinh cht, 7. Bo t, 8. Tin mao, 9. Khun mao, 10. Khun mao gii tnh, 11. Bao nhy, 12. Tng dch nhy, 13. Ribosome, 14. Th n nhp, 15. Plasmid. 3.1. Mng nhy (capsule) Nhiu vi khun c bao bc bn ngoi bng mt lp mng nhy c bn cht ha hc l polysaccharide ca mt loi gc ng (homopolysaccharide) hoc ca nhiu gc ng khc nhau (heteropolysaccharide), mt s vi khun trong v ny cn cha mt t lipoprotein. Khi lm kh, ngi ta xc nh c 90 - 98% trng lng ca mng nhy l nc. Mng nhy c tc dng hn ch kh nng thc bo, do tng cng c lc i vi vi khun gy bnh, do cu trc ha hc ca mng nhy l polysaccharide v t lipoprotein nn c lin quan n tnh khng nguyn ca vi khun gy bnh. Mt khc, khi mi trng ngho cht dinh dng mng nhy c th cung cp mt phn cc cht sng cho t bo, trong trng hp mng nhy teo i. Mt khun lc gm cc vi khun c mng nhy nhn thy dng nhn bng - dng S (smooth), cn khun lc gm cc vi khun t hay khng hnh thnh mng nhy s c dng nhn

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nheo - dng R (rough), i vi nhng vi khun trong iu kin mi trng qu d tha carbon, nh rnh cc nh my ng, th mng nhy rt dy v to ra khun lc nhy nht - dng M (mucoid). V d nh my ng thng gp Leuconostoc mesenteroides c nhiu mng nhy dy gp 20 ln chiu ngang ca t bo vi khun. Nhng mng nhy nh vy c gi l nhng mng nhy ln (macrocapsule) cn nhng mng nhy nh hn 0,2m (ngha l khng nhn thy trn knh hin vi thng, m ch nhn thy trn TEM hay SEM) c gi l mng nhy nh (microcapsule). C nhng vi khun ch hnh thnh mng nhy trong cc iu kin nht nh, v d Bac. anthracis (vi khun gy bnh nhit than) ch hnh thnh mng nhy trong mi trng c protein ng vt, Diplococcus pneumoniae (gy bnh vim mng phi) ch hnh thnh mng nhy khi xm nhp vo c th ngi v ng vt. Mun quan st mng nhy ngi ta lm tiu bn m vi mc nho hoc nhum n bng thuc nhum kim. 3.2. Thnh t bo Ngy nay khoa hc bit kh r v thnh t bo cc c th nhn s. Da vo s nghin cu cu trc phn t ca thnh t bo, kiu trao i cht m ta c th xp chng vo hai gii: vi sinh vt c vi loi thnh t bo c bit v kiu trao i cht khc thng v vi khun vi ba nhnh tin ha: nhnh c thnh t bo dy l cc vi khun Gram dng, nhnh c thnh t bo mng l cc vi khun Gram m v nhm tiu gim, khng c hay thnh t bo rt mng. Nh cc phng php tch ly t bo nh lc dch huyn ph vi khun vi cc ht thy tinh c ng knh 0,1 mm hoc p dch huyn ph qua mng c l nh hn ng knh t bo, hoc a t bo vo mi trng u trng gy co nguyn sinh ri sau qua siu ly tm, ngi ta c th thu c lp thnh t bo kh tinh khit. Thnh t bo ca cc vi sinh vt c rt khc bit vi thnh t bo ca vi khun v hon ton khc vi c th nhn chun. i vi vi khun, thnh t bo chim khong 20 - 30% trng lng kh ca t bo, c bit Corynebacterium diphteria thnh t bo chim ti 76 - 78% trng lng kh ca t bo. Thnh t bo vi khun Gram dng dy 150 - 800A0 Cn vi khun Gram m lp thnh murein mng hn: t 50 - 180A0. Hp cht c bn ca thnh t bo vi khun l hai cht d cao phn t (heteropolyme): glucopeptid v acid teichoid. Glucopeptid (hay cn gi l peptidoglucan, mucopeptid, murein) l khung rn chc gi vng hnh dng vi khun, khi thy phn glucopeptid ta s oc 2 hp cht vi s phn t

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Gram nh nhau l: N-acetyl glucosamin v acid N- acetyl muramic, chng lin kt vi nhau qua lin kt 1,4 glucozid. Enzyme lysozim ct lin kt glucozid gia C1 cn li ca acid Nacetyl muramic v C4 cn li ca N-acetyl glucosamin (lin kt 1,4). Cc gc N- acetyl muramic lin kt vi nhau qua dy ni peptid, to ra mng li chng cht nh t ong, trong thnh phn ca N- acetyl muramic c mt ca cc acid amin vi trng lng phn t Gram: 2 D.L.alanin, 1 D. glutamic v 1 acid diamin. Acid diamin ny Sta. aureus l lizin, E.coli l acid L diaminpimelic (ADP), cc acid diamin ny c th kt hp vi mch peptid ca chui bn, do m hnh thnh mt mng li murein chc chn. i vi nhm vi khun tiu gim thnh t bo (Mycoplasmatales, Mollicustes, Tenericutes) ngi ta khng tm thy hp cht murein, nhng vi khun ny khng c thnh rn chc cho nn chng c th thay i hnh dng, phn ln chng l nhng c th a hnh nh cc dng PPLO, Mycoplasma (vi khun nh nht c th nui cy trn mi trng). Trong thnh t bo vi khun cn c mt loi hp cht c bit l acid teichoic, hp cht thy nhiu vi khun Gram dng (c th chim ti 50% trng lng kh ca thnh), vi khun Gram m hin cha tm thy hp cht ny. Acid teichoic lin kt vi acid muramic qua mch phosphodieste. Acid teichoic c hai loi ribiteichoic v glycerin teichoic. Thnh t bo vi khun c chc nng quan trng l gi hnh dng n nh ca t bo , tham gia vo vic duy tr p sut thm thu, s phn bo, tham gia vo qu trnh nhum Gram...
Vi khun Gr+ Vi khun Gr-

Hnh 2.3: M hnh cu trc thnh t bo vi khun Gram(+) v Gram(-)

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3.3. Mng sinh cht Mng sinh cht ca vi khun v cc vi sinh vt c c th thy c nh co nguyn sinh, lp mng ny di knh hin vi i pha l lp c dy khong 7,5nm, nm ngay di lp thnh hoc cc lp mng ngoi, n bao bc ton b khi cht nguyn sinh. Khoa hc gi n l mng c s v c cu to ging hu ht cc mng trong t bo nh mng nhn, mng ty th, lc lp, mng li ni cht, mng bo tng Phn tch ha sinh cho thy mng sinh cht gm 3 loi phn t: lipid (ch yu dng phospholipid - chim khong 30 - 40%), protein (gm rt nhiu h enzyme, trong c cc h permease, thnh phn protein ny c th chim ti 60 - 70%) v mt t hp cht glucid.Cc phospholipid c th l phosphatidiglycerol v/hoc l phosphatidylethanolamin. Cc phospholipid di knh hin vi in t gm 2 lp nm gia, trong sut trong khi cc lp protein bn ngoi c mu m ti. Cc phn t phospholipid c u a nc (hydrophyle) gm c choline - phosphate v glycerol cn ui k nc (hydrophone) l cc phn t acid bo. Do cu trc cc phn t phospholipid nh trn nn chng phi sp xp cc ui k nc vi nhau v u a nc quay ra pha ngoi v pha trong sinh cht, chnh cch sp xp ny c li nht cho chc nng vn chuyn cc cht (a cc dng proton nh cc enzyme), chc nng h hp Chc nng ch yu ca mng sinh cht l tm bnh phong ngn tr dng cc cht ra cng nh cho i qua cc hp cht t pha ngoi vo. N m bo t bo hp th c cc cht dinh dng, cc nguyn t c li cho qu trnh trao i cht. N la chn cho i qua c cc phn t cht hu c loi nh ng thi ngn cn cc hp cht phn t ln. i vi cc phn t b k nc nh O2, N2, CH4, N2O, H2 hoc cc phn t c cc nhng khng tch in nh H2O, urea, CO2,mng sinh cht th hin nh mt mng vt l cho i qua theo quy lut vt l hc. i vi cc phn t c kch thc ln quan trng i vi t bo v a nc th mng cho i qua theo c ch khuch tn theo nng cht ha tan, c th l khuch tn th ng t nng cao n nng thp hoc khuch tn tch cc ch ng nh cc enzyme vn chuyn permease, nh m c th tp trung vo trong cc hp cht cn thit cho t bo.

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C mt cch vn chuyn khc l do s thay i v tr cc nhm chc nng, v d khi phn t glucose i vo c phosphoryl ha v gii phng vo bn trong mng t bo l hp cht glucoso - phosphate. Mng sinh cht cha cc enzyme sinh tng hp kim sot cc khu kt thc tng hp lipid ca mng v cc hp cht kin to thnh t bo. Cui cng, mng sinh cht l ni nh v ca nhiu enzyme tham gia tng hp ATP. cc c th nhn chun, cc enzyme ny c mt trong ty th. Cc chui h hp ca mng sinh cht ca vi khun v vi sinh vt c lm chc nng tng t nh mng trong ca ty th. 3.4. Sinh cht v Ribosome Sinh cht ca c th nhn s gm c 80 - 90% l nc, nc c th trng thi t do (chim phn ln) lm nhim v ha tan cc cht v to nn dung dch keo vi cc cht cao phn t. Nc trng thi kt hp (chim phn nh) thng lin kt trong cc vi cu trc nh protein, lipid v hydratcarbon. Phn cn li ca sinh cht l lipoproteid (chim 10 20%). H keo ca sinh cht bao gm 2 pha: pha th nht l dung dch mui khong v cc hp cht ha tan c bn cht l lipoproteid, pha th 2 l pha huyn ph gm cc ht nucleoprotein, lipid v nhiu loi ht c kch thc rt khc nhau. Khi cn non, ang sinh trng, cht nguyn sinh c cu to ng nht v bt mu ging nhau. Khi trng thnh, trong cht nguyn sinh xut hin cc vt th n nhp, khng bo kh lm cho sinh cht c dng huyn ph ln nhn, bt mu khng ng u v c tnh chit quang khc nhau. Sinh cht ca vi khun c pH bnh thng l 7 - 7,2. nghin cu sinh cht, ngi ta dng siu li tm cao tc tch cht nguyn sinh v cc cu trc siu hin vi ring ra. * RNA v Ribosome Mt t bo vi khun cha trung bnh khong 18.000 ribosome, ht 70S (S l ch u ca Sverberg - 10-3 cm/giy trong siu li tm) vi ng knh t 10 - 30nm, trng lng phn t 3.106 daltons. Mi ribosome, khi gim nng Mg2+ ca dung dch s tch ra thnh 2 tiu phn trong siu li tm: tiu phn ln (50S) v tiu phn be (30S). tiu phn ln lin kt vi tiu phn b bng mi lin kt trung gian RNA - protein v protein protein. Cc ribosome cha phn ch yu l RNA (63%) v phn kia l protein (37%). Ngoi RNA v protein, ribosome c th cn cha mt lng nh lipid v enzyme ribonuclease, lepxinaminopeptidase, galactosidase v mt t cht khong giu Mg v ngho Ca.

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S t hp ca tiu phn ln vo tiu phn nh bi lin kt bn trong ribosome, lin kt RNA - protein, y c 2 site (cht) c bit c vai tr quan trng trong qu trnh dch m (translation) t chui RNA sang protein. Cc site ny gi l site "P" (nh peptidyl) v "A" (nh aminoacyl). Ribosome l c quan tng hp protein ca t bo, nhng ch c mt s nh ribosome (khong 5 - 10% tng s ribosome) dng lin kt vi RNAm i hi phi c s tham gia ca nhiu yu t lm di chui polypeptide. Mt trong cc yu t l EF-2 thng thy cc vi sinh vt c v c th nhn chun, nhng khng thy vi khun, cc yu t ny thay i v tr phin m ca histidine (cho diphtamide) lm mn cm vi c t diphteria, y l mt s khc bit na gia cc vi sinh vt c v vi khun. 3.5. Cc ht d tr vi khun cui pha trng thnh, trong t bo vi khun xut hin nhng ht c ln v thnh phn ha hc khc nhau. Kch thc v s lng cc ht ny ty thuc vo loi vi khun v iu kin nui cy chng. Trong khi sinh trng, vi khun tch ly dn cc cht d tr hu c v v c, cc cht d tr ny t n kch thc nht nh th hnh thnh nn ht d tr (vt th n nhp) c th thy di knh hin vi. Cc cht d tr carbon o vi khun thng thy l glycogen, tinh bt, poly-- hydroxy butyrate Cc ht d tr carbon d dng nhn thy khi nhum bng dung dch c iod, hp cht ny nhum cc cht ang trung hp (polymer) khng phn nhnh ca glucose (tinh bt) thnh mu xanh thm v cc hydratcarbon phn nhnh (glucogen) thnh nu. Cc hp cht poly-hydroxy butyrate c nhum mu bi Soudan en nh mu cc git m. - Cc ht d nhim sc (metachomatic granulation) hay ht volutin c tm thy ln u xon khun Spirillum volutans. Chng bt mu vi thuc nhum kim nh xanh methylene, xanh toluidine thnh mu ta trong khi sinh cht ca vi khun li c mu xanh. Cc ht volutin c dng hnh trn, ng knh c th t ti 0,3m, c th tan trong nc nng 800C, trong dung dch kim hoc dung dch NaCl 5%, trong dung dch HCl 1Nkhng tan trong ru, ester, chlorofor. Ht volutin l nhng ht d tr polyphosphate v c. - Cc vi khun Chromatium, Beggiatoa c th tch ly lu hunh bn trong t bo do oxy ha H2S gii phng ra S. Cc vi khun oxy ha st c th tch ly st trong sinh cht di dng Fe3O4.

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- Cc ht carboxysome thng thy Cyanobacteria, mt s vi khun mu ta, vi khun nitrat ha, vi khun oxy ha lu hung nh Thiobacillus, l nhng ht ribulose diphosphat carboxylase (hay carboxydis mutase) c bao bc bi s gp np ca mng sinh cht, c ng knh khong 50 - 100nm. - Tinh th dit cn trng thng thy mt s loi vi khun sinh bo t nh Bacillus thuringiensis, Bacillus dechrolimus, tinh th ny xut hin khi hnh thnh bo t, chng c bn cht l protein v ngy nay c dng dit su hi. 3.6. Cc th mang mu v sc t vi khun quang hp c quan thc hin qu trnh quang hp l cc th mang mu (chromatophores) ( to v thc vt l lc lp chlorophaste) v cu trc siu hin vi ca cc th mang mu khc vi lc lp. Mt khc bn cht ca cc sc t quang hp cng khc bit, to v thc vt l dip lc t (chlorophyll) trong khi vi khun quang hp l cc sc t gn vi chlorophyll m ngi ta gi l khun dip lc t (bacteriochlorophyll). Halobacterium halobium (c th sng trong mi trng mn) c cha bacteriorhodopsine mng sinh cht, sc t gn ging vi sc t vng mc mt. Ngoi ra, chng ta c th gp cc sc t sau: - Vitamine K2 (hp cht quinon) Bacillus subtilis v Bacillus cereus. - Sc t carotenoid chng tia t ngoi Corynebacterium. - Pyocyanine, violaceine Chromobacterium violaceum, sc t c hot tnh khng sinh. - Mt s sc t to thnh mu c trng ca khun lc nh zeaxanthine (carotenoid), sc t vng Staphylococcus aureus; xanthophyll v sarcinaxanthine (carotenoid) l loi sc t Sarcina. - Sc t pyocyanine xanh pyoverdine xanh lc hung quang P. aeruginosa. - Dn xut sc t pyrolic Serratia marcescens. 3.7. Khng bo kh y l mt loi ti cha y kh thng gp nhiu loi thuc 3 nhm vi khun quang hp: vi khun lam (Cyanobacteria), vi khun ta v vi khun lc. Khng bo kh gip vi khun quang hp tri l lng trong nc v ni ln mt nc.

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3.8. Cht nhn ca vi khun DNA ca t bo vi khun chim khong 1 - 2% trng lng kh ca chng, l hp cht cha thng tin di truyn ch yu ca t bo. Cht nhn ca vi khun khng c mng bc, hnh dng rt khc nhau v ch c 1 si gm 2 mch DNA. Chiu di ca n trong t bo E.coli o c l 1mm, tc l gp 500 - 1000 ln chiu di ca vi khun. Vng th nhim sc c nh v ti mt im trn mng sinh cht lc sp phn chia. ln DNA vo khong 5.106 pb (cp base nit) vi trng lng phn t vo khong 3.109 dalton (4,5.108 i vi Mycoplasma, 1.109 i vi Acholeplasma). Khong thy protein histone kiu t bo nhn chun m ch c cc polyamine nh specmidin v specmine lm chc nng cng c n nh DNA. Tuy nhin, nhng nghin cu gn y cho thy Thermoplasma (mt loi vi sinh vt c - Archaea) tm thy histone. Vng DNA xon kp ca vi khun thng c gi l th nhim sc vi khun hay genophore. Nhng nghin cu trn knh hin vi in t cho thy genophore ca vi khun c nhiu vng, mi vng nh c khong 400 cp base nit, nhiu vng to thnh bi xon. E.coli c 50 bi xon v c gn 500 vng trong mt bi, iu gii thch ti sao chui DNA ca vi khun di ti 1mm v gm khong 5.106 pb. - E. coli tm thy t nht l 4 loi protein lin kt vi DNA, l nhng protein kim, bn nhit nn rt gn gi vi histone ca cc sinh vt nhn chun: + Hu c hnh thnh bi 2 tiu phn v (nh phn protein ProteinII) nn rt gn vi histone H2B. + H gn ging vi histone H2A. + H1 v HLP1, nhng protein lin kt DNA ny c chc nng iu ha khng c hiu trong qu trnh sao m. S c mt ca cc protein trn lm cho cu trc th cp DNA ca E. coli vng chc hn. Mc d ch c mt th nhim sc i vi mt t bo vi khun, nhng trong mi trng nui cy lin tc khi c s sinh sn nhanh, c th dn ti s c mt ca 2 thm ch 4 th nhim sc trong mt t bo vi khun ang phn chia. - Ba loi DNA polymerase c chit t E. coli: + DNA polymerase I: lm chc nng khi phc, sa cha b sung, xc tc b sung desoxyribonucleotide vo u ca chui DNA. Phn ng ch xy ra khi DNA c inh v mng sinh cht.

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+ DNA polymerase II c hot tnh ngoi nhn, n cn thit cho hot ng ca RNAm. + DNA polymerase III l loi c hot tnh nht trong 3 loi polymerase. - Cc DNA-ligase xc tc hnh thnh cc u ni 2 mch DNA - cu phosphodiester gia nhm hydroxyl 3' u mch v nhm phosphate 5' u mch kia. DNA-ligase c th thc hin cc chc nng sau: + Sa cha, ct b mt on ca mch trong DNA xon kp. + ng v to thnh vng cc phn t DNA xon kp mch thng. + Hn cc on DNA trong qu trnh ti t hp di truyn. + Tham gia vo qu trnh nhn DNA cng vi DNA polymerase. Cc enzyme tho mch ca chui xon kp lm cho cc mch DNA trong chui xon kp c tch ra nhn ln. t hai mch lm khun tng hp hai mch mi b sung to thnh hai chui DNA xon kp hon ton ging nhau. y l c ch nhn i DNA bo m tnh di truyn ca cc th h vi khun. Cc thng tin di truyn t DNA c sao m (transcription) sang mRNA v sau c dch m (translation) thnh chui polypeptide hay enzyme. Bng phng php phng x t ghi (autoradiogragh) khoa hc c th chp c qu trnh nhn ln ca nhim sc th E.coli v nhiu c th nhn s khc. DNA ca c th c cu to c trng, s lng adenine bng s lng thymine v s lng guanine bng s lng cytosine. T l (G+X)/(A+T) l mt ch s sinh ha quan trng gip phn loi n ging v loi trong phn loi sinh ha (Chemotaxonomy), v d ch s Chargaff ca Micrococcus luteus l 4 v ca Clostridium perfringens l 0,34. 3.9. Plasmid Ngoi cc gen nm trong genophore ra, t bo vi khun (v mt s loi nm men) c th cha cc yu t di truyn ngoi th nhim sc, chng c th t nhn ln. V vy m nm 1952, Lederberg gi chng l plasmid ch tnh cht c lp ca chng vi cc gen nm trn th nhim sc. Khi nim vt cht di truyn ngoi th nhim sc bao gm DNA ca cc ty th, lc lp (nu c), cc plasmid, cc yu t gii tnh, cc yu t "dit", mt s prophage Khoa hc xc nh plasmid c rt nhiu loi vi khun nh E. coli, cc trc khun ng rut v vi khun Gram m, t cu khun

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Staphylococcus aureus, x khun Streptomyces coelicolor v Rhizobium melitoli Plasmid l phn t DNA vng kn 2 mch, him thy 2 mch thng, nm ngoi th nhim sc, c kch thc nh (bng khong 1/100 th nhim sc ca vi khun, gn ging mt prophage), c kh nng t nhn ln c lp vi t bo v chng c phn sang cc t bo con khi nhn ln cng vi s nhn ln ca t bo. Cc plasmid c th tng ln hoc gim i khi c yu t bt li nh nhit , thuc mu, khng sinh, cc cht dinh dng Cc plasmid c th trng thi ci vo th nhim sc, c kh nng tip hp hoc khng tip hp, c th c mt hoc nhiu bn sao cng loi ngay trong mt t bo vi khun. Cc plasmid khng phi l yu t nht thit phi c i vi s sng t bo, nhng khi c mt, chng em li cho t bo nhiu c tnh chn lc qu gi nh c thm kh nng phn gii mt s hp cht, chng chu vi nhit bt li, chng chu vi cc khng sinh Thng th cc plasmid nhn ln khi t bo nhn ln hay t bo tip hp, nhng khng c ngha s nhn ln ca plasmid ph thuc vo s nhn ln ca t bo. trong trng hp cng nhn ln, chc phi c c ch kim sot m bo ng thi s nhn ln, s lng bn sao v s phn chia ng u cc bn sao cho hai t bo con, cho n nay c ch ny cn cha sng t. C nhng plasmid c th chuyn t t bo cho sang t bo nhn nh tip hp (tip hp c), ngc li c plasmid khng tip hp c. Cc plasmid qua t cu khun Staphylococcus ch c th chuyn sang t bo nhn nh ti np. Mt s plasmid (v d yu t gii tnh F) c th xm nhp vo th nhim sc v tr c bit c trt t nucleotide b tr vi on nucleotide trn plasmid, nhng site nh vy gi l nhng yu t gia nhp (IS), nhng IS ny gm khong 1000bp v thy E. coli nh IS 1 (768bp) v IS 2 (1327bp). Nh vy, cc yu t gia nhp (yu t in vo - IS) l on nucleotide nh (700 - 1500bp) thng ch m ha s chuyn v (transposition) (to ra cc site - t ct t trc ca DNA). Cc yu t di truyn vn ng (Transposon - Tn) l nhng on nucleotide di hn (vi ngn n vi vn bp) m ha kh nng thay i tnh cht (nh chng chu) thng nh v bn cnh IS. Cc gen vn ng ny c th di chuyn trong ni b mt th nhim sc hoc gia cc th nhim sc. Cc yu t Tn thy E.coli nh Tn5 (5700bp) chng chu Kanamycine, Tn681 (2100bp) sinh c t rut, Tn2571 (23000bp) chng chu Chloramphenicol, Tn3 (4597bp) chng chu Ampicillin. Tn s ca s chuyn v (Transposition) cng nh tn s ca cc t bin t pht ngu nhin (10-5 - 10-7).

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3.10. Tin mao (Flagelles), tim mao (Cils) v nhung mao (Pili) Tin mao thng thy Vibrio, Spirillum v nhiu loi vi khun Gram m. S lng tin mao c th t 1 - 30 si ty thuc vo loi vi khun. Mt tin mao c chiu di t 6 - 30m v ng knh 10 30nm (12nm Proteus, 20 - 25nm Vibrio v Pseudomonas). Khi tin mao ngn th ngi ta thng gi l tim mao. Tin mao c cu to t mt loi protein gn vi Keratin m ngi ta gi l flagelline, protein c trng lng phn t khong 40.000 (trong c cc acid amin ch yu l arginine, lysine, acid aspartic, acid glutamic v tyrosine), nhng protein ny c tnh khng nguyn (H, khng nguyn ng nhit). Tin mao gip cho vi khun chuyn ng, khi vi khun di ng tin mao xoy vo nc hoc mi trng lng (c th ti 100 vng trong 1 giy), trong khi tim mao chuyn ng nh que gt. Mc d vy, trong vi sinh hc ngi ta thng dng tin mao v tim mao vi cng mt ngha, chng khc vi nhung mao (pili) l nhng si mnh v ngn hn nhiu, thng c xung quanh t bo Gram m v t thy t bo vi khun Gram dng. Ngi ta chia nhung mao (pili hoc fimbria) lm 2 loi: loi nhung mao ph thng (Type I) v loi nhung mao gii tnh (Type II). Loi nhung mao ph thng phn b vi s lng ln trn b mt t bo vi khun (vi trm), ngi ta cho rng loi nhung mao ny c lin quan n tnh cht kt dinh mu ca vi khun, loi nhung mao ny ngn hn nhung mao gii tnh (loi II c th di ti 10m), s lng nhung mao gii tnh rt t, khong 1 - 4, u cng c ch phnh ra, nhung mao loai II c vai tr quan trng trong qu trnh tip hp (conjugation) gia 2 t bo vi khun. Trong s cc si (tim mao v nhung mao) trn b mt t bo vi khun th tin mao c nghin cu k hn v ngi ta chia chng lm 2 nhm phn b: phn b cc (polaire) v phn b xung quanh (peritriche). S chuyn ng ca t bo vi khun c th t ti 10m/s (tc l trong 1 giy vi khun c th i c khong cch di gp 10 ln n) nn i hi mt nng lng rt ln (khong 2% nng lng trao i cht ca t bo). B my xoay tin mao gc nm trong mng sinh

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cht v thnh t bo gm 2 vnh khuyn, vnh trong gm 16 n phn protein c th dch chuyn rt nhanh nh dng proton (H+) i vo. Cch sp xp tin mao l mt tiu chun trong phn loi hnh thi (Morphology taxonomy) vi khun: - n mao cc (Monotriche polaire hay parapolaire) c trng cho nhiu loi Vibrio hoc chm mao cc (Lophotriche) c trung cho nhiu loi Pseudomonas, Chromatium v Thiospirillum. - Lng cc (Amphitriche hay Cephalotriche) c th chm mao lng cc nh cc loi Spirillum. - Chu mao (Peritriche) hay tin mao sp xp xung quanh thn t bo vi khun c trng cho nhiu loi Prote 3.11. Ni bo t vi khun (Endospores) Mt s vi khun cui giai on sinh trng, khi cht dinh dng mi trng cn kit v cht qua trao i c hi qu nhiu, hoc do c s thay i t ngt cc iu kin sinh trng c kh nng hnh thnh bo t bn trong t bo, c gi l ni bo t (endospores). Mi t bo vi khun ch to mt ni bo t nn loi bo t ny khng phi l bo t sinh sn, khc vi cc loi nh bo t (conidiospores), bo t ti (ascospores) hay bo t m (basidiospores) nhiu loi nm, chng l nhng bo t sinh sn v tnh hay hu tnh. Nhng vi khun c kh nng hnh thnh ni bo t gm nhiu loi thuc cc ging Bacillus, Clostridium, Desulfotomaculum, Sporosarcina, Sporolactobacillus, Thermoactinomyces. V bo t c trng bng s c mt ca acid dipicolinic (di dng dipicolinate calcium), hp cht ny c th chim ti 10 - 15% trng lng kh ca bo t. Vai tr ca hp cht dipicolinate calcium lm cho bo t chng chu c nhit cao, khi th nghim ngi ta thay calcium bng strontium th kh nng chu nhit ca bo t thu c km i. Mt hp cht khc ca v bo t mi tm thy l acid L.N-succinylglutamic khng c trong t bo sinh dng v ch c tng hp khi hnh thnh bo t, ngay giai on u khi hnh thnh vch ngn DNA mi vi mt t nguyn sinh cht, hp cht ny gip cho bo t bn nhit. Khi hnh thnh bo t, t bo c th mt i n 70% nc. Mt s vi khun khi hnh thnh bo t c th tn ti rt lu trong t nhin, v d Bac. anthracis c th sng tim sinh trong nhiu nm. Trong mt s cht c t bo vi khun b cht rt nhanh nhng bo t c th tn ti c kh lu, v d trong phenol c th vn sng trong 15 ngy, trong HgCl2 1% tn ti

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trong 2 ngy. Thi gian hnh thnh bo t khc nhau ty loi (t vi gi n 20 gi). Khi a bo t vo mi trng thun li pht trin, bo t s c hng lot thay i tch cc v mc mm, qu trnh mc mm gm 3 giai on ch yu: hot ha, nt v v mc ra. mun quan st c bo t ngi ta dng phng php nhum n hoc kp. 4. X khun v vi khun ni bo Nhng vi khun thuc b Actinomycetales (nhm bc thp v bc cao) l nhng vi khun c hnh si Gram dng, si c th phn nhnh, thng gp chng trong t v nc. Nhng x khun rt gn vi nm v hnh dng h si pht trin trong c cht nh b "r" hp th cht dinh dng, h si mt c cht to nn khung ca khun lc v h si kh sinh tn cng bng nhng cung mang bo t. l nhng loi thuc ging Streptomyces, mt ging khai thc c khong 50% s khng sinh hin bit, ng knh si khong 1- 2m (nh hn nhiu so vi ng knh si nm 7 - 9m). X khun cng ging nh vi khun l cha c nhn c bao bc bng mng, thnh t bo x khun khong cha cellulose hay chitine m cha hp cht in hnh ca vi khun l glucopeptide. X khun cng c qu trnh sinh sn phn bo theo kiu amitose (phn bo khng t), loi vi khun ny cng cha c gii tnh, ngha l cha c s phn ha thnh t bo c, ci v c th hnh thnh hp t tng phn nh tip hp, ti np v bin np, x khun cng b phage tn cng v gi l Actinophage. Streptomyces l mt ging x khun bc cao c Waksman v Henrici t tn nm 1943. Chng c v tr tin ha cao trong s cc ging thuc vi khun Gram dng, c tr s % mol Guanine v Xytozin rt cao (69 - 77% (G + X) trong DNA). X khun sinh sn v tnh bng bo t. Trn u si kh sinh ca Streptomyces hnh thnh cung bo t v chui bo t. Si mang bo t c nhng hnh dng khc nhau ty theo loi: thng-ln sng (Retiflexibilis), xon (Spirales) hoc c mc, vng (Retinaculiaperti) on tn cng ca cung mang bo t bng phng php ct khc (segmentation) hay kt on (fragmentation) m hnh thnh nn chui bo t. Bo t x khun (Arthrospores bo t t) hnh bu dc, hnh lng tr hoc hnh cu vi ng knh khong 1,5m. Mng bo t x khun c th nhn (smooth), gai (spiny), tc (hairy), khi u (warty), np nhn (rugose) ty thuc vo loi x khun. Nhng tiu tit ny c th thay i theo quy lut trn mi trng nui cy, thng th x khun c tiu tit

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c th c mng nhn trn mi trng c ngun nit hu c, trn mi trng c m v c v glucose cc bo t d biu hin cc tiu tit r nht. Mu sc ca khun lc v h si kh sinh cng rt khc nhau ty theo nhm Streptomyces, mu sc ny cng c th bin i khi nui cy trn mi trng khc nhau, chnh v vy m y ban quc t v phn loi x khun (ISP) nu ra cc mi trng chun v phng php chung phn loi nhm vi sinh vt ny. Hin nay khoa hc m t c khong 600 loi x khun bc cao ny. - Rickettsia l mt ging ca Rickettsiaceae, Rickettsiales, y l vi khun k sinh ni bo, c 3 nhm (Tribus): Rickettsiae, Ehrlichiae v Wolbachiae vi 2 ging ch yu l Rickettsia v Coxiella, k sinh ng vt c xng. Nhng vi khun ny cng c 2 loi acid nucleic, thnh t bo cng c hp cht c trng ca thnh t bo vi khun, sinh sn bng cch chia i khi trong t bo ng vt. - Chlamydia l mt ging ca Chlamydiaceae, thuc b Chlamydiales, l nhng vi khun k sinh bt buc, Gram m v c nhiu im ging Rickettsia, trong ng vt c xng sng chng khng to ra ATP ring m s dng ATP ca vt ch, kch thc ca chng rt nh (0,3 - 0,45m) v th trong mt thi gian di trc y ngi ta coi chng l mt loi virus (virus kim tnh Van Royen). - Mycoplasma thuc h Mycoplasmaceae, h c nht ca b Mycoplasmales (i khi l Mycoplasmatales - Freundt, 1955). Chng l nhng vi khun khng c thnh t bo, thn mm d bin hnh, bt ng. Trong b Mycoplasmales c cc dng vi khun Mycoplasma dng L v PPLO. y l nhng vi khun thuc Tenerecutes, lp Mollicutes, l lp vi khun tiu gim thnh, vi khun c kch thc nh nht khoa hc hin bit (0,1 - 0,3m) v vy chng d qua mng lc vi khun, chng l tc nhn gy cc bnh h hp, tit niu - sinh dc. 5. Vi sinh vt c (Archaea) Cc vi sinh vt c m trc y gi l cc vi khun c (Archaeobacteria) l nhm c th nhn s c sm nht (khong 4 t nm trc y), c Woese R. v Woese C.R. tch ra thnh mt nhnh tin ha ring. Nhng c th cn li hin nay thng sng trong cc iu kin khc thng. Nhng nghin cu gn y v nhit v pH ti u ca vi khun a nhit v Archaea cho php chia vi sinh vt c thnh 2 nhm: Crenarchaeota v Euryarchaeota. Nhm Crenarchaeota l nhng vi sinh vt c k kh bt buc, a nhit v a acid (Thermoacidophiles, Thermophiles anaerobies stricts).

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Nhm Euryarchaeota l nhng vi sinh vt c a mn, sinh methane (methanogenes) v mt vi loi k kh a nhit.
Bng 2.3: So snh mt s tnh cht gia vi khun v vi sinh vt c Tnh cht Bacteria Archaea Thnh phn thnh t bo Murein Pseudomurein, protein, polysaccharide Lipid ca mng Glycerol, acid Glycerol, ete bo, aster isopranyl Hnh dng t bo c loi + (hoc khng) hnh vung v dt Ni bo t (endospore) + (hoc khng) Pseudouridine hoc Cht " nhnh" ca RNAt Ribothymidine L.methylpseudouridine Hnh thnh cht kch thch + methyonyl RNAt Cc intron trong gen + C Polymerase - RNA loi + nhn chun C coenzyme c bit + Nhit sinh trng ti a 900C 1100C C quang hp phc tp C th C th sinh methane + Chu trnh Calvin c s C th dng khi c nh CO2

Nhng vi sinh vt c (s liu ch yu da trn methanogenes) l nhng n bo nh khong 1m c thnh t bo gm nhng hp cht c bit (thay v murein y c acid talosaminuronic, ester ca acid bo mch khng thng v glycerol, s phn nhnh ca chui dang ether (-O-) m khng phi dng ester (-CO-O-) (lipide ether-phytanyl), c nhiu lin kt mui v hydro trong protein, c pseudouracil v tr uracil trong RNAt, khng c dihydrouracil, thymine trong vng bn ca RNAt, cht nhn phn tn trong sinh cht vi lng nh (ch bng khong 1/3 so vi cht nhn ca E.coli). DNA ca chng giu hm lng G + X (mi lin kt 3 hydrrogen), ribosome loi c th nhn s, c intron, c coenzyme c bit (M), hu nh khng c s n nh v quinon v cytochrome. Chnh nhng c im gip cho vi sinh vt c sng trong nhng iu kin khc thng: nhit cao (c loi sng c 2500C) di 265 atm, chng chu vi lysozyme C 3 nhm sinh l v sinh thi quan trng l: * Cc c th sinh methane (methanogenes), y c l l nhnh c xa nht, cc lp nc su, y, k kh, mt s loi tm thy trong

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ng tiu ha ca ng vt nhai li. Phn loi c th sinh methane da ch yu vo kh nng v c ch sinh methane ca chng, gm c 5 b: - Methanobacteriales, c cc loi: Methanobacterium ivanovii, M. thermoautotrophicum, M. smithii, M. fervidus, M. sociabilis. - Methanococcales, c cc loi Methanococcus voltae, M. vannielii, M. thermolithotrophicus. - Methanomicrobiales, c loi Methanomicrococcus sp. - Methanosarcinales, c cc loi Methanosarcina mazeii, M. barkeri, M. thermophila, M. acetivorans, Methanothrix sp. - Methanopyrales c loi Methanopyrus sp. Nhng c th sinh methane c th thc hin nhng c ch khc nhau ty theo loi (hu ht l c th k kh bt buc): CO2 + 4H2 CH4 + 2H2O (G10 = -135,4 KJ/mol) CH3COOH CH4 + CO2 (G10 = -32,5 KJ/mol) HCOOH + 3H2 CH4 + 2H2O CH3OH + H2 CH4 + H2O (G10 = -112,5 KJ/mol) CH3NH2 + H2 CH4 + NH3 (CH3)2S + 2H2 2CH4 + H2S Ngi ta cho rng nhng c th sinh methane cng vi c th dinh dng methane (methanotrophes) lm tun hon nguyn t carbon trong thi k kh quyn khng c hoc rt t oxy. S bin i t CO2 thnh CH4 c bit vi sinh vt c c 4 giai on: + 2H 2H+ 2H+ 2H+ 2e2e2e2eCO2 HCOOH HCHO CH3OH CH4 acid formic H2O formandehyde methanol H2 O Trong khi , qu trnh oxy ha methane c th dinh dng methane (methanotrophes) c thc hin theo c ch nh sau: CO2 CH4 2H+ H CH3OH PQQ HCHO HCOOH CO2

PQQH2 NAD+ NAD+H+ NAD+ NAD+H+2eC th ng ha

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* Cc c th a mn (halophiles) nh Haloarcula, Halobacterium. Nhng c th ny sng trong mi trng c nng mui cao ( bin, cc m mui), hp thu cht dinh dng ch yu l do chnh lch gradient nng mui to ra, qu trnh quang hp y kh c bieet nh Bacteriorhodopsine, hp cht lin kt trong mng sinh cht ch khng phi l khun dip lc t (bacteriochlorophyll). * Cc c th a nhit, a acid (Thermoacidophiles), l nhng c th sng ngun t - nc nng, vng ni la, chng l nhng c th hiu kh hoc hiu k kh.V d: Sulfolobus acidocaldaricus c nhit sinh trng ti a l 900C v ti u l 750C, pH ti u l 2,5. Thermoplasma acidophilum c nhit sinh trng ti a l 650C v ti u l 600C, pH ti u l 1,5. 6. Nguyn tc phn loi c th nhn s C th nhn s ngy cng c pht hin nhiu hn, v vy nhn bit v s dng chng ta cn phi t tn cho n v phi phn loi chng. Taxonomy l khoa hc nghin cu s a dng ca vi sinh vt v mi lin h vn c tn ti gia chng. Khoa hc nh loi ny bao gm 3 lnh vc khc nhau: Classification (xp loi), Nomenclature (t tn) v Identification (nhn bit).
Bng 2.4. Bng nh loi cc nhm ln c th nhn s (Da ch yu vo Gergey's Manual, 1984, tn siu gii c xut) Tn nh loi Nhm c th + Siu gii nhn s (Super C th nhn cha c mng kingdom procaryote) A. Gii vi khun (Kingdom C th c thnh t bo vi hp cht murein Bacteria) I. Ngnh I: Gracilicutes C th nhn s c thnh c trng vi khun Gram m Lp 1: Scotobacteria 1. Vi khun ha dng v c (Chimiolithotrophes) 2. Myxobacteria 3. Vi khun c bao (Trichome). 4. Vi khun dnh kt v vi khun c chi 5. Spirochaete 6. Vi khun Gram m, hiu kh v vi hiu kh 7. Vi khun Gram m, k kh khng bt buc

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8. Vi khun Gram m, k kh bt buc 9. Rickettsia v Chlamydia Lp 2: Vi khun quang hp 10. Vi khun quang dng khng thi oxy (Onoxyphotobacteria) Lp 3: Vi khun quang hp thi 11. Cyanobacteria oxy (Oxyphotobacteria) II. Ngnh II: Firmicute C th nhn s c thnh vi khun gram dng Lp 1: Fimibacteria 12. Vi khun Gram+, khng sinh bo t 13. Vi khun Gram+, sinh bo t Lp 2: Thallobacteria 14. Vi khun tia v ch (Actinomycetales) (x khun) III. Ngnh III: Tenericutes 15. Mycoplasmes B. Gii sinh vt c (Kingdom Archaea) Mendosicutes Lp 1: Archaea C th nhn s khng c thnh, hoc c thnh vi hp cht c trng Pseudomurein, kiu sinh l rt c bit. tng chim a s trn hnh tinh cch y 3 t nm v trc, hin nay ch cn li vi nhm: Methanogens, Halophiles v Thermoacidophiles

S xp loi (Classification) thit lp cc nhm nh loi (cc taxon) cn c vo nhng tieu chun hnh thi v phn t cu to t bo. S t tn (Nomenclature) l a ra mt tn cho mt nhm, mt c th theo nguyn tc ca Linaeus gm 2 t, mt t latin ch ging v t k tip ch loi. V d tn ca trc khun c kh l Bacillus subtilis. S nhn bit (Identification) ch nh cc loi nghin cu vo mt trong cc loi (cc taxon) m t. Phn loi hc vi khun ngy nay s dung phng php nh loi hnh thi (Taxonomy phenetic), hay s dng phng php nh loi s (Taxonomy numeric), phng php ny s dng mt lng ln cc tnh trng v so snh cc tnh cht rt ra s ging nhau gia hai c th, trong ch s Jaccard l d hiu v thng dng nht trong nghin cu i cng. SAB = h s ging nhau gia A v B nS+ SAB = nS+ = s cc c im ging nhau nS+ + nd nd = s cc c im khc nhau.

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Vi khun hc cn s dng phng php nh loi di truyn (Taxonomy genetic), 3 tiu chun c s dng y l ch s (G + X)% (h s Chargaff), t s lai DNA - DNA, s lng v trt t cc nucleotide trong 16sRNAt v 5sRNAt. ch s lai DNA - DNA l mt tiu chun bt buc phn tch v tra cc loi vi khun, cho n nay cc loi ca h vi khun ng rut Enterobacteriaceae cho lai vi nhau v thit lp c mi lin h chng loi pht sinh gia chng. Theo Woese (1981) trt t nucleotide trong 16sRNAt l mt ch s quan trng xc nh s gn gi ca hp cht protein v t quy nh mi quan h chng loi pht sinh gia cc nhm vi khun. Chnh nh phn tch 16sRNAt m hnh thnh mt hng nh loi mi - nh loi chng loi pht sinh (polygenetic taxonomy), nh c th tch cc vi sinh vt c ra khi vi khun v chng minh n l nhnh tin hoa c lp khc xa x khun. Cc phng php nh loi sinh ha (chimiotaxonomy) ngy nay c s dng rng ri, nh cc hp cht polymer ca thnh t bo vi khun (bao gm vi khun Gram dng, Gram m v Cyanobacteria) l peptidogulcan (murein), hp cht c trng ny khng thy nhm vi khun tiu gim thnh Mycoplasmes v cc vi sinh vt c. Cc vi sinh vt c c hp cht thnh c trng l pseudomurein, trong acid muramic c thay bng acid talosaminuromic. Cc mc n v phn loi dng trong vi sinh vt hc l: Clone biotype species genus familia order classe division kingdom - Dng t bo (clone): l mt qun th vi khun (hay vi sinh vt) c c t mt t bo b m ban u nh s phn chia v tnh. - Dng (biotype): mt nhm c th c chung c im di truyn tuyn chn v nhng im ch yu, dng c c t tuyn chn cc dng, y l n v hay dng trong cng ngh vi sinh. - Chng (ni - souche): l t thng dng trong k thut thc hnh, l taaoj hp nhng c th c c do s cy chuyn t mt khun lc vi khun. - Loi (species): tp hp nhng c th ging nhau v hnh dng, cch sng, c th sinh sn bnh thng gia chng v thng khng c kh nng ny vi cc c th thuc loi khc. - Ging (genus): tp hp cc vi khun trong bng xp loi di h, trn loi, tp hp cc loi rt gn nhau. - H (familia): tp hp cc ging c mt s c im chung.

43

- B (order): mt nhnh trong bng xp loi vi khun, bao gm mt s h gn nhau nm di lp. - Lp (class): mt nhnh trong bng xp loi vi khun, bao gm vi khun trong mt s b gn nhau. - Ngnh (division): tp hp cc vi khun mt s lp c chung mt s c im c bn. II. Vi sinh vt nhn chun. 1. Vi nm Cc c th nm vi cu to thnh t bo, kiu trao i cht v h enzyme khc bit vi cc c th nhn chun khc (thc vt, ng vt) v khc xa vi c th nhn s nn t lu c xp thnh mt gii ring l gii nm (Kingdom Fungi). C 3 danh t thng dng ch nm l: nm men (Levures, Yeasts), nm mc (Moisissures) v nm ln hay nm c qu th. Trong vi sinh vt hc, vi nm c hiu l cc nm c c th hin vi, bao gm hai nhm: - Nm n bo hay nm men. - Nm si hay nm mc. 1.1. Nm men (Levures, Yeasts) Nm men l nhm nm c th n bo, nhn chun, hin vi. Nm men c th thuc v 3 lp nm l nm ti (Ascomycetes), nm m (Basidiomycetes) v nm bt ton (Deuteromycetes). Trong s 75.000 loi nm hin bit th c hn 500 loi nm men thuc khong 50 ging. Nm men c hnh trng, qu da chut ng ring l hoc tp hp thnh si d gy, kch thc t bo t 7 - 10m. Trn mi trng sng, c nhng loi nm men sinh cc sc t c trng nh , vng Nm men sinh sn v tnh bng m chi hoc phn chi, gia qu trnh ny c th sinh sn hu tnh. Nm men c 3 dng chu trnh sinh hc: - Chu trnh n bi - lng bi nh loi Saccharomyces cerevisiae. - Chu trnh u th lng bi nh loi Saccharomycodes ludgyzii. - Chu trnh u th n bi nh loi Schizosaccharomyces octosporus. Nh knh hin vi in t khoa hc thy c s khc nhau v thi gian hnh thnh thoi v sc trong sinh sn v tnh nm men phn i v nm men m chi.

44

Nm men nhn i th trong chu k phn bo cng c cc giai on G1, S, G2 v M nh sinh vt nhn chun. Trong khi mng nhn cha c pht tn, cc thoi vo sc ca mitose c hnh thnh trong nhn v th nhim sc c a v hai cc, sau mi hnh thnh vch ngn to thnh hai t bo ring. nm men m chi c cc pha G1 v S bnh thng, nhng thoi v sc y c hnh thnh rt sm ngay cui pha S lm cho pha G2 khng bnh thng (ngn li) v trong khi cha hnh thnh hai nhn thnh t bo bt u gp li. C ch tch cc th nhim sc phn bo c t ca nm men l nh cc thoi v sc m cc th nhim sc trt v hai u c din ra trong nhn. Trong khi phn bo v t ca cc vi khun, cc th nhim sc con c hnh thnh vn nh ln mng sinh cht v ch c tch ra nh s pht trin v phn vch ca mng. So snh cu to t bo nm men v t bo vi khun ta thy c s tin ha nhy vt t c th nhn s chuyn thnh c th nhn chun. Cng vi s tin ha v nhn v c ch phn chia nhn (nhn c mng, c cc th nhim sc, phn bo c t) c th nhn chun xut hin nhiu c quan t khng thy cc c th nhn s nh ty th, lc lp ( c th nhn chun quang hp) Nghin cu cc c im ca c quan t ta thy rt nhiu im ging c th nhn s nh DNA mch vng (DNA nhn mch thng), khng c protein histone (th nhim sc trong nhn c histone), ribosome 70 S (trong t bo l 80S) v phn bo trc phn trong khi t bo phn bo gin phn. Mt khc, khoa hc cn tm thy mt t loi ng vt nguyn sinh nh Microsporidia v Giardia l nhng n bo nhn chun k kh cn li cho n nay, khng c ty th, nhng c th ny i din cho mt nhnh tin ha tiu gim khng ty th ca cc c th nhn chun, k kh so vi nhnh tin ha pht trin ca cc c th nhn chun hiu kh. Con ng tin ha hnh thnh ty th trong cc c th nhn chun hiu kh c thc y nhanh hn khi hm lng oxy trong kh quyn tng ln. Ngi ta cho rng mt con ng tin ha hnh thnh ty th trong t bo nhn chun l do s tip xc ca cc vi khun ni cng sinh (endosymbiotic bacteria), nhng t bo nhn chun mang trong mnh vi khun hiu kh ni cng sinh, sau nhng thi k ni cng sinh ko di, c s chuyn gen t vi khun vo nhn v ch cn li mt s gen t tr trong ty th, l t bo nhn chun hiu kh mang ty th nh ngy nay. Ngoi nhng cu to c bit ca nm men so vi vi khun, thnh t bo ca nm cng l mt nt rt c trng. Thnh t bo nm c n 80%

45

l polysaccharide mang tnh khng nguyn, trong cc hp cht mannan, glucan v chitine l rt quan trng, phn cn li l protein c th n 10 20%, trong gn mt na l mannoprotein, chng to thnh 3 lp ca thnh t bo nm men. Cc polymer ca thnh t bo c thay i cht t ph thuc vo tng nhm nm.
Bng 2.5: Bng phn nhm n gin cc nm men
Lp (Classes) B H Saccharomycetaceae H ph Schizosaccharum vcetoideae Nadsonioideae Lipomycetoideae Saccharomycetoideae Ging Schizosaccharomyces Hanseniaspora Lipomyces Debaryomyces Hansenula Kluyveromyces Pichia Sacharomyces Coccidiascus Filobasidium Leucospiridium Sirobasidium Tremalla
Cryptococcoideae

Nm ti (Ascomycetes)

Endomycetales

Spermophthoraceae Filobasidiaceae Leuvures Sirobasidiaceae Tremellaceae Cryptococcaceae

Nm bt ton Nm m (Deuteromy-cetes) (Basidiomycetes)

Ustilagi -nales Tremellales

Rhodotoruloideae
Trichospororoideae

Sporobolomycetaceae

Brettanomyces Candida Cryptococcus Torulopsis Rhodotorula Trichosporon Sporobolomyces

Nm men chim mt v tr c bit trong cng nghip thc phm: lm n bt m, nu ru, lm ru vang, lm pho mt, sn xut sinh khi ch protein (trong nm men c th cha n 40% m ca trng lng kh). Ring sn xut bnh m hng nm th gii tiu th 1,7 triu tn nm men bnh m.

Blastomycetales

46

1.2. Nm mc Nm mc l loi nm si in hnh. Cng nh nm men, nm si l nhng c th d dng, mt s sng cng sinh vi thc vt, khi cng sinh vi to n bo hoc tp hp n bo th hnh thnh a y (Lichens). Mt s nm k sinh trn ng vt v thc vt gy nn cc bnh nm rt kh cha. Nhiu nm sng hoi sinh s dng rc thi hu c ng vt v thc vt hoc ph hoi thc n, vt dng hng ngy. Chng thng c nhng enzyme phn gii rt mnh nh h enzyme phn gii cellulose, phn gii pectin, cc enzyme amylase, protease, lipase Con ngi t lu bit s dng mt c li ca nm mc trong vic ch tng, nc chm, sn xut khng sinh, to cc enzyme
Bng 2.6: Bng phn loi n gin mt s ging nm mc
Ngnh Amastigomycota Ngnh ph Zygomycotina (Zygomycetes) Ascomycotina Lp Zygomycetes Plectomycetes Pyenomycetes Hemiascomycetes Basidiomycotina (Basidiomycetes) Hemibasidiomycetes B Mucorales Eurotiales Spahaeriales Endomycetales (nm men) Urenidales Ustilaginales Ging Mucor Rhizopus Emericella (A. nidulans) Neurospora (N. grassa) Eremothecium Puccinia Ustilago Candida Geotrichum Aspergillus (A. flavus, A. niger) Penicillium P. votatum, P. camenbertii, P. roquefortii)

Deuteromycotina (Deuteromycetes)

Hyphomycetes (dng nm men = Blastomycetes) Hyphomycetes

Moniliales

Si nm c th c vch ngn nh cc lp nm bc cao (Ascomycetes, Basidiomycetes, Deuteromycetes) hoc hnh ng trong si c nhiu nhn m ngi ta gi l si cng bo (coenocytis). Nhng loi nm si khng vch ngn thuc v cc nm bc thp nh Oomycetes

47

v Zygomycetes. Cc vch ngn khng ngn cch hon ton gia cc t bo ca si m chng thng lin h vi nhau qua l vch. Mt bo t khi ri vo mi trng thun li s ny mm v to thnh khun lc gm h si pht trin su vo c cht ht thc n (si c cht - SM), si c cht to thnh khung ca khun lc v si kh sinh mang cc cung bo t (si kh sinh - AM) Cc nm bc thp c th sinh ra cc ng bo t mt roi (chytridiomycetes) hoc hai roi (Oomycetes) trong chu trnh sinh sn ca mnh. Cc nm bc cao nh loi Aspergillus v Penicillium c th hnh thnh cu tip hp gia hai t bo ca hai si (+ v -), l hin tng sinh sn cn tnh. Cc cung mang bo t pht trin t mt loi si kh sinh, c th phn nhnh hoc khng, trn u cung bo t c th hnh thnh ti mang bo t (Mucor, Rhizopus) vi cc bo t ti sinh sn v tnh hoc trn u cung bo t bng phng php m chi m sinh ra cc bo t nh (conidie hay conidiospore), cc cung sinh bo t c th tp hp li thnh thng hay khoang nh bo t (pycnide). i khi cc bo t c hnh thnh bng cch phn t ca si (Geo trichum) m ngi ta gi l bo t t (athrospore). Cc bo t hu tnh c hnh thnh nh qu trnh hu tnh kt hp cc t bo c v ci (cc giao t) hoc cc si khc gii tnh, hoc do s hp nht hai nhn trong si cng bo (coenocytis) hnh thnh hp t, sau nh gim nhim m hnh thnh cc ti bo t vi cc bo t ti. cc nm m (Basidiomycetes) qu trnh hnh thnh m v cc bo t m l giai on cui cng, qu th nhn thy c bng mt thng, trong khi phn ln chu trnh pht trin dng si mc. cc loi nm m qu trnh hp nhn xy ra mun hn so vi qu trnh hp cht nguyn sinh. Giai on si lng nhn (mt t bo c 2 nhn) tn ti kh lu, ch giai on hnh thnh m mi c t bo c nhn l 2n. * Tng qut v nm:
Bng 2.7: Cc lp nm thng gp
Lp nm Oomycetes Loi si khng c vch ngn (coenocytic) Bo t v tnh bo t chuyn ng Bo t hu tnh Oospora Ni chnh sng V d Allomyces

thy sinh, nhiu loi gy bnh cho c, mc sng khoai ty t, phn gii cht hu c thc vt

Zygomycetes

coenocytic

bo t ti, i khi bo t nh (conidia)

Zygospora (bo t tip hp)

Mucor, Rhizopus

48

Ascomycetes

c vch ngn, mt s n bo c vch ngn, mt s n bo c vch ngn, mt s n bo

nh bo t (m chi)

bo t ti (ascospore)

t, phn gii cht hu c thc vt t, phn gii cht hu c thc vt t, thc vt v trn c th ng vt

Neurospora, Saccharomyces, Morchella Agaricus, Amanita Candida, Trychophyton, Epidermophyton

Basidiomycetes

Uredospora, (m chi)

conidia

bo t m (basidiospore) cha thy

Deuteromycetes

bo t nh, bo t t (arthrospora)

Bng 2.8: Cc kiu bo t nm


V d ging nm Saprolegnia Aspergillus, Penicillium Mucor, Rhizopus Coccidioides Candida Kiu bo t Bo t v tnh (ng bo t) Zoospores) nh bo t (Conidiospore) Tnh cht Bo t n, c roi, chuyn ng Bo t n hoc tp hp thnh chui c hnh thnh trn cung bo t (Conidiospore) Bo t c hnh thnh trong ti (bo t v tnh) Bo t c hnh thnh bng cch chia t cc si kh sinh Thnh dy, bo t n, c hnh thnh bng phn i hay chi ging nm men c hnh thnh trong ti (bo t hu tnh), thng 4 - 8 bo t trong mt ti Pht trin tn cng ca m (thng 4 bo t) Bo t ln c hnh thnh trong mt thnh dy (bo t hu tnh) Bo t pht trin trong Oogonium (nguyn bo trng)

Bo t ti v tnh (Sporangiospores) Bo t t (Arthrospores) Bo t dy (Chlamydospore), bo t mm (Blastospore) Bo t ti

Saccharomyces, Neurospora Agaricus Rhizopus Saprolegnia

Bo t m (Basidiospores) Bo t tip hp (Zygospores) Bo t non (Oospore)

Cc loi nm m n c nh cc ging nm m (Agaricus bisporus), nm rm (Volvariella volvacea), mc nh (Auricularia), nm hng (Lentinus), nhn nh (Tremella)ang tr thnh i tng ch yu trong cng ngh nui trng nm n. Phn loi nm mc ch yu da vo cc tnh trng hnh thi: cu to si mang bo t, cu to bo t v mt s tnh trng sinh l sinh ha.

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2. Vi to Vi to l to hin vi c sc t quang hp. Vi to n gin nht l c th n bo, hoc tp hp n bo, c th c roi nh Clamydomonas, Peridium v Euglena (to mt), hoc khng c roi nh Chlorella (to lc), Diatomia (to ct). Cc vi to thng gp hn l cc c th a bo hoc tp hp n bo, nh cc tp on Volvox, Pediastrum, Scenendesmus (thuc nhm Archethalle) hoc phc tp hn c b phn nh bm v b phn dng ng nh cc si mnh phn nhnh hoc khng (c th c vch ngn to thnh cc t bo tng i c lp hoc khng c vch ngn nh mt ng cng bo (coenocytic). Nhng to ny sinh sn bng cch phn chia nhng t bo l gia hoc bng cch rng t bo u cng (Sphacelaria, Ectocarpus), chng sinh sn hu tnh bng tip hp ng giao (hai giao t bng nhau) hoc d giao (hai giao t khc nhau). Cc sc t quang hp v h tr cc nhm to khc nhau th khc nhau, hin nay ngi ta bit 6 nhm to vi cc sc t c nghin cu tng i k. Bn ging to lc l Clamydomonas (n bo 2 roi), Gonium (tp hp n bo 2 roi), Pandorina (tp hp n bo, pha ngoi cn c 2 roi, pha trong mt roi) v Volvox (tp hp n bo, pha ngoi c 2 roi lm chc nng di ng cho c tp on, pha trong t bo mt roi lm chc nng quang hp, h hp) l mt v d r nt chng minh s tin ha t t chc n bo ln t chc a bo phn ha th s.
Bng 2.9: Sc t v mt s tnh cht ca cc nhm to khc nhau
Ngnh to Chrysophy Euglenoph Chlorophy ta (to vng, yta (to mt) ta (to lc) to silic) Dip lc a v b Carote -noid v mt t Oxycaroten Lutein, Zeaxanthine, Neoxanthine, Violaxanthine Astaxanthine, Neoxanthine Mt s tnh cht T bo 2 roi, sinh sn vo tnh bng chia i hoc sinh sn hu tnh, cht d tr l tinh bt, thnh t bo ch yu l cellulose n bo c 1 roi (mt s c 2 3 roi), sinh sn v tnh bng chia i hoc hu tnh, cht d tr l m v loi tinh bt paramylon, khng c thnh t bo Phn lp l n bo, mt s nh dng si c 1 - 2 roi, sinh sn v tnh hoc hu tnh, cht d tr l du v lecucosin vi silic, thnh t bo thm pectin, silica

a v b

Lutein, Fucoxanthine, Diadinoxanthine, Diatoxanthine

a v c

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Phaeophyta (to Pyrophyta nu) (To la, to gip)

a v c

Dinoxanthine, Diadinoxanthine, Peridinine Tucoxanthine, Lutein, Diatoxanthine, Xanthophylls

n bo 2 roi bn, sinh sn v tnh bng phn i, cht d tr l tinh bt, thnh t bo l cellulosse

Rhodophyta (to )

a bo, kch thc ln, hai roi khc bit bn, sinh sn v tnh bng ng bo t, sinh sn hu tnh bng giao t chuyn ng, cht d tr l Laminarian, thnh t bo c cellulosse v acid alginic Phycocyanine, Hu ht a bo, kch thc ln, Phycoerythrine, bt ng, sinh sn v tnh bng Neoxanthine, Lutein, bo t, sinh sn hu tnh bng Zeaxanthine, giao t, cht d tr l tinh bt, Violaxanthine thnh sinh cht ch yu l cellulosse

a v c

3. ng vt n bo Ton b ng vt c chia lm hai mc t chc: ng vt n bo (Protozoa) v ng vt a bo (Metazoa) (theo phn gii truyn thng th l hai gii ph). ng vt n bo (i khi ngi ta gp vo nhm ny c nhng ng vt hin vi dng si nhiu nhn) l nhng c th n bo nhn chun, thng dinh dng hu c, mt s nh quang dng. Nhng ng vt n bo u tin c Leeuwenhoek A.V pht hin ra ngay t th k XVII nhng c nghin cu vo th k XVIII bi Joblot L. v nhiu tc gi khc. Theo h thng phn loi hin nay th ng vt dn bo c chia lm 4 nhm: 1. Sarcomastigophora vi cc nhnh Sarcodina hay Rhizopodes (Rhizopodes v Actinopodes), nhnh Mastigophora hay Flagelles, nhnh Opalinata (cng c ti liu hp nht Rhizopodes v Flagelles vo dng Rhizoflagelles). 2. Sporzoa (k sinh trn ng vt, mt hoc nhiu vt ch). 3. Cnidospora (k sinh trn ng vt c xng v khng xng). 4. Ciliophora hay cilie (roi ngn - cils, c hai loi nhn: nhn to v nhn b). Vi hn 30.000 loi c m t, ng vt n bo sng t v nc, nhiu ng vt n bo c vai tr quan trng cc lp bn hot tnh ti cc trm lc nc thi.

a v d

51

Bng 2.10: Mt s nhm ng vt n bo v tnh cht ca chng


Mt s tnh cht Ni sng V d Mt hoc nhiu roi Nc ngt, k sinh Trypanosoma, (flagella), t bo c th trn ng vt Giardia, chia dc. Leishmania Sarcodina Dng amip, gi tc, Nc ngt v mn, Amoeba, khng roi, chia i. k sinh trn ng vt Entamoeba Sporozoa Thng bt ng, mt s K sinh s cp trn Plasmodium (gy c th trn, b, chia ng vt chn t, bnh st rt cn i, k sinh ng vt, su tc nhn truyn bnh Malaria), b. k sinh Toxoplasma Ciliophora Nhiu roi ngn (tim Nc ngt v mn, Paramecium, mao-cilia), chia i k sinh trn ng vt, Balantidium ngang, mi t bo trong d con ca thng c 2 nhn, nhn ng vt nhai li ln v nhn b lm chc nng khc nhau. Cnidophora Hnh thnh chui bo t K sinh trn ng vt Nosema gy bnh nh si phnh ra v ct c xng v khng tm gai (Pebrina) khc. xng. Sau y l so snh mt s tnh cht ca cc nhm vi sinh vt Bng 2.11: So snh mt s tnh cht ca cc nhm vi sinh vt Tnh cht Vi khun Nm To ng vt n Ghi ch bo Loi t nhn s nhn chun nhn chun nhn chun bo Kiu dinh ha d dng ha d dng quang t ha d dng tnh cht dng (mt s quang hu c dng hu c s ng dng) a bo, n bo a bo (tr mt s n n bo n bo nm men) bo v mt s a bo Cch sp ring l, mt n bo, si n bo, tp ring l, tp vch hp si v hp xp t bo s hnh thnh khng ngn v si c bt u hnh tp hp vch ngn thnh m Phng quang hp, hp th, thc tnh cht hp th php thu hp th hp th bo s ng nhn thc n Tnh cht phn bo v bo t hu sc t quang chuyn ng c trng t (trc phn) tnh v v hp v sc t tnh h tr Thnh t Murein Hemicellulose cellulose khng c hoc bo v chitine lipoproteid pH ti u 6,5 - 7,5 3,8 - 5,6 gn trung trung tnh tnh cht Nhm Mastigophora

52

Nhu cu O2 Cht d tr chnh S loi hin bit

k kh n hiu kh cc loi polysaccharide 4000

hiu kh glucogen 80.000 (tt c gii nm)

tnh hiu kh tinh bt 15.000 (ch tnh to n bo)

hiu kh glucogen v nhiu loi polysaccharide 30.000 (ch tnh ng vt n bo)

s ng tnh cht s ng

Cu hi n tp chng 2
1. V s cu to t bo vi khun, trong ghi r cc cu to bt buc phi c v cu to khng thng xuyn ph thuc vo nhm vi khun. 2. So snh cu to thnh t bo vi khun v vi sinh vt c. Ni r vai tr ca thnh trong hot ng sng v trong phng php nhum Gram. 3. V s cu to mng sinh cht ca vi khun, ni r chc nng vn chuyn cc cht qua mng. 4. Bn cht ca cc vt th n nhp, cu to ca chng v kh nng nhum mu. 5. Cht nhn ca vi khun, nhng pht hin mi trong vn genophore ca c th nhn s. 6. Plasmid c th nhn s, vai tr v chc nng. 7. Mng nhy v tin mao, cu to v chc nng cc loi. 8. Ni bo t, cu to v nhum mu. 9. Nu mt s v d vi khun sinh bo t v khng sinh bo t, ng dng ca chng trong cng ngh vi sinh. 10. V s cu to t bo nm men. 11. Cu to thnh t bo nm. 12. Cc chu trnh sinh hc ca nm men, i din nm mc. 13. Nguyn tc v phng php phn loi vi sinh vt. 14. nh ngha v cho v d cc khi nim sau: bo t v tnh, bo t hu tnh, ni bo t, bo t nh, bo t ti, bo t m, si nm c vch ngn, si cng bo, si hai nhn. 15. Nu mt s nm c li v gy hi. 16. Cc nhm to, cu to t bo v thnh t bo. 17. ng vt n bo, cu to c trng khc vi vi khun. 18. So snh tng qut s khc bit ca vi khun, nm, to n bo v ng vt n bo.

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Chng 3

Cc khi nim c bn v virus


Virus l cc tc nhn rt nh c th gy bnh mi c th sng. Do cu to rt n gin nn mun nhn ln chng bt buc phi k sinh trong t bo v nh b my tng hp ca t bo.

I. c im ca virus
Kch thc nh. Virus c kch thc rt nh t 10nm n 300nm trong khi kch thc ca vi khun khong 1000nm v kch thc ca hng cu l 7500nm. V vy virus ch c th quan st c trn knh hin vi in t.

Hnh 3.1: Hnh thi ca mt s virus


1.i xng a din: [A] polio-, wart-, adeno-, rota-; [B] herpet. 2.i xng xon: [C] khm thuc l; [D] cm;[E] si, quai b, parainfluenza; [F] di; 3.i xng hn hp:[G] poxvirus; [H] phage T chn

Genome virus ch cha mt loi acid nucleic, c th l DNA hoc RNA, c th dng thng hoc khp kn, chui n hoc chui kp. Genome phn on hoc khng phn on. L dng sng khng c hot tnh trao i cht. Virus khng c ribosome hot ng hoc khng c b my tng hp protein. Cho nn mc d mt s virus c enzyme ring cu mnh nhng virus ch c th nhn ln

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trong t bo sng, iu khin b my tng hp ca t bo phc v cho mnh to thnh cc ht virus mi.

II. Cu trc virus


Virus c cu to rt n gin, bao gm li l acid nucleic, tc genome nm pha trong cn pha ngoi c bao bc bi v protein, v protein bo v genome khi s tc ng ca cc yu t mi trng v d nh nuclease trong mu. V protein c gi l capsid. Capsid c cu to bi cc n v hnh thi l capsome. Capsome li c cu to bi cc n v cu trc l protome. Protome c th l monome (ch c mt phn t protein) hoc polyme (nhiu phn t protein). Capsid v acid nucleic c gi l nucleocapsid.

Hnh 3.2. Cu trc c bn ca virion


Li l acid nucleic, v l capsome l protein, hp li thnh nucleocapsid. Nucleocapsid c bao bc bi lp v ngoi (lipoprotein) vi cc gai. A.S virus a din n gin nht, mi mt hnh a din l tam gic u. nh do 5 cnh hp thnh. Mi cnh cha 3 capsomer B. S ca virus hnh que vi cu trc i xng xon (virus khm thuc l). Capsomer sp xp xon xung quanh si acid nucleic dng xon c.

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Mt s virus cn cha v ngoi, bao bc bn ngoi capsid. V ngoi c bn cht l lipoprotein cha khng nguyn ca virus. V ngoi mt phn bt ngun t mng sinh cht ca t bo ch khi virus chui ra ngoi theo li ny chi. mt s virus, v ngoi c ngun gc t mng nhn ca t bo. Ht virus nguyn vn cn c gi l virion. Virus c 3 kiu cu trc Cu trc hnh khi. Capsid c cu trc hnh khi 20 mt tam gic u. Cu trc xon. Nucleocapsid dng ko di. Cc capsome sp xp xung quanh theo chiu xon ca acid nucleic. a s virus c cu trc xon c v ngoi bao bc nucleocapsid xon. Cu trc phc tp. Cu trc hn hp va dng khi va dng xon. V d phage c u dng khi, ui dng xon trng nh con nng nc.

III. Nui cy virus


Do virus ch sinh sn bn trong t bo sng nn phi c cc phng php c bit nui cy chng. C 3 h thng chnh dng nui cy virus trong phng th nghim. Nui cy m t bo. Cc t bo c ngun gc t cc m ca ngi hay ng vt c nui trong bnh cha mi trng nhn to, cho pht trin v dng lm ngun nguyn liu cy virus. Phi g. Mt s virus c th nhn ln trong t bo phi g 6- 13 ngy. Ngy nay phng php nui ny c thay th bi t bo nui cy m. Tuy nhin trong sn xut mt s loi vaccine, phng php ny vn c s dng. ng vt thc nghim. Trc y phng php ny c dng ph bin phn lp v nghin cu virus. Cc ng vt c s dng l chut, th, kh, chn...Tim hn dch nghi l c virus vo ng vt v quan st bnh cnh lm sng. Hin nay phng php ny vn c dng phn lp mt s virus. IV. nh hng ca virus ln t bo Virus c th tc ng ln t bo theo 4 cch sau: Gy cht t bo. Kt qu ca vic nhim virus l lm cho t bo b hu hoi, dn n lm cht t bo (CPE- Cytopathic effect).

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Chuyn dng. T bo b nhim virus nhng khng cht m chuyn t trng thi bnh thng sang trng thi c bit, thnh cc t bo u hoc ung th. Nhim tim tng. Virus tn ti bn trong t bo trng thi hot ng tim n nhng khng nh hng r rt n chc nng ca t bo. Gy ngng kt hng cu. Mt s virus trn b mt v ngoi c cha protein gy ngng kt hng cu (Haemaglutinin) gn trn b mt cc t bo nhim. Khi thm hng cu vo th hng cu s b kt dnh bi cc t bo nhim.

V. Phn loi virus


Virus c phn loi da theo c im hnh thi, bn cht ca genome (DNA hay RNA), c hay khng c v ngoi, v tr lp rp... U ban quc t phn loi virus quy nh: H virus c tip v ng l viridae, h ph virinae v chi- virus. Sau y l mt s virus gy bnh.
Bng 3.1. Virus gy bnh cha genome DNA Nhm virus Pox Herpes Tn virus Variola Molluscum (u mm) Herpes simplex Varicella zoster Cytomegalo EB ( Epstein- Barr), HH6 Virus adeno Vim gan B Papiloma Virus JC B19 Tn bnh u ma u mm ly Herpes Thu u zona (shingles) Nhim trong tho hip min dch Bnh bch cu n nhn ly nhim Bnh ngoi ban t ngt Vim hng Vim kt mc Vim gan Mn cc Vim cht trng no nhiu tin trin Ban truyn nhim, cn bt sn

Adeno Hepadna Papova Parvo

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Nhm virus Orthomyxo Paramyxo Corona Rhabdo Picorna Calici Toga Flavi Bunya Reo Arena

Bng 3.2. Virus gy bnh cha genome RNA Tn virus Tn bnh Virus cm cm;Hp bo h hp Si Quai b Virus corona Cm Vim nhim ng h hp Si Quai b Gy nhim ng h hp SARS Virus di Bnh di Entero Vim no, bi lit Rhino Cm lnh Vim gan A Vim gan SRSV (virus c cu trc dng Vim d dy, rut trn nh- small round structure virus) Alpha (virus arbo nhm A) Vim no Rubi St xut huyt Rubeon (si c) Flavi (virus arbo nhm B) Vim no St xut huyt Vim gan C Vim gan Mt s virus arbo Vim no St xut huyt St, vim thn Rota Bnh ng tiu ho Vim mng no m ri mng Vim mng no mch lympho bo Virus Machupo Virus Junin Virus lassa St xut huyt Ung th t bo T HTLV-I, II U lympho Lit AIDS HIV- 1, 2 Virus Marburg St Marburg Virus E bola St xut huyt Ebola

Retro

Filo

VI. nh hng ca tc nhn vt l, ho hc n virus


- Nhit cao: a s virus b bt hot 560C trong vng 30 pht, hoc 1000C trong vi giy.

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- Nht thp: a s virus bn nht lnh nn c th bo qun lu 700C. Mt s virus b bt hot trong qu trnh lm ng lnh hoc tan bng. - Kh hn: Kh nng chu kh hn ca virus khc nhau tu loi. Mt s sng st, mt s b bt hot nhanh iu kin kh hn - Bc x t ngoi: Virus b bt hot bi tia t ngoi - Chlorofoc, ete v cc dung mi khc: Cc virus c v ngoi cha lipid s b bt hot, cn khng cha lipid s bn vng. - Cc cht oxi ha v cht kh. Virus b bt hot bi di tc dng ca formaldehyt, clo, iot v H2O2. - propiolacton v formaldehyd l cc ho cht c dng bt hot virus trong sn xut vaccine, song a s virus khng b bt hot bi phenol. - Cht kh trng virus: Tt nht l dng dung dch hypoclorua (mt cht n mn) v glutaraldehyt (l cht c th gy mn cm v kch thch gy kh chu chy nc mt cho ngi dng).

VII. Cc bnh do virus


Virus l tc nhn gy bnh quan trng cho ngi, ng vt, cy trng v vi sinh vt. a s cc bnh thng gp ngi l do virus. Hu ht chng gy bnh th nh, bnh nhn t bnh phc sau mt thi gian nht nh. Nhiu loi tn ti thm lng trong c th. Chng nhn ln nhng khng gy bt k triu chng no. Tuy nhin vic nhim virus thng th nh, nhng i khi c th gy bnh trm trng nhng ngi mn cm bt thng. Mt s do virus gy bnh rt nng v thng c t l t vong cao.

VIII. Con ng ly nhim ca virus vo c th


Virus vo c th theo 4 con ng chnh: - Ht th: Qua ng h hp - n ung: Qua ng tiu ho (d dy- rut) - Xm nhp qua da, vt xc nim mc (qua quan h tnh dc), truyn mu, tim chch, phu thut cy ghp hay do cn trng hoc ng vt cn. - Bm sinh: Do m truyn qua nhau thai sang con

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IX. S xm nhp C ch gy bnh ch yu ca virus l s xm nhp. Bnh sinh ra do virus lan truyn trc tip ti cc m v c quan trong c th. S sinh sn ca virus trong t bo s git cht t bo (tuy nhin c trng hp khng git t bo). Tc ng gy hu hoi t bo c gi l CPE (cytopathic effect) s dn n tn thng v hu hoi chc nng ca cc m v c quan, ri t biu hin ra cc du hiu, triu chng.

X. Cc qu trnh nhn ln ca virus


Virus khng c hot tnh trao i cht m s dng b my trao i cht ca t bo tng hp cc thnh phn thit yu ca mnh, sau lp rp to ra cc ht virus con ging nh nguyn bn. V vy ngi ta thng s dng thut ng nhn ln (nhn bn) ca virus thay cho t sinh sn. S nhn ln ca virus c th lm tan t bo gi l chu trnh sinh tan hoc c th gn genome ca mnh vo nhim sc th ca t bo, tn ti lu di di dng tim n m khng l cht t bo gi l chu trnh tim tan. Qu trnh nhn ln ca virus din ra theo 7 giai on 1. Hp ph a. Gn th th c hiu ca mnh ln th th nm trn mng sinh cht ca t bo. V c tnh c hiu cao nn ch c virus nht nh mi gn ln c cc t bo nht nh. b. S hp ph c tng cng khi c mt ca ion Mg2+ hoc Ca2+ 2. Xm nhp a. Thng thng virus xm nhp vo t bo theo c ch m bo. - Virus khng c v ngoi : Mng t bo lm vo bao ly virus to khng bo tm thi. Tip khng bo dung hp cng vi mng li ni cht gii phng nucleocapsid. - Virus c v ngoi: V ngoi ca virus dung hp vi mng sinh cht ri y nucleocapsid vo trong m khng to khng bo. V ngoi virus ho vi mng sinh cht m khng chui vo t bo cht . - Mng t bo lm vo bao ly virus cng v ngoi, to khng bo. Sau mng khng bo (c ngun gc t mng t bo) dung hp vi v ngoi ca virus ri y nucleocapsid vo t bo cht.

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Hnh 3.3: Cc phng thc xm nhp ca virus vo t bo


1-Virus c v ngoi: a) Dung hp vi mng sinh cht, y nucleocapsid vo t bo. V ngoi virus nm trn mng sinh cht (v d virus paramyxo, herpes). b) Xm nhp theo li m bo, to khng bo. Dung hp vi mng li ni cht hoc endosom tiu ha ri gii phng nucleocapsid (v d virus cm, toga v rabdo) 2.Virus khng c v ngoi. Xm nhp theo li m bo, to khng bo, dung hp vi endosom tiu ha, tin hnh ci v v gii phng acid nucleic (v d virus polio, adeno v reo).

3. Ci v Enzyme tiu ho ca t bo t lysosome tin hnh phn gii v protein cu virus gii phng acid nucleic hoc genome vo t bo cht. 4. Phin m - To thnh mRNA ca virus hoc phn t dng sao chp ca genome (RF- replicative form) - Thc hin nh enzyme ca t bo hoc ca virus - Din ra theo c ch iu khin phc tp: Kiu phin m trc v sau khi sao chp acid nucleic hon ton khc nhau.

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Nhiu genome virus cha promoter v enhancer c tc dng kch thch qu trnh phin m. Bn phin m u tin thng c ct ni (splicing) loi b cc on intron nm xen gia cc exon. Phin m i khi xy ra theo c ch chng lp. Trong cng mt gen c nhiu im khi u v nhiu im kt thc, nh to ra nhiu protein t cng mt on acid nucleic - V c bn mRNA ca virus c nhng c im sau y (nhng cng c th khc): Cha trnh t khi u C gn m u 5 u 3 c gn ui polyA 5. Tng hp cc thnh phn ca virus 5.1. Tng hp protein ca virus mRNA ca virus c phin m trn ribosome ca t bo to ra 2 loi protein - Protein cu trc l protein capsid, protein v ngoi v protein trong li. - Protein khngcu trc l enzyme cn cho sao chp genome. Protein khng cu trc tm thy trong ht virus, tr mt s trng hp c bit v d enzyme phim m ngc c trong virus HIV hoc virus vim gan B cha DNA polymerase, mt s virus RNA cha RNA polymerase 5.2.Tng hp acid nucleic ca virus - Genome ca virus con c sao chp t genome ca virus m. Trong trng hp genome l mch n th khun l mch b sung mi to thnh ca genome m. - Phn ln qu trnh sao chp c thc hin nh polymerase (replicase) do virus m ho. i vi mt s virus DNA th qu trnh tng hp c thc hin nh enzyme ca t bo. 6. Lp rp - Genome v protein mi c to thnh lp rp vi nhau to nn ht virus mi. a s trng hp protein capsid lp rp to thnh cu trc

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rng gi l tin capsid (procapsid) sau do chuyn ng Brao, acid nucleic chui vo cu trc ny ri hn kn li. - Lp rp c th xy ra trong nhn t bo, trong t bo cht hoc ngay st mng sinh cht (i vi a s virus c v ngoi). Mng sinh cht bao ly nucleocapsid to v ngoi. 7. Gii phng Virus c th lm tan t bo chui t ra ngoi hoc i vi virus c v ngoi th chui ra t t theo li ny chi.

XI. Genome virus


C th l DNA hoc RNA m khng bao gi cha c hai. C dng chui n hoc chui kp. Dng thng hoc khp kn (ng vng). Virus cha genome DNA kp thng c kch thc ln, virus cha genome DNA n thng c kch thc nh. Genome RNA kp tt c u phn on (trong mi ht virus c nhiu on). Cn phn ln genome RNA n khng phn on (tr virus cm v HIV). Genome RNA n c trnh t nucleotit ging trnh t ca mRNA th c quy c l genome (+), cn ngc li c gi l genome (-). Genome ca HepDNAaviridae (v d virus vim gan B) khi sao chp phi thng qua RNA trung gian (DNA RNA- DNA). Acid nucleic ca virus c kch thc ln. C trng lng phn t ln. M ho cho nhiu protein. M cho nhiu enzyme cn cho qu trnh nhn ln. Acid nucleic ca virus c kch thc nh. C trng lng phn t nh. Do d kh nng m ho to protein hn ch. Nhiu trng hp virus phi c gen chng lp. nhn ln phi dng mt s enzyme ca t bo.

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Kh nng gy nhim. i vi nhiu loi virus, acid nucleic ch c kh nng ly nhim khi c a vo trong t bo. V d khi c gii phng khi v capsid, bn thn acid nucleic c th t thc hin qu trnh gy nhim t bo, bt u chu trnh gy nhim hon chnh nhn ln. Genome ca cc virus cha RNA (-) khng c kh nng gy nhim. Mun nhn ln chng phi phin m thnh RNA (+).

XII. Protein ca virus


Tu thuc vo thi gian to thnh m protein ca virus c chia lm 3 loi Protein c tng hp ngay sau khi nhim c gi l protein sm tc protein khng cu trc. y l enzyme cn cho sao chp acid nucleic . Protein c tng hp mun hn gi l protein mun, thng l protein cu trc to capsid, v ngoi v protein li. Protein phn gii, thng l lyzozym gip virus gii phng ra khi t bo. Ba loi protein c iu ho tng hp mt cch hp l. Protein sm l enzyme xc tc nn ch cn mt lng nh, cn protein cu trc th phi tng hp mt lng ln.

XIII. Cc phng thc nhn ln ca virus


Qu trnh nhn ln ca virus trong t bo ht sc phc tp. Mi nhm virus c cch nhn ln ring. Virus c chia lm 6 nhm da vo genome v cch thc tng hp mRNA. Sau y l vi nt n gin ho qu trnh nhn ln nu bt s khc nhau gia cc nhm. Cc virus c genome DNA 1. Nhm 1. virus DNA kp V d virus vaccinia, herpes simplex, adeno, papiloma. 1.1. Dch m C 2 loi mRNA c tng hp . mRNA sm: c to thnh trc khi tng hp DNA virus, ch yu m ho cho cc enzyme tham gia vo qu trnh tng hp acid nucleic ca virus,

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mRNA mun: to thnh sau khi tng hp DNA virus , ch yu m ho cho cc protein cu trc nh v capsid, v ngoi 1.2. Tng hp DNA virus - Cc enzyme Enzyme sao chp DNA ch yu l DNA polymerase ph thuc DNA.Cc virus c kch thc ln hn (v d vaccinia, herpes simplex) th t tng hp DNA polymerase ring cho mnh.Cc virus c kch thc nh (v d adeno, papiloma) th s dng DNA polymerase ca t bo - Khun: DNA ca virus con c tng hp trn khun genome DNA ca virus m. - V tr tng hp: trong nhn t bo (tr virus pox). - DNA mi to thnh Dng lm khun to mRNA mun cn cho tng hp protein mun. Lm khun to mch tngb cn cho tng hp nhiu genome ca virus mi. 1.3. Tng hp protein virus Qu trnh gm 2 giai on tu thuc vo s tng hp mRNA. - Tng hp protein sm: y l cc enzyme (DNA polymerase ph thuc DNA ) cn cho sao chp DNA. - Tng hp protein mun Din ra sau khi tng hp DNA Ch yu l cc protein cu trc to v capsid v v ngoi V tr tng hp : Protein c tng hp trn ribosome trong t bo cht , sau c vn chuyn ti v tr lp rp. 1.4. Lp rp Qu trnh lp rp genome vi protein to thnh ht virus mi xy ra cc v tr khc nhau bn trong t bo. - Trong nhn t bo: V d virus Herpes simplex, adeno, papiloma. Ring virus herpes hnh thnh v ngoi bng cch ny chi qua mng nhn. Mng nhn trc c gn glycoprotein do virus m ho ti

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cc v tr c hiu. Virus c bao bi mng nhn khi ra khi nhn, sau kt hp vi mng li ni cht sn hoc b my Golgi ra khi t bo. - Trong t bo cht: V d virus vaccinia nhn ln hon ton trong t bo cht ti cc cm ribosome. 1.5. Virus c genome DNA kp c bit Virus vim gan B (HBV) c chu trnh nhn ln c bit v phc tp. Genome gm 2 mch khng bng nhau. Mch (-) di, mch (+) ngn. Cha enzyme DNA polymerase. Sau khi nhim, DNA c gii phng vo nhn. Phin m xy ra trong nhn nh RNA polymerase ca t bo to ra nhiu loi mRNA trong c RNA kch thc ln c coi l tin genome mt dng trung gian to genome. Cc RNA i ra t bo cht tng hp protein ca virus nh protein li v polymerase. Enzyme ny c 3 hot tnh ( DNA polymerase, enzyme phin m ngc v ribonuclease H). Tip RNA tin genome lin kt vi DNA polymerase v protein li to ra ht li (virus cha hon chnh). Enzyme phin m ngc tin hnh chuyn RNA tin genome thnh mch DNA (-), sau hu ht RNA tin genome b phn hu nh ribonuclease H. Ch mt on RNA c gi li dng lm mi cho DNA polymerase tng hp mch DNA (+) t khun DNA (-) to chui DNA kp. Tip l hon thin nucleocapsid. 2. Nhm 2. Virus DNA n. Ch c mt h duy nht l Parvoviridae. Cc virus cha DNA n thng c genome nh. Tin hnh sao chp trong nhn nh DNA polymerase ca t bo to DNA kp trung gian, gi l dng sao chp (RF-replicative form). RF va dng lm khun tng hp DNA n genome va dng phin m to mRNA sau dch m tng hp protein . Mt s virus c khim khuyt nn mun nhn ln cn s h tr ca cc virus khc. Cc virus c genome RNA. Do vt liu di truyn l RNA nn virus c c ch sao chp khng ging vi cc c th sng khc. Genome RNA ca virus c cc dng phin m sau y.

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- RNA n, (+): Genome ca virus chnh l mRNA. - RNA n, (-): mRNA c phin m t genome ca virus m . - RNA kp, phn on: mRNA ca virus c phin m ring t mi on RNA genome ca m s dng transcriptase lin kt vi mi on. Virus RNA c cc c im sau: Do t bo ch khng c RNA polymerase ph thuc RNA nn enzyme ny bt buc phi c m ho bi genome virus v thng c mt trong ht virus trng thnh. Gen m ho cho RNA polymerase thng l gen ln nht trong genome v c lp hon ton vi nhn t bo ch trong sao chp v phin m. Do vy khc vi virus DNA gy nhim eucaryota, rt nhiu virus tin hnh nhn ln hon ton trong t bo cht. Cc enzyme RNA polymerase ph thuc RNA hot ng khng chnh xc nh polymerase ph thuc DNA v khng c kh nng c sa (proofreading), nn c tn s t bin rt cao, khong 10-3- 10-4 base, qua mi chu k sao chp xut hin mt t bin, gp 3-4 ln so vi virus DNA. iu ny dn n 3 h qu: Tn s t bin virus rt cao nn nu virus c chu k nhn nhanh th s bin i khng nguyn cng din ra nhanh, v th tnh c cng pht trin rt nhanh. iu ny c c l do virus RNA thch nghi rt nhanh vi s thay i ca iu kin mi trng hoc t bo ch. Mt s virus RNA t bin nhanh n ni chng to thnh v tn ti cc qun th cha cc genome khc nhau ngay trong mt vt ch. Vic xc nh chng mc phn t ch c th da vo cc trnh t chim a s hoc trung bnh. Do nhiu t bin c hi cho s nhn ln ca virus, nn tn s t bin t gii hn cao hn kch thc genome RNA, vo khong 10 nucleotit. Sau y l mt s v d v qu trnh nhn ln ca virus RNA. 3. Nhm 3. Virus RNA n, (+) V d virus gy bnh bi lit polio. Lc u khng c qu trnh phin m, v RNA genome c trnh t nucleotid ging vi trnh t ca mRNA nn lm lun chc nng ca mRNA.

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C kh nng gy nhim v genome m ho c cho tt c cc protein cn thit ca virus. 3.1. Dch m Genome RNA (+), dng lm mRNA tng hp polypeptit tin cht ln sau nh protease phn ct thnh cc phn t nh c chc nng khc nhau nh: Protein cu trc to capsid. RNA polymerase ph thuc RNA cn cho qu trnh sao chp (t bo khng c enzyme ny) Protease phn ct peptit ln. Protein gn vo u 5' ca genome (thay cho m) gi l protein VPg, cn cho khi u sao chp. 3.2. Tng hp RNA ca virus S to thnh genome mi xy ra trn khun RNA dng sao chp (RF). Dng ny c to thnh bng cch tng hp RNA (-) b sung trn khun RNA (+) m. Genome RNA (+) c tng hp trn khun RNA (-) ca dng sao chp RF

Hnh 3.3: Chu trnh n gin ho qu trnh nhn ln ca virus RNA (-), n

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RNA polymerase ph thuc RNA tham gia tng hp c RF v c genome RNA (+) mi . RNA (+) mi c tng hp c nhng chc nng sau: Lm khun tng hp nhiu RF ri t tng hp nhiu RNA genome. Dng lm genome cho cc virus mi Dng lm mRNA 3.3. Lp rp Protein c to thnh sau khi phn ct polypeptit tin cht s lp rp vi RNA genome mi trong t bo cht to ra virus mi Vi virus polio th s nhn ln hon ton xy ra trong t bo cht 13.3.4. Gii phng Virus ph v t bo t ngt t ra ngoi . 4. Nhm 4. Virus RNA n, (-) V d virus cm hoc cm. Chu trnh nhn ln c minh ho hnh 3.4

Hnh 3.4: Chu trnh n gin ho qu trnh nhn ln ca virus RNA (+), n

c im qu trnh phin m c th tm tt nh sau:

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Virus RNA (-) lun mang theo RNA polymerase ph thuc RNA v t bo khng c enzyme ny. mRNA c tng hp trong nhn t bo t genome RNA (-) ca m nh enzyme RNA polymerase ph thuc RNA c trong ht virus . Virus cm cha genome nhiu on do mi mRNA to thnh c dng tng hp mt loi protein. 4.1. Tng hp RNA genome. Khc vi phin m to mRNA, mun tng hp genome RNA (-) trc ht phI tng hp chui RNA trung gian lm khun. Qu trnh ny khng cn mi. Tip tng hp RNA (-) genome trn mch khun trung gian. 4.2. Tng hp protein. Protein ca virus bao gm: Enzyme transcriptase (RNA polymerase ph thuc RNA ). Protein v ngoi gm 2 loi u c gn glucoz (glycosyl ho). Mt loi c hot tnh haemaglutinin hoc neuraminidase v mt loi c hot tnh dung hp hoc tan mu. 4.3. Lp rp Nucleocapsid ca virus c lp rp mng nhn t bo v to v ngoi khi ny chi qua mng sinh cht. Virus cm l virus RNA nhiu on, n. Mi on phin m cho mt m RNA ring t tng hp thnh cc phn t protein c chc nng khc nhau. RNA mi ca virus c tng hp trong nhn t bo. Qu trnh ny cn c s tham gia phin m cu t bo ch. Protein virus c tng hp trong t bo cht ri di chuyn ti b mt t bo. Ti y glycoprotein v ngoi cng c gn vo cc v tr nht nh. Genome RNA mi c vn chuyn n b mt t bo v lp rp vi protein mi tng hp ca virus. Ht virus c bao v ngoi khi ny chi qua mng sinh cht 5. Nhm 5. Virus RNA kp Nhm ny bao gm cc virus Reo v Rota. Tt c cc virus RNA kp u c genome nhiu on. Mi on phin m cho mt mRNA tng hp mt protein ring.

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Ht virus cha RNA polymerase ph thuc RNA . Phin m thc hin theo c ch bo th v khng i xng, ngha l ch to thnh mRNA. Mi mRNA li c sao mt ln to chui RNA kp, cng chnh l genome (s nhn ln c minh ho hnh sau ).

Hnh 3.5: Chu trnh n gin ho qu trnh nhn ln ca virus Retro (RT: Enzyme phin m ngc)

Hnh 3.6: Chu trnh n gin ho qu trnh nhn ln ca virus RNA kp (RdRp: Enzyme RNA polymerase ph thuc RNA)

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6. Nhm 6. Virus Retro cha genome DNA n, (+) Rt nhiu nhng khng phi tt c virus retro c kh nng to khi u. Khi nhn ln khng git cht t bo m c th chuyn dng thnh t bo ung th. Genome l 2 phn t RNA n, (+), gn vi nhau pha u (dng dime). Cha 3 gen chnh l gap (m cho protein li), gen pol m cho polymerase phin m ngc (RT) v gen env m cho protein v ngoi. Ngoi ra cn c mt s gen iu ho. 6.1.Qu trnh nhn ln Picorma v flavivirus

1
i genome RNA (-)

Genome RNA (+)

2
Enzyme sao chp

Tin polyprotein

Protein cu trc v khng cu

Virus mi
Hnh 3.7. S nhn ln ca virus RNA chui dng, khng to thnh cc mRNA genome. Sau khi xm nhp v ci v (bc 1), RNA genome c s dng trc tip tng hp cc polyprotein kch thc ln, cc protein cu trc v khng cu trc (bc 2 v 3). Protein khng cu trc (enzyme) xc tc sao chp RNA thng qua s tng hp RNA chui m (i genome) (bc 5 v 6). Qu trnh sao chp to ra nhiu genome RNA mi phc v cho tng hp cc protein ca virus (bc 6 v 7) v cho lp rp to virus mi (bc 8). Mi tn m th hin s tng hp mc cao hn.

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Gm 2 giai on: - Giai on u Phin m nh enzyme phin m ngc to chui lai RNA/ DNA. Chuyn chui lai RNA/DNA thnh chui DNA kp. Enzyme RT c hot tnh ribonuclease H phn gii mch RNA. Cn mch cDNA dng lm khun tng hp mch DNA b sung. Ci xen phn t DNA kp mi tng hp vo nhim sc th ca t bo to ra provirus. - Giai on hai. RNA ca virus phin m nh enzyme ca t bo. Bn sao RNA c 2 chc nng: va l m RNA tng hpj protein virus, va l genome ca virus mi. 6.2. Tng hp protein mRNA c phin m t DNA provirus tin hnh tng hp protein trn ribosome nm trong t bo cht, bao gm: Enzyme phin m ngc, protein li, protein v ngoi. 6.3. Lp rp Nucleocapsid c lp rp t genome RNA mi to thnh vi protein di tr ra b mt t bo. V ngoi c hnh thnh khi virus ny chi qua mng sinh cht.

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Atro, calici, toga, corona v arterivirus

1
Di genome mRNA

i genome RNA (-)

5 2
Enzyme sao chp

Genome RNA (+)

7
Protein cu trc v tin polyprotein

Tin polyprotein khng cu trc

4 8

10

Protein khng cu trc

Protein cu trc

11

Virus mi

Hnh 3.8. S nhn ln ca virus RNA dng, km theo tng hp RNA di genome. Sau khi xm nhp v ci v (bc 1), RNA genome c dng trc tip lm mRNA tng hp protein khng cu trc, trong c enzyme RNA polymerase ph thuc RNA (RdRp) ( bc 2 v 3). RdRp xc tc sao chp RNA, tng hp chui RNA m (i genome) c kch thc (tng ng RNA genome) ( bc 4 v 5). T chui m lm khun tin hnh sao chp to ra nhiu chui RNA genome mi (bc 5 v 6). mRNA di genome (c kch thc nh hn genome) tin hnh dch m to protein cu trc ( bc 7 v 8). Genome mi to thnh tin hnh dch m to nhiu protein khng cu trc (bc 9 v 10) v sau lp rp vi protein cu trc to virus mi ( bc 11). Ch thch: Bc 7. Dch m ca mRNA di genome; Bc 8. Phn ct nh protease v x l sau dch m; Bc 10. Phn ct nh protease

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Phabdo, filo, borna, paramyxo, orthomyxo v mt s bunya

1
RNA (+) i genome

5 2
Protein capsid v

RNA genome

mRNA

3 4

7 8

Protein cu trc v khng cu trc

Virus mi

Hnh 3.9. S nhn ln ca virus RNA chui n, m . Sau khi xm nhp v ci v mt phn( bc 1), RNA tin hnh phin m nh RNA polymerase ph thuc RNA do virus mang theo to mRNA (bc 2) cho tng hp protein cu trc v khng cu trc (RdRp) ( bc 3). Enzyme RdRp tin hnh sao chp RNA thng qua bc trung gian l to RNA chui dng (i genome) (bc 4 v 5). i genome (chui dng) c dng lm khun tng hp nhiu RNA genome (bc 5). Mt phn RNA genome c dng tin hnh phin m mnh, to ra mRNA cn cho tng hp protein (bc 7), mt phn dng lp rp vi protein to virus mi (bc 8).

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Virus Reo v Birna

1
mRNA trong ht di mc virus

9 5

Genome RNA kp trong ht di mc virus

4 8

mRNA virus

7 10

Protein cu trc v khng cu trc

Virus mi

Hnh 3.10. S nhn ln ca virus RNA chui kp. Sau khi xm nhp v ci v tng phn (bc 1), cc on RNA chui kp vn nm trong li tin hnh phin m nh enzyme RdRp gn li tng hp mRNA (bc 2) ri t tng hp protein (bc 3). Cc protein ny lp rp quanh mRNA to ht di mc virus (bc 4). Sau mRNA trong cc ht ny tin hnh sao chp to RNA genome chui m tham gia to thnh RNA genome chui kp (bc 5). Cc ht di mc virus c to thnh li tip tc tham gia vo qu trnh tng hp m RNA (bc 6), tng hp protein (bc 7), sao chp (bc 8 v 9), lp rp vi protein cu trc to thnh virus mi (bc 10) Ch thch: Bc 1. Xm nhp v ci v Bc 4. Lp rp to ht di mc virus. Bc 10. Lp rp to virus mi.

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Virus Retro

1
DNA kp

cDNA (i genome)

RNA genome

4
DNA ci xen (provirut)

RNA cha ch bin

6
RNA virus sau ch bin

7 8

Cc bn sao DNA kp ca virus

Protein cu trc, khng cu trc v protein gy ung th

Virut mi Hnh 3.11. S nhn ln ca virus Retro.


Sau khi xm nhp v ci v tng phn (bc 1), RNA genome c phin m thnh cDNA (i genome), sau thnh DNA chui kp nh enzyme phin m ngc (bc 2 v 3). Nh enzyme integrase, DNA ca virus ci xen vo nhim sc th t bo ch (bc 4). C hai loi enzyme trn u nm trong li ca virus. Nh RNA polymerase ca t bo, provirus phin m to RNA (bc 5) l tin cht ca mRNA (bc 6) tng hp protein (bc 7) v lp rp to virus mi (bc 8).

Cu hi n tp chng 3
1.Hy gii thch cc thut ng sau y: capsome, capsid, nucleocapsid, capsid c cu trc xon, khi a din, virus c cu trc phc tp, v ngoi. 2.Virus khc vi plasmit v khc vi t bo im no. 3.Virus c th tc ng ln t bo nh th no ? 4.Nu cc giai on nhn ln ca virus.

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5.Ti sao virus ch c th nhn ln trong cc t bo nht nh ? 6.Trnh by cc phng thc xm nhp ca virus vo t bo ch. 7.Hy nu cc loi genome ca virus, Th no l RNA(+), RNA().Th no l genome chng lp. 8.Dng sao chp (RF) khc vi genome im no ? 9.Ti sao cc virus RNA(-) nht nh phi mang theo enzyme RNA polymerase. 10.Hy tm tt qu trnh nhn ln ca cc virus AND n, AND kp,RNA(+), RNA(-).

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Chng 4

Dinh dng, sinh trng v pht trin ca vi sinh vt


I. Dinh dng
Trong qu trnh sng, t bo vi sinh vt tin hnh trao i cht khng ngng vi mi trng chung quanh. T bo vi sinh vt tuy rt nh, nhng v hp thu cc cht dinh dng v thi ra cc sn phm trao i cht qua ton b b mt, cho nn cng trao i cht ca chng l rt ln. Cc cht dinh dng vo t bo qua mng v c chuyn ho to thnh nhng cht ring bit cn thit cho vic xy dng t bo. Nh qu trnh ng ho cc t bo mi c th sinh trng, pht trin tng sinh khi, ng thi to ra cc sn phm trao i cht. S bin i cc cht dinh dng bao gm nhiu phn ng ho sinh khc nhau nh h enzyme theo con ng trao i cht, hoc to ra nhng cht l thnh phn ca t bo (qu trnh ng ho) hoc to ra nng lng sinh hc cn thit cho hot ng sng (qu trnh d ho). Nhng cht dinh dng l nhng hp cht phn t nh c th i qua mng vo bn trong t bo vi sinh vt v tham gia vo hai loi phn ng sinh ho: - Bin i d ho lm xut hin nhng sn phm c cu trc n gin hn. Nhng bin i d ho ny cung cp cho vi sinh vt nng lng chuyn ho dng ATP hoc nhng hp cht giu nng lng khc. Mt s nhng sn phm d ho c thi i, mt s khc lm vt liu hoc lm tin cht cho cc phn ng ng ho. - Bin i ng ho, m bo s tng hp ca thnh phn mi c cu trc phc tp hn v phn t lng cao hn. Qu trnh ny thng c gi l ng ho hoc sinh tng hp. iu kin ch yu sinh tng hp cc thnh phn t bo vi sinh vt l cung cp mt lng thch hp ca nhng hp cht c phn t lng nh, nh cc acid hu c hoc amino acid, c th lm nguyn liu hay tin cht cho cc phn ng ng ho hay phn ng sinh tng hp. Khi trong mi trng c nhng hp cht - vt liu th vi sinh vt s trc tip s dng. Nhng khng phi bao gi trong mi trng cng c sn nhng hp cht - vt liu cn cho qu trnh sinh tng hp. Mun c t bo vi sinh vt bt buc phi t sn xut ly bng cch t bin i nhng thnh phn ca mi trng nui cy. nhng vi sinh vt d dng sng bng cht hu c,

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mt s tin cht c hnh thnh trong cc phn ng d ho, n sn sinh ra ATP cng mt s ln cc hp cht khc nhau ca carbon. Cc cht dinh dng ca vi sinh vt ch yu ly mi trng xung quanh. Cc mi trng dinh dng nhn to cn cung cp y nng lng, cc vt liu xy dng t bo v m bo cho hiu sut sinh tng hp cao. Thnh phn mi trng gm c cc ngun n carbon, nit, cht khong, cc nguyn t vi lng v cc cht kch thch sinh trng. Vic la chn cc ngun dinh dng v nng ca chng trong mi trng ph thuc vo c tnh sinh l ca tng chng tng loi vi sinh vt v iu kin nui cy chng.

II. Mi trng dinh dng v iu kin sinh trng


1. Mi trng dinh dng 1.1. Ngun thc n carbon Ty nhm vi sinh vt m ngun carbon c cung cp c th l cht v c (CO2, NaHCO3 , CaCO3... ) hoc cht hu c. Gi tr dinh dng v kh nng hp th cc ngun thc n carbon khc nhau ph thuc vo thnh phn ha hc, tnh cht sinh l ca ngun thc n v c im sinh l ca tng loi vi sinh vt. Hu nh khng c hp cht carbon no m khng b hoc nhm vi sinh vt ny hoc nhm vi sinh vt khc phn gii. Khng t vi sinh vt c th ng ha c c cc hp cht carbon rt bn vng nh cao su, cht do, du m, parafin, kh t nhin. Nhiu cht hu c v khng tan c trong nc hoc v c khi lng phn t qu ln cho nn trc khi c hp th, vi sinh vt phi tit ra cc enzyme thy phn (amylase, cellulase, pectinase, protease, lipase...) chuyn ha chng thnh cc hp cht d hp th (ng, amino acid, acid bo...). Ngi ta thng s dng ng lm ngun thc n carbon khi nui cy phn ln cc vi sinh vt d dng. nui cy cc loi vi sinh vt khc nhau ngi ta dng cc nng ng khng ging nhau. Vi vi khun, x khun thng dng 0,2 - 0,5% ng cn i vi nm men, nm si thng dng 3 - 10% ng. Hu ht vi sinh vt ch ng ha c cc loi ng dng ng phn D. Nhng phn ln cc ng phn ca ng n trong t nhin u l thuc loi D. Cc hp cht hu c cha c C v N (peptone, nc tht, nc chit ng, nc chit nm men, nc chit i mch, nc chit gi u...) c th s dng va lm ngun C va lm ngun N i vi vi sinh

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vt. Vi vi sinh vt d dng, ngun thc n carbon lm c hai chc nng, ngun dinh dng v nng lng. 1.2. Ngun dinh dng nit (N) ngha ch yu ca cc cht dinh dng cha nit i vi vi sinh vt l cung cp N to thnh cc nhm amin (-NH2) v nhm imin (=NH) trong phn t amino acid, cc nucleotide, purine v pirimidine, trong nhiu vitamin... Ngun nit d hp th i vi vi sinh vt l NO3- v NH4+. Mui nitrate l ngun thc n nit thch hp i vi nhiu loi to, nm si v x khun nhng t thch hp i vi nhiu loi nm men v vi khun. Sau khi vi sinh vt s dng ht gc NO3- cc ion kim loi cn li (K+, Na+, Mg2+..) s lm kim ha mi trng. trnh hin tng ny ngi ta thng s dng mui NH4NO3 lm ngun nit cho nhiu loi vi sinh vt. Tuy nhin gc NH4+ thng c hp th nhanh hn gc NO3-. Vi sinh vt cn c kh nng ng ha rt tt nit cha trong cc thc n hu c. Cc thc n ny s va l ngun carbon va l ngun nit cung cp cho vi sinh vt. Vi sinh vt khng c kh nng hp th trc tip cc protein cao phn t. Rt nhiu vi sinh vt c kh nng sn sinh protease thy phn protein thnh cc hp cht phn t thp c kh nng xm nhp vo t bo vi sinh vt. Ngun nit hu c thng c s dng nui cy vi sinh vt l peptone (l ch phm thy phn khng trit ca mt ngun protein no ) chng khc nhau v lng cha cc loi polypeptide v lng cha amino acid t do. Ngoi ra phn t N2 t do cng c mt nhm nh vi sinh vt s dng gi l nhm vi sinh vt c nh nit (nitrogen fixing microorrganisms). Chng c h enzyme nitrogenase c th b gy ni ba ca nit phn t iu kin nhit v p sut bnh thng. 1.3. Ngun dinh dng khong Khi s dng cc mi trng thin nhin nui cy vi sinh vt thng khng cn thit b sung cc nguyn t khong. Trong nguyn liu dng pha ch cc mi trng ny (khoai ty, nc tht, sa, huyt thanh, peptone, gi u...) thng c cha cc nguyn t khong cn thit i vi vi sinh vt. Ngc li, khi lm cc mi trng tng hp (dng nguyn liu l ha cht) bt buc phi b sung cc nguyn t khong cn thit. Nhng nguyn t khong m vi sinh vt i hi phi c cung cp vi liu lng ln c gi l cc nguyn t i lng. Cn nhng

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nguyn t khong m vi sinh vt ch i hi vi nhng liu lng rt nh c gi l cc nguyn t vi lng. Phosphorus (P) bao gi cng chim t l cao nht trong s cc nguyn t khong ca t bo vi sinh vt (nhiu khi P chim n 50% so vi tng s cht khong). P c mt trong cu to ca nhiu thnh phn quan trng ca t bo (acid nucleic, phosphoprotein, phospholipid, ADP, ATP, UDP, UTP, CDP, CTP, NAD, NADP, flavin...; mt s vitamin nh thiamine, biotin...). m bo ngun dinh dng P ngi ta thng s dng cc loi mui phosphate v c. Vic b sung phosphate vo cc mi trng dinh dng ngoi tc dng cung cp P cn c tc dng to ra tnh m ca mi trng. Lu hunh (S) cng l mt nguyn t khong quan trng trong t bo vi sinh vt. S c mt trong mt s amino acid (cysteine, cystine, methionine), mt s vitamin (biotin, thiamine...) cysteine, cystine v mt tripeptide l glutation khng ch tham gia vo cu trc protein m cn c vai tr quan trng trong cc qu trnh xy ha kh. Cc hp cht hu c c cha S dng xy ha thng c tc dng c i vi t bo vi sinh vt. Trong khi cc mui sulphate v c vi nguyn t S trng thi xy ha li c c th vi sinh vt ng ha rt tt. Mt s vi sinh vt c th dng c thiosulphate lm ngun thc n S. Mt s vi sinh vt khc li i hi cc thc n cha S dng kh (H2S, cystine, cysteine...). Fe l nguyn t rt cn thit gip vi sinh vt c th tng hp mt s enzyme loi porphiryl cha st (nh cytochrome, cytochrome oxydase, peroxydase, catalase...). Mt s vi sinh vt t dng quang nng cn s dng Fe tng hp ra cc sc t quang hp c cu trc porphiryl (chlorophyll, bacteriochlorophyll). Mg l nguyn t c vi sinh vt i hi cung cp vi lng kh cao. Mg mang tnh cht mt cofactor, chng tham gia nhiu phn ng enzyme c lin quan n qa trnh phosphoryl ha. Mg2+ c th hot ha cc enzyme nh hexokinase, ATP - ase, carboxylase, cc enzyme trao i protein, cc enzyme xy ha kh ca chu trnh Krebs... Mg cn c vai tr quan trng trong vic lin kt cc tiu phn ribosome vi nhau. Ca mc du l nguyn t t tham gia vo vic xy dng nn cc cht hu c nhng n c vai tr ng k trong vic xy dng cc cu trc tinh vi ca t bo. Ca ng vai tr cu ni trung gian gia nhiu thnh phn quan trng ca t bo sng (nh gia ADN v protein trong nhn, gia cc nucleotide vi nhau, gia ARN v protein trong ribosome). Ca rt cn thit i vi vic hnh thnh cc cu trc khng gian n nh ca nhiu bo quan nh ribosome, ti th, nhn...

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Zn cng l mt cofactor tham gia vo nhiu qu trnh enzyme. Zn c tc dng ng k trong vic hot ha cc enzyme nh carbonanhydrase, enolase, pyrophosphatase, phosphatase kim ... Cng c mt s nguyn t ha hc chng ta cha hiu r v vai tr sinh l ca chng, trong s phi k n l K. K l nguyn t chim t l kh cao trong thnh phn khong ca t bo vi sinh vt, nhng cho n nay ngi ta cha tm thy K tham gia vo bt k thnh phn no ca nguyn sinh cht, cng cha tm thy bt k enzyme no c cha K. K thng tn ti dng ion K+ mt ngoi ca cu trc t bo. Mt phn ng k K tn ti trng thi lin kt ha l khng bn vi protein v cc thnh phn khc ca nguyn sinh cht. K lm tng ngm nc ca cc h thng keo do nh hng n cc qu trnh trao i cht, nht l cc qu trnh sinh tng hp. K cn c th tham gia vo qu trnh tng hp mt s vitamin v c nh hng n qu trnh h hp ca t bo vi sinh vt. Na v Cl cng l nhng nguyn t m nhiu vi sinh vt i hi vi mt lng khng nh, nhng cho ti nay ngi ta vn cn bit rt t vai tr sinh l ca chng. Hm lng Na v Cl c bit cao trong t bo vi sinh vt a mn sng trong nc bin, t, vng ven bin hoc trn cc loi thc phm p mn. 1.4. Yu t sinh trng i vi vi sinh vt th yu t sinh trng l mt khi nim rt linh ng. N ch c ngha l nhng cht hu c cn thit i vi hot ng sng m mt loi vi sinh vt no khng t tng hp c t cc cht khc. Nhng cht c coi l yu t sinh trng ca loi vi sinh vt ny hon ton c th khng phi l yu t sinh trng i vi mt loi vi sinh vt khc. Hu nh khng c cht no l yu t sinh trng chung i vi tt c cc loi vi sinh vt. c im ca mi trng sng mt mt nh hng n kh nng tng hp yu t sinh trng ca vi sinh vt, mt khc nh hng n c im trao i cht ca chng. Chnh thng qua cc nh hng ny m mi trng sng ca tng loi vi sinh vt gp phn quyt nh nhu cu ca chng v yu t sinh trng. Khi sng lu di trong cc mi trng thiu yu t sinh trng, vi sinh vt s dn dn to ra c kh nng t tng hp yu t sinh trng m chng cn thit. Cng mt loi vi sinh vt nhng nu nui cy trong cc iu kin khc nhau cng c th c nhng nhu cu khc nhau v yu t sinh trng. Chng hn nm mc Mucor rouxii c chng minh l ch cn biotin v

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thiamine khi pht trin trong iu kin k kh. Khi nui cy trong iu kin hiu kh, chng s t tng hp ra c yu t sinh trng ny. S c mt ca mt s cht dinh dng khc c khi cng nh hng n nhu cu v yu t sinh trng ca vi sinh vt. Chng hn vic i hi acid pantotenic ca mt s vi sinh vt (v d vi khun bch hu Corynebacterium diphtheriae) c th tha mn khi ch cn cung cp cho chng - alanine, chng c th t tng hp c acid pantoic (acid pantotenic cu to t acid pantoic v - alanine). Thng thng cc cht c coi l yu t sinh trng i vi mt loi no c th thuc v mt trong cc loi sau y: cc gc kim purine, pirimidine v cc dn xut ca chng, cc acid bo v thnh phn ca mng t bo, cc vitamin thng thng... Mt khc, nhiu loi vi sinh vt c kh nng tng hp vitamin. Sinh khi vi sinh vt thng cha hu ht cc loi vitamin ch yu vi nhng hm lng kh cao. Ngi ta s dng thnh cng mt s loi vi sinh vt sn xut quy m cng nghip mt s loi vitamin tinh khit dng trong y hc. ng ch nht l acid ascorbic (vitamin C), riboflavin (vitamin B2), cyanocobalamin (vitamin B12). 2. iu kin sinh trng Sinh trng v trao i cht ca vi sinh vt lin quan cht ch vi cc iu kin ca mi trng bn ngoi. Cc iu kin ny bao gm hng lot cc yu t khc nhau, tc ng qua li vi nhau. a s cc yu t u c mt c tnh tc dng chung biu hin ba im hot ng l ti thiu, ti thch v cc i. 2.1. C ch tc dng ca cc yu t bn ngoi ln vi sinh vt Cc yu t ca mi trng bn ngoi tc dng ln t bo thuc ba loi l yu t vt l ( m, nhit , p lc, tia bc x...), yu t ha hc (pH mi trng, th xy ha kh, cc cht dit khun...) v yu t sinh hc (cht khng sinh, khng th). D l yu t no nhng khi tc dng bt li ln t bo th thng trc ht gy tn hi n cc cu trc quan trng cho s sng ca t bo. Nhng tn hi dn n ph hy chc phn hot ng ca cc cu trc v lm cht t bo. Chng no t bo vi sinh vt c th sng st chnh l do chng thch ng vi yu t cho bng nhng thay i v sinh l hoc di truyn. Tc dng c hi ca cc yu t bn ngoi t bo th hin ch yu nhng bin i sau y: 1. Ph hy thnh t bo: mt s cht nh enzyme lysozyme (cha trong l lch, bch cu, lng trng trng, ui thc khun th...) c kh

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nng phn hu thnh t bo vi khun dn n to thnh cc nguyn lp ch yu vi khun Gram dng v cc cu lp vi khun Gram m. 2. Bin i tnh thm ca mng t bo cht: mt s cht khng nht thit phi xm nhp t bo, nhng vn gy tc dng khng khun. Do tc dng ln mt hoc mt chc phn sinh l ca mng t bo, cht ny s lm vi khun mt kh nng sinh sn. Rt c th, trong trng hp nh vy, hng ro thm thu tn ti trong mng t bo cht b h hi. Tc dng khng khun ca cc cht xy ha v cc cht kh (H2O2, cc halogen ...) l do nh hng ca chng ln cc thnh phn ca mng t bo cht. Cng c th xp vo nhm cc hp cht ny l ru, phenol, cc cht ty ra tng hp, cc mui mt v mt s khng sinh (polymyxin, thyroxydin, nistatin). 3.Thay i c tnh keo ca nguyn sinh cht: cc yu t vt l cng nh ha hc u c th gy nn tc dng ny. Chng hn nhit cao lm bin tnh protein v lm chng ng t do kh nng kh nc. 4. Km hm hot tnh: mt s cht tc ng vo cc h thng sinh nng lng ca t bo, cyanite km hm cytochrome - oxydase, fluoride ngn cn qu trnh ng phn, cc hp cht ho tr ba ca acsenic bao vy chu trnh Krebs, dinitrophenol km hm qu trnh phosphoryl ho xy ha. Cc cht xy ha mnh (H2O2, halogen) ph hy cc h thng t bo lm tn hi n chc phn trao i cht. Cc enzyme khc c th b bt hot khi lin kt vi cc yu t kim loi nh thy ngn. 5. Hy hoi cc qu trnh tng hp: trong s c mt ca mt s cht tng t v mt cu trc vi cc cht trao i t nhin, gi l cc cht khng cht trao i (antimetabolism), qu trnh sinh tng hp c th b c ch. C ch tc dng ca cc cht khng cht trao i khng ging nhau. Mt s gn vi trung tm hot ng ca enzyme lm cho enzyme mt hot tnh phn hy c cht; mt s khc c th tham gia vo phn ng enzyme v c lp vo sn phm ca phn ng nhng sau khng c s dng trong trao i cht vi cng mc nh trong trng hp ca c cht thc. 2.2. Cc nhn t nh hng * Cc yu t vt l - m: hu ht cc qu trnh sng ca vi sinh vt c lin quan n nc, do m l mt yu t quan trng ca mi trng. a s vi khun thuc cc sinh vt a nc ngha l chng cn nc dng t do, d hp th. Ch mt s x khun c th xp vo bn a kh. Nu lm lnh t bo ng thi lm kh trong chn khng ta c th bo qun c sc

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sng ca t bo trong mt thi gian di. y l nguyn tc ca phng php lm ng kh vi khun. Do vi khun cn m nht nh sinh trng nn bng cch phi kh hoc sy kh ta c th bo qun c lu di nhiu loi sn phm (hoa qu kh, c, ruc tht kh). - Nhit : hot ng trao i cht ca vi sinh vt c th coi l kt qu ca cc phn ng ha hc. V cc phn ng ny thuc ph thuc cht ch vo nhit nn yu t nhit r rng nh hng su sc n cc qu trnh sng ca t bo. Nh ni trn, hot ng ca vi sinh vt b gii hn trong mi trng cha nc dng c th hp th. Vng ny ca nc nm t 2oC n khong 100oC gi l vng sinh ng hc. Hu ht t bo sinh dng ca vi sinh vt b cht nhit cao do protein b bin tnh, mt hoc hng lot enzyme b bt hot. Cc enzyme h hp c bit l cc enzyme trong chu trnh Krebs rt mn cm vi nhit . S cht ca vi khun nhit cao cng c th cn l hu qu ca s bt hot ha ARN v s ph hoi mng t bo cht (ni chung, cc acid nucleic t mn cm vi nhit so vi cc enzyme). Nhit thp (di vng sinh ng hc) c th lm bt hot qu trnh vn chuyn cc cht ha tan qua mng t bo cht do thay i cu hnh khng gian ca mt s permease cha trong mng hoc nh hng n vic hnh thnh v tiu th ATP cn cho qu trnh vn chuyn ch ng cc cht dinh dng. - p lc: p sut thm thu v p sut thy tnh c th nh hng n cu trc ca t bo vi khun. Mng t bo cht ca t bo vi khun l bn thm do cc hin tng thm thu v vic iu chnh thm p qua cc h thng permease u c lin quan n mng ny. Trong mi trng u trng t bo mt kh nng rt nc v cc cht dinh dng ha tan bao quanh, t bo chu trng thi kh sinh l, b co sinh cht v c th b cht nu thi gian ko di. Trong thc t ngi ta ng dng tc dng co sinh cht ca cc nng mui cao (10 - 15%) hoc ng cao (50- 80%) bo qun thc phm (mui da, c, p tht c), hoa qu (lm mt) v a s vi sinh vt rt mn cm vi thm p cao ca mi trng. Ngc li, khi vi khun vo dung dch nhc trng nc s xm nhp t bo, p lc bn trong s tng ln. Tuy nhin, do c thnh t bo cng vi khun khng xy ra ra hin tng v sinh cht nh t bo thc vt. a s vi khun sinh trng tt trong mi trng cha t hn 2% mui, nng mui cao hn c hi cho t bo. Nhng cng c mt s vi khun li sinh trng tt nht trong mi trng cha ti 30% mui, ta gi

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l cc vi khun a mui (halophilic). Nhiu vi khun bin thuc nhm ny, chng khng c kh nng pht trin nhng nng ng cao. - Cc tia bc x: nh sng c th gy ra nhng bin i v do c th gy tn thng sinh hc nu c t bo hp th. Mc gy hi ty thuc vo mc nng lng trong lng t nh sng c hp th. Cc tia c th gy nhng bin i ha hc l nh sng mt tri, tia t ngoi, tia X, tia gamma v tia v tr. nh sng mt tri c th gin tip tc ng ln t bo do lm bin i mi trng. Tia t ngoi c tc dng mnh nht bc sng 260nm, y l vng hp th cc i ca acid nucleic v nucleoprotein gy dimer ha thimine, dn n c ch s nhn i ca ADN. Nhng sau khi chiu tia t ngoi, nu a vi khun ra nh sng ban ngy th vi khun c th phc hi kh nng sinh trng v phn chia. Hin tng ny gi l quang ti hot (photoreactivation). - m thanh: sng m thanh, c bit trong vng siu m (trn 20 kHz) c nh hng ln n sinh trng ca vi khun, cc t bo sinh dng b cht nhanh chng. * Cc yu t ha hc - nh hng ca pH mi trng: pH ca mi trng c ngha quyt nh i vi sinh trng ca nhiu vi sinh vt. Cc ion H+ v OH- l hai ion hot ng ln nht trong tt c cc ion, nhng bin i d nh trong nng ca chng cng c tc ng mnh m. Cho nn vic xc nh pH thch hp ban u v duy tr pH cn thit trong thi gian sinh trng ca t bo l rt quan trng. Cc gi tr pH (cc tiu, ti thch, cc i) cn cho sinh trng v sinh sn ca vi khun tng ng vi cc gi tr pH trung bnh cn cho hot ng ca nhiu enzyme. Gii hn pH hot ng i vi vi sinh vt trong khong 4 - 10. a s vi khun sinh trng tt nht pH trung tnh (7,0) nh nhiu vi khun gy bnh (mi trng t nhin l mu v bch huyt ca c th ng vt c pH khong 7,4). Cc vi khun nitrate ha, vi khun nt sn, x khun, vi khun phn gii urea li a mi trng hi kim. Mt s vi khun chu acid (vi khun lactic, Acetobacter, Sarcina ventriculi), mt s khc a acid nh Acetobacter acidophilus, Thiobacillus thiooxydans (xy ha lu hunh thnh H2SO4) c th sinh trng pH <1. Nm mc v nm men a pH acid (pH 4 - 6). pH mi trng khng nhng nh hng mnh m n sinh trng m cn tc ng su sc n cc qu trnh trao i cht. - nh hng ca xy: xy c vai tr ht sc quan trng trong hot ng sng ca vi sinh vt. Tu thuc vo nhu cu i vi xy m ngi ta

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chia vi sinh vt thnh cc nhm sau: + Hiu kh bt buc: thuc nhm ny l cc vi sinh vt ch c th sinh trng c khi c mt xy phn t (O2), chng c chui h hp hon chnh, dng O2 lm th nhn hydro cui cng. Trong cc t bo c cha enzyme SOD (superoxid dismutase), catalase v peroxidase c tc dng chuyn ho cc gc O22- v O2- gii c cho t bo. Tuyt i a s vi nm v s ng vi khun thuc nhm ny. + Hiu kh khng bt buc: thuc nhm ny l cc vi sinh vt c th sinh trng c c trong iu kin c xy ln khng c xy. Trong t bo c cha SOD v peroxidase, khi c xy chng sinh trng tt hn. Phn ln nm men v nhiu vi khun thuc nhm ny. C th k n cc loi nh Saccharomyces cerevisiae, E. coli, Enterobacter aerogenes, Proteus vulgaris. + Vi hiu kh: thuc nhm ny l cc vi sinh vt ch c th sinh trng c iu kin p lc xy rt thp. Chng cng thng qua chui h hp v dng xy lm th nhn hydro cui cng. C th k n cc loi nh Vibrio cholerae, Hydrogenomonas spp, Zymononas spp, Bacteroides spp... + K kh: vi cc vi sinh vt thuc nhm ny s c mt ca xy phn t l c hi. Chng khng sinh trng c trn mi trng c hoc bn c khi trong khng kh hay trong khng kh c cha khong 10% CO2. Chng ch sinh trng c lp th dch su, ni khng c xy, c cc qu trnh ln men, qu trnh phosphoryl ho quang hp, qu trnh methane ho... Trong t bo ca cc vi sinh vt ny khng c SOD, cytochrome-oxidase, phn ln khng c hydrogenperoxidase. C th k n rt nhiu loi trong cc chi Clostridium, Fusobacterium, Butyrivibrio, Desulfovibrio, Veillonella... - Cc cht dit khun (st trng): thng s dng l phenol v cc dn xut ca phenol, alkol, halogen, kim loi nng, H2O, x phng, thuc nhum v cc cht ty ra tng hp ca cc mui ammon bc bn. * Cc yu t sinh hc Trong cc yu t sinh hc nh hng c hi ln cc qu trnh sng ca vi sinh vt cn k n khng th v khng sinh. Cht khng sinh c th c t nhiu ngun gc khc nhau nh tng hp ho hc, chic xut t thc vt, ng vt nhng ch yu l c tng hp t vi sinh vt. y l cc cht c hiu m nng thp cng c kh nng c ch hoc tiu dit vi sinh vt mt cch chn lc.

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Khng th l nhng cht c sn trong mu hoc c xut hin khi c khng nguyn xm nhp vo c th, c kh nng lin kt c hiu vi cc khng nguyn lm mt hot lc ca khng nguyn (xem thm phn khng sinh v min dch ).

III. Cc kiu dinh dng ca vi sinh vt


Cn c vo dng nng lng s dng ngi ta c th chia lm cc loi vi sinh vt: - Cc c th quang t dng (photoautotrophic) hay quang dng v c (photolithotrophic) c th chuyn nng lng nh sng thnh nng lng trao i cht (ATP) v cht c tnh kh (NADPH2). CO2 l ngun carbon ch yu, H2O hay H2S l cht cho in t. C th gp nhng vi khun quang hp thi xy ging nh cy xanh, i din l cc loi vi khun lam (Spirulina, Anabaena, Nostoc). Cc to n bo quang hp khng thi xy nh cc vi khun lc thuc h Chlorobacteriaceae (Chlorobium, Thiosulfatophilum...), y l nhng vi khun quang dng v c k kh vi H2S hoc S2O32- lm cht cho in t. Cc vi khun lu hunh mu ta thuc b Thiorhodobacteriales nh Chromatium, nhng vi khun ny c kh nng pht trin trn mi trng mui v c nh nhiu loi cy xanh, y l nhng vi khun k kh quang dng v c khi c mt trong mi trng S hoc H2S. - Cc vi khun quang d dng (photoheterotrophic) hay quang dng hu c (photoorganotrophic), s dng nh sng lm ngun nng lng v hp cht hu c lm ngun carbon, nh cc vi khun mu ta khng lu hunh thuc b Athiorhodobacteriales, h Athiorhodaceae, cc loi thuc ging Rhodospirillum. Nhng vi khun ny thin v vi hiu kh, l nhng vi khun quang dng hu c, khng c kh nng pht trin vi H2S l cht cho electron c nht, cht cho electron ca chng l acetate, succinate hay hydro. - Cc vi khun ha dng v c (Chemolithotrophic) hay ha t dng (Chemoautotrophic) s dng nng lng c to thnh t cc phn ng ha hc v CO2 lm ngun carbon. Nng lng c c l nh xy ha cc hp cht v c nh NH4+, NO2-, H2, H2S, S, S2O32-, Fe2+... Chng hnh thnh mt nhm rt hn ch tham gia vo cc chu trnh vt cht sng trong t v nc nh Hydrogenomonas xy ha hydro, Nitrosomonas xy ha NH3, Nitrobacter xy ha nitrite, Thiobacillus xy ha cc hp cht kh ca lu hunh... - Cc c th ha d dng (Chemoheterotrophic) hay ha dng hu c (Chemoorganotrophic) bao gm cc vi khun, vi nm, ng vt nguyn

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sinh, ng vt, chng s dng nng lng ha hc v mt c cht hu c lm ngun carbon ch yu. Mt nhm ln cc vi khun ha dng hu c l nhng vi sinh vt gy bnh rt c ch trong Y hc, cc vi khun tp nhim vo thc n, cc vi khun c s dng trong cng nghip tng hp cc cht khng sinh, cc loi vitamin, cc amino acid...

IV. Sinh l hc ca s sinh trng


1. Cc phng php xc nh s lng v sinh khi vi khun 1.1. Xc nh sinh khi vi khun Vic chn phng php xc nh sinh khi vi khun ty thuc vo mc nh nghin cu. Chng hn, mun nh gi sn lng t bo th cn sinh khi ti hoc kh sau khi li tm t bo; mun xc nh cng trao i cht hay hot tnh enzyme, ngi ta li tnh hm lng protein hoc nit. Nh vy, trong thc t c th s dng cc phng php trc tip hoc gin tip. Cc phng php trc tip gm c: 1. Xc nh sinh khi ti hoc sinh khi kh (phng php ny km chnh xc). 2. Xc nh hm lng nit tng s (phng php Kjelhdal) hay hm lng carbon tng s (theo Vslike-Folch), cc phng php ny cho chnh xc cao. 3. Trong thc t ngi ta thng xc nh hm lng protein ca vi khun. C th dng phng php buire ci tin hoc phng php so mu khc. Cc phng php vi lng da vo vic o s lng cc thnh phn c trng ca protein nh threonine, tryptophan (theo Lowry hoc FolinCiocalteu) cng cho kt qu tt. C th ni xc nh hm lng protein trong sinh khi l phng php thch hp nht v mt mt protein l thnh phn ch yu ca cht kh, mt khc l nhng thnh phn hot ng trong sinh khi (hu ht protein ca t bo l enzyme). * Cc phng php gin tip o sinh khi vi khun: 1. o c ca dch treo t bo, y l phng php rt thun li. Trong thc t ta thng o mt quang hc ca dch treo, trong mt s trng hp ngi ta cng xc nh s khuch tn nh sng. 2. o cc ch s cng trao i cht nh hp th O2, to thnh CO2 hay acid, v cc ch s ny lin quan trc tip vi sinh trng. D nhin ta ch dng cc phng php ny trong trng hp khng s dng c cc phng php khc, v d khi mt sinh khi rt nh.

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1.2. Xc nh s lng t bo Trong trng hp xc nh s lng t bo sng ngi ta thng m s khun lc to thnh bi cc vi khun sng trong iu kin sinh trng thun li. Dch treo vi khun c pha long v mt th tch nht nh c a ln mi trng thch trong hp Petri. Sau khi nui cy ngi ta m s khun lc mc ln. Nu gi d rng mi t bo to thnh mt khun lc th m s khun lc ta s m c s t bo sng. Tt nhin iu ny khng ng i vi cc vi khun mc thnh chui, thnh i hay thnh m hoc i vi cc dch treo vi khun khng ng nht. S khun lc cng rt ph thuc vo thnh phn mi trng. T bo mc thnh khun lc trn mi trng ny khng nht thit cng cho xut hin khun lc trn mi trng khc. Vi mt s loi vi khun kh pht trin trn mi trng thch ngi ta c th kim tra s lng bng phng php pha long lin tip sau cy t mi pha long 1ml vo tng ng ng mi trng ch th (nu c 1 vi khun a vo cng cho phn ng dng tnh sau khi nui cy). Phng php ny gi l phng php s lng c kh nng nht (MPN = Most Probable Number). C th cy vo 5 ng v ly s liu 3 pha long cui (c phn ng dng tnh) ri tra bng tm ra s lng gn ng mt vi khun. Hoc c th lc mu qua mng lc vi khun ri t mng lc ln a mi trng thch kim tra s khun lc mc v suy ra mt vi sinh vt trong mu. Nu kch thc ca t bo kh ln c th m trc tip trn phng m (thng hay dng cc loi phng m Thomas hay Goriaev). 2. Sinh trng ly tha v thi gian th h Sinh trng v pht trin l thuc tnh c s ca vi sinh vt. Sinh trng l s tng kch thc v khi lng t bo, cn pht trin (sinh sn) l s tng s lng t bo. iu cn ch l khi ni v sinh trng v pht trin ca vi khun tc l cp n sinh trng v pht trin ca mt s lng ln t bo ca cng mt loi. Bi v, vic nghin cu mt c th t bo vi khun qu nh l rt kh. Tuy nhin s tng s lng t bo khng phi bao gi cng din ra song song vi s tng sinh khi. Chng hn, khi cht dinh dng trong mi trng cn, vi khun tuy cn phn chia 1 - 2 ln nhng cho 2 - 4 t bo nh hn t bo bnh thng; trong pha m u, sinh khi vi khun tng ln, nhng s t bo khng thay i, ngc li, vo cui pha logarid kch thc t bo gim i nhng s t bo vn cn tng.

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Gi s trong mt bnh kn cha mt lng ln mi trng dinh dng, ta cy vo mt t bo vi khun. Nu thnh phn mi trng hon ton ph hp vi nhu cu ca t bo vi khun s sinh trng, tng khi lng v th tch, tng hp cc thnh phn ca t bo (thnh t bo, mng t bo cht, ADN, ARN, protein...) cho n khi kch thc ln gp i v vi khun sphn chia cho 2 t bo. Hai t bo ny li tip tc sinh trng v phn chia cho 4, 8, 16 t bo Nu s t bo ban u khng phi l 1 m l No th sau n ln phn chia ta s c s t bo tng cng l N : N = N0 . 2n (1) Cc gi tr N v N0 c th xc nh nh phng m hoc tnh s khun lc mc trn mi trng c. Cn gi tr n (s th h) c th tnh nh logarid thp phn: logN = logN0 + n. log2 n=

log(N No) log2

(2)

Gi s vi khun phn chia n ln sau thi gian t, khong thi gian gia 2 ln phn chia lin tip (hoc thi gian cn cho vic phn i t bo) gi l thi gian th h v c biu th bng g: (t t1 ).log 2 t g= = (3) log N log N 0 n y t = t2 t1 biu th s sai khc gia thi gian u (t1) v thi gian cui (t2) tnh bng gi. Gi tr o nghch ca thi gian th h hay l s ln phn chia sau mt n v thi gian (tc sau 1 gi) gi l hng s tc phn chia C: C=

log N log N0 n = (t2 t1 ).log2 t

(4)

R rng thi gian th h cng ngn, vi khun sinh trng v sinh sn cng nhanh. T cng thc (4) ta c: n = ct (5) Thay gi tr ca n vo phng trnh (1) ta c: N = N0.2ct (6) Hng s tc phn chia C ph thuc vo mt s iu kin: loi vi khun, nhit nui cy, mi trng nui cy. 3. Sinh trng ca vi khun trong nui tnh

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3.1 Qu trnh sinh trng Nui cy tnh l phng php nui cy m trong sut thi gian ta khng thm vo cht dinh dng cng khng loi b i cc sn phm cui cng ca trao i cht (qun th t bo b gii hn trong mt khong khng gian nht nh). S sinh trng trong mt h thng ng hay h kn nh vy tun theo nhng quy lut bt buc khng nhng i vi cc c th n bo m c i vi cc c th a bo. th biu din s ph thuc ca logarid s lng t bo theo thi gian gi l ng cong sinh trng (hnh 4.1).
Pha n nh
12

10

lo g N

Pha t vong Pha logarid

Pha lag
4

t( h )

Hnh 4.1 ng cong sinh trng ca vi khun

Qu trnh sinh trng ca t bo vi sinh vt trong h kn tri qua cc giai on : * Pha lag (pha m u = pha tim tng) Pha ny tnh t lc bt u nui cy n khi vi khun t c tc sinh trng cc i. Trong pha lag vi khun cha phn chia (ngha l cha c kh nng sinh sn) nhng th tch v khi lng t bo tng ln r rt do qu trnh tng hp cc cht, trc ht l cc cao phn t (protein, enzyme, acid nucleic...) din ra mnh m. di ca pha lag ph thuc trc ht vo tui ca ng ging v thnh phn mi trng. Thng thng t bo cng gi th pha lag cng di. R rng nguyn nhn ca pha lag l s khc bit gia cc t bo pha n nh (hoc bo t) vi cc t bo ang sinh trng logarid. Trong pha lag din ra vic xy dng li cc t bo ngh thnh cc t bo sinh trng logarid (hoc sinh trng theo ly tha).

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Khi chuyn cc t bo ang sinh trng logarid vo mi trng mi khc vi mi trng trc ta vn thy c s xut hin pha lag. Nguyn nhn ca pha lag trong trng hp ny chnh l s thch ng ca vi khun vi iu kin nui cy mi; s thch ng c lin quan n vic tng hp cc enzyme mi m trc y t bo cha cn. Cc enzyme mi ny c tng hp nh s cm ng ca cc c cht mi. V d: Nu chuyn cc t bo ang sinh trng logarid t mi trng khong - glucose sang mi trng khong - maltose ta s thy xut hin pha lag, y l thi gian cn cho vic hnh thnh enzyme maltase (glucozidase). c trng ca hin tng sinh trng kp l ng cong sinh trng gm hai pha lag v hai pha log. Sau khi kt thc pha log th nht t bo li m u pha lag th hai v tip tc pha log th hai (hnh 4.2).

Hnh 4.2 ng cong sinh trng kp E. coli trong mi trng hn hp glucose - sorbitol (bn tri) Aerobacter aerogenes trong mi trng hn hp mui ammon v

Hin tng sinh trng kp thng gp khi nui cy vi khun trong mi trng cha ngun carbon gm mt hn hp ca hai cht hu c khc nhau. Khi sinh trng t bo s ng ha trc tin ngun carbon no m chng a thch nht. ng thi c cht th nht ny km hm cc enzyme cn cho vic ng ha c cht th hai. Ch sau khi ngun carbon th nht cn th ngun carbon th hai mi c th cm ng tng hp nn cc enzyme cn trong vic chuyn ha n. Hin tng sinh trng kp khng ch hn ch cc ngun carbon v nng lng m thy cc ngun nit v phtpho. Chng hn Aerobacter aerogenes c th s dng ngun nit l amino acid, mui ammon v nitrate. Nu khi cy chuyn vi khun t mi trng nc tht sang mi trng cha mui ammon hoc nitrate th pha lag s biu hin.

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Nu t mi trng nc tht sang mi trng cha hn hp c hai loi mui s thy c hin tng sinh trng kp. Cc ion NH4+ c ng ho trc ng thi kim ch vic tng hp cm ng enzyme nitrate reductase. Ch sau khi ht mui ammon trong mi trng th mui nitrate mi c s dng. Sinh trng kp l hin tng ph bin v c th gii thch bng c ch kim ch ni chung v c bit bng hiu ng glucose. * Pha logarid (pha log) Trong pha ny vi khun sinh trng v pht trin theo ly tha, ngha l sinh khi v s lng t bo tng theo phng trnh: N = No.2ct hay X= Xo.eut Kch thc ca t bo, thnh phn ha hc, hot tnh sinh l... ni chung khng thay i theo thi gian. T bo trng thi ng hc v c coi nh l nhng t bo tiu chun. * Pha n nh (pha cn bng) Trong pha ny qun th vi khun trng thi cn bng ng hc, s t bo mi sinh ra bng s t bo c cht i. Kt qu l s t bo sng khng tng cng khng gim. Tc sinh trng ph thuc vo nng c cht. Cho nn khi gim nng c cht (trc khi c cht b cn hon ton) tc sinh trng ca vi khun cng gim. Do vic chuyn t pha log sang pha n nh din ra dn dn. Nguyn nhn tn ti ca pha n nh r rng l do s tch ly sn phm c ca trao i c cht (cc loi ru, acid hu c) v vic cn cht dinh dng (thng l cht dinh dng c nng thp nht). Nguyn nhn th nht rt phc tp v kh phn tch, nguyn nhn th hai c nghin cu k hn. * Pha t vong Trong pha ny s lng t bo c kh nng sng gim theo ly tha (mc d s lng t bo tng cng c th khng gim). i khi cc t bo b t phn nh cc enzyme ca bn thn. cc vi khun sinh bo t qu trnh phc tp hn do s hnh thnh bo t. Thc ra cha c mt qui lut chung cho pha t vong. S cht ca t bo c th nhanh hay chm, c lin quan n s t phn hay khng t phn. Do sc sng ln bo t b cht chm nht (trong nhng iu kin thch hp nh kh v nhit thp bo t c th c kh nng sng vi trm nm). Nguyn nhn ca pha t vong cha tht r rng, nhng c lin

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quan n iu kin bt li ca mi trng. Trong trng hp mi trng tch ly cc acid nguyn nhn ca s cht t bo tng i d hiu. Nng cht dinh dng thp di mc cn thit cho hu qu lm gim hot tnh trao i cht, phn hy dn dn cc cht d tr v cui cng dn n s cht ca hng lot t bo. Ngoi c tnh ca bn thn chng vi khun, tnh cht ca cc sn phm trao i cht tch ly li cng nh hng n tin trnh ca pha t vong. Mt s enzyme th hin hot tnh xc tc cc i trong pha t vong nh deaminase, decarboxylase, cc amylase v protease ngoi bo. Ngoi chc phn xc tc mt s qu trnh tng hp nhng enzyme ni trn ch yu xc tc cho qu trnh phn gii. Tc t vong ca t bo c lin quan trc tip n thc tin vi sinh vt hc v k thut, l vn bo qun cc chng vi sinh vt quan trng v mt l thuyt (cc chng v cc bin chng c bit) v k thut (cc chng sinh cht khng sinh, amino acid, vitamin... vi sn lng cao). Ngoi kh nng sng ta cn cn bo qun c cc c tnh di truyn ca vi khun vi nhiu phng php bo qun khc nhau, nhng tt c u nhm lm gim trao i cht n ti thiu ch yu bng cch gim nhit v m 4. Cc thng s ca ng cong sinh trng Sn lng t bo c tnh bng hiu gia khi lng cc i (Xmax) v khi lng ban u (X0) khi nui cy vi khun (tnh bng gam): X = Xmax - X0 T s gia sn lng t bo (X) vi lng c cht c dng (S) (tnh bng n v trng lng) gi l h s kinh t (Y). Y=
X S

Nu sn lng tnh bng gam v c cht dng tnh bng mol th gi l h s kinh t mol (Ym). H s kinh t mol (Ym) cho php ta lin h sn lng t bo vi s lng ATP thu c do phn gii mt lng c cht no . Ta c th tnh c sn lng t bo (tnh bng gam) cho 1 mol ATP tiu th. i lng ny gi l h s nng lng (YATP). H s nng lng c tnh d dng nu ta bit con ng phn gii ca loi c cht v nng lng ATP sinh ra do s phn gii y. Hng s tc sinh trng ca t bo vi sinh vt trong pha log c tnh theo cng thc:

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ln X t ln X 0 = (t t0 )

lg X t lg X 0 lg e(t t0 )

Trong cng thc: X0 v Xt l mt huyn ph t bo ti cc thi im t0 v t, v lge = 0.43429. Thi gian ca pha lag (T1). y l mt thng s quan trng xem xt tnh cht ca vi khun v mi trng nui cy c thch hp hay khng. Thng s ny c xc nh bng hiu gia thi im tr (ti y huyn ph t bo c mt xc nh no xr) v ti (ti y khi t bo c th t n mt m sau nu em nui cy th chng bt u pha log ngay). T1 = tr ti = tn -

ln xr ln x0

V T1 ch thun li so snh 2 ging vi cng tc sinh trng log ging nhau, nn ngi ta thng biu th thi gian ca pha lag khng phi thi gian tuyt i, m l thng qua thi gian sinh l (thi gian ca 1la g). Hiu gia s sinh trng thc t v l thuyt l L = T1 x v. i lng L ch cho bit ging pht trin trong thc t chm hn pht trin theo l thuyt bao nhiu th h ti thi im cui pha lag v chuyn vo u pha log. Thng s ny thng dng so snh cc s liu ni v nh hng ca cc cht dinh dng khc nhau, cc cht c ch sinh trng v iu kin nui cy. 5. Sinh trng ca vi khun trong nui lin tc Trong phng php nui cy tnh ni trn, cc iu kin mi trng lun lun thay i theo thi gian, mt vi khun tng ln cn nng c cht gim xung. Vi khun phi sinh trng v pht trin theo mt s pha nht nh, sinh khi t c khng cao. Tuy nhin trong nhiu nghin cu v thc tin sn xut ta cn cung cp cho vi sinh vt nhng iu kin n nh trong mt thi gian di chng vn c th sinh trng trong pha log. D nhin trong mt mc no c th cy chuyn t bo nhiu ln (qua nhng khong thi gian ngn) vo mi trng dinh dng mi. Nhng n gin hn, ngi ta a mi trng dinh dng mi vo bnh nui cy vi khun ng thi loi khi bnh mt lng tng ng dch vi khun. y chnh l c s ca phng php nui cy lin tc trong chemostas v turbidostas. C s ca phng php nui lin tc: gi s ta c mt bnh nui cy trong vi khun ang sinh trng pht trin. Cho chy lin tc vo bnh mi trng mi c thnh phn khng i. Th tch bnh nui cy c gi

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khng i, ngha l bnh mi trng i vo c b bi dng mi trng i ra vi cng mt tc . Ta gi th tch bnh l v (lt), tc dng i vo l f (lt/gi). Do tc pha long (cn gi l h s pha long) D = f/v (i lng D biu th s thay i th tch sau mt gi). Nu vi khun khng sinh trng v pht trin, chng s b rt khi bnh nui cy vi th tch: v- = dX = D.X dt

Nh vy mt vi khun trong bnh gim, ta c cng thc tnh: X = X0. e-Dt Tc sinh trng ca qun th vi khun trong bnh biu th bi phng trnh: v+ =
dX = .X dt

Nh vy mt vi khun trong bnh tng theo phng trnh: X = X0. et Tc thay i cui cng (tng hoc gim) mt vi khun trong nui cy lin tc l s sai khc gia tng v+ v gim v-: v = v+ - v- = Nu > D => v > 0: mt vi khun trong bnh tng. Nu < D => v < 0: mt vi khun trong bnh gim. Nu = D => v = 0: mt t bo khng tng khng gim theo thi gian, qun th vi khun trong trng thi cn bng ng hc. Nu bnh th nghim c thit b duy tr lun lun bng D ta s thu c qun th vi khun sinh trng v pht trin theo ly tha, thng xuyn mt mt t bo khng i v khng ph thuc v thi gian. Trong trng hp nh vy khng nhng kch thc trung bnh ca t bo, trng thi sinh l ca chng m c mi trng nui cy u khng i v khng ph thuc vo thi gian. iu ny, mt mt to iu kin cho vic nghin cu sinh trng v sinh l ca t bo vi khun, mt khc ci thin qu trnh sn xut vi sinh vt qui m cng nghip. Chemostas v turbidostas l hai thit b nui cy trong ta c th duy tr c iu kin = D. Nui cy tnh c xem nh h thng ng, qun th t bo sinh trng phi tri qua cc pha m u, logarid, n nh v t vong. Mi pha
dX = ( - D).X dt

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sinh trng c c trng bi nhng iu kin nht nh. Vic iu khin t ng kh thc hin. Nui cy lin tc, tri li, l h thng m c khuynh hng dn n vic thit lp mt cn bng ng hc. Yu t thi gian y trong phm vi nht nh b loi tr. T bo c cung cp nhng iu kin mi trng khng i, nh vic iu chnh t ng (hnh 4.3).
Chemosta Turbidost

Hnh 4.3 Nui cy lin tc trong chemostas (tri) v turbidostas (phi)

V. S km hm sinh trng v s dit khun


1. Cc phng php kh trng 1.1. Kh trng bng nhit - Kh trng bng phng php Pasteur C s ca phng php ny l lm nng vt liu nhit di 1000C nhm tiu dit tt c cc vi sinh vt trong cc mi trng d b hng khi un si nh sa, bia, ru vang... Kh trng kiu Pasteur c th 600C trong 30 pht hoc 750C trong 15 pht hay 800C trong 10 pht. - Kh trng bng cch un si Ngi ta dng ni Koch hay ni Arnola kh trng. Ni hp c cu to bng thng hai v kim loi. Nc thng ngoi c un si, hi nc s vo thng trong. Nhit hi nc si khng vt qu 1000C.

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nhit ny t bo sinh dng b tiu dit, nhng bo t vn cn kh nng ny mm. Ngoi phng php trn ngi ta cn c th un si trc tip cc dng c bng kim loi, cao su, kim tim v xylanh trong thi gian khong 10 pht. - Kh trng bng phng php hp gin on Cc mi trng dinh dng, cc ng bng cao su v mt s dng c d b hng khi kh trng nhit cao. V vy cn phi kh trng bng cch un si lp li 3 ln trong 3 ngy k tip nhau. Ln un th nht trong 30 - 45 pht lm cho cc t bo sinh dng u b cht, nhng bo t vn cn sng. Sau mi trng, dng c kh trng vo t m 28 - 300C. nhit ny trong mi trng thch hp, cc bo t s ny mm. Trong ln kh trng th hai cc t bo va mi ny mm li b tiu dit. m bo hon ton v trng ta tip tc kh trng ln th 3 trong ngy th 3 k tip. - Kh trng bng ni hp p lc (Autoclave) Phng php ny da trn nguyn tc lm nng mi trng bng hi bo ha p sut cao hn 1atm. Ni hp p lc cao lm bng kim loi, c hai v v c kh nng gi p sut cao. V trong l thng kh trng, ni cc dng c, mi trng cn kh trng. Thng kh trng c np van thot kh, c p k xc nh p sut hi nc, c van bo him x hi khi p sut qu mc cn thit v ni khi b n. Khong trng gi hai lp v l ngn cha hi nc c ni vi phu. Nc trong ni phi n mc quy nh khc trn ng o nc, pha trn ca thng c l a hi nc vo thng trong. Ni hp c y kn bng cc kha van xung quanh. Chun b mi trng kh trng: cc dng c cha mi trng ch c cha n 2/3 th tch v nt kn bng nt bng. Nt bng khng c cht qu khi ngn cn s cung cp xy cho vi sinh vt v khng lng qu d b vi sinh vt bn ngoi xm nhp vo mi trng, y nt bng phi gn trn khng di qu hoc ngn qu. Khi phn phi mi trng tuyt i khng mi trng dnh vo ming ng nghim, bnh hay nt bng. Dng c xp vo ni kh trng phi ngay ngn, khng xp qu st nhau, cho hi nc d i qua. Khi kh trng bng ni p lc cao, p sut trong ni tng ln lm cho nhit tng theo. Khi chn ch kh trng phi ch pH ca mi trng, pH di 5,5 thch b thy phn v khi ngui khng c kh nng to gel. Nu mi trng kim, khi kh trng s gy kt ta st, caramen ha ng. trnh cc hin tng trn, trong khi kh trng phi mi trng c pH trung tnh, sau khi kh trng phi trung ha li mi trng n mc pH

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cn thit. Mi trng sau khi kh trng c gi trong t m 300C trong 2-3 ngy kim tra v trng. - Kh trng bng sc nng kh Cch kh trng ny tin hnh trong t sy nhit 160 - 1700C trong 2 - 3 gi. iu kin ny cc t bo sinh dng v bo t ca vi sinh vt u b tiu dit. Cch kh trng ny c s dng kh trng cc dng c thy tinh. ng nghim, bnh tam gic trc khi kh trng cn c nt kn bng nt bng. Cc pipette, hp petri, que gt phi c gi kn bng giy bo, trc khi s dng mi c m ra. - Kh trng bng cch t trn ngn la Cch kh trng ny dng kh trng que cy vi sinh vt, cc dng c bng st (kim, kp, gp, dao, ko...), mt s dng c thy tinh (a thy tinh, que gt...). Mun kh trng ta a i a li nhiu ln dng c trn ngn la n cn, bng cch ny mi vi sinh vt bm trn b mt dng c u b tiu dit. 1.2. Kh trng khng bng nhit - Dng nn lc vi khun Mt s cht hu c nh huyt thanh, albumine trong mi trng d b bin tnh khi kh trng bng nhit cao nn phi kh trng bng cch lc qua cc dng c lc vi khun. Cc l ca mng lc nh hn kch thc ca t bo vi khun. Loi dng c thng dng hin nay l loi lc seltz, loi ny c 2 b phn. Mt b phn hnh vin tr pha trn, dng cha dch lc, mt b phn hnh phu pha di. Hai b phn c gn vo nhau v c c nh li nh 3 inh c. Mi ln lc, ngi ta t vo gia 2 b phn mt mng lc trn bng amian hay bng nitrocellulose dy khong 3 - 5cm (mng lc ch dng mt ln). - Kh trng bng ha cht Ha cht c dng kh trng bo qun nguyn vt liu v lm sch phng th nghim, bnh vin. Khi s dng ha cht cn lu ti nng thch hp, khng gy c i vi con ngi. Sn nh, bn gh trong phng c lau bng cc dung dch st trng nh chloramine 0,5 3%, hoc nc phenol 3 - 5%. S dng cn st trng lau cc dng c thy tinh nh que gt, phin knh, l knh, a thu tinh... - Kh trng bng tia t ngoi, nh sng mt tri Tia t ngoi c bc sng 13 - 400 nm u c tc dng dit khun. kh trng bung nui cy, phng th nghim, ngi ta chiu tia t ngoi trong khong 30 - 40 pht.

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Tia sng mt tri c bc sng 330 - 400 nm cng c tc dng dit khun. V th trong mt s trng hp ngi ta c th phi nng cc dng c ca phng th nghim 2 - 3 gi sau khi c ra sch. 2. Cc phng php bo qun 1. Cy truyn thng xuyn trn thch nghing hoc trch su vo thch. Sau khi sinh trng vi khun c gi trong t lnh +40C. Phng php ny n gin nht v thng c dng nhng km hiu qu nht. 2. Bo qun di du v trng: du paraffin va ngn cn mi trng kh va lm gim trao i cht gy cn tr s xm nhp ca xy. 3. Bo qun trong ct hoc t st v trng: do cu trc l - ha ct v t st u l nhng vt cht tt mang cc t bo vi sinh vt, ch yu l cc bo t. Sau khi lm kh khng kh ct (hoc t st) cng vi vi khun c th bo qun t bo rt lu. 4. ng kh: l phng php hon thin v c hiu qu nht. Vi khun c trn vi mi trng thch hp (sa, huyt thanh...) ri lm lnh v lm kh nh bng kh. Sau my nm bo qun t bo vn gi c kh nng sng m khng b bin i v di truyn. 5. Bo qun trong glycerine (10%) v gi trong t lnh su (-600C hay - 800C): y l phng php rt thch hp nhng cn mua c loi ng nha chu nhit (khi kh trng).

Cu hi n tp chng 4
1. Cc kiu dinh dng ca vi sinh vt? 2. Cc c cht dinh dng cn thit cho hot ng sng ca vi sinh vt? 3. C ch v tc dng ca cc yu t bn ngoi ln sinh trng v pht trin ca vi sinh vt? 4. Cc pha sinh trng ca vi sinh vt, im khc bit ca phng php nui cy tnh v nui cy lin tc? 5. ngha ca vic nghin cu ng cong sinh trng, hin tng sinh trng kp?

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Chng 5

Trao i cht vi sinh vt


I. Cc con ng phn gii hexose
1. Con ng Embden - Meyerhof - Parnas (EMP hay ng phn) 1.1 C ch (hnh 5.1) Glucose (1) Glucose - 6 P (2) Fructose - 6P ATP (3) ADP Fructose - 1,6 DP (5) (4) (AlPG) Glyceraldehyd - 3P H3PO4 (6) Dioxy acetone P (DAP) NAD

NADH2 a. 1,3 diphosphoglyceric (a. 1,3DPG) ADP (7) ATP a.3 P - Glyceric (A3PG) (8) a.2P- Glyceric (A2PG) (9) Phosphoenolpyruvic (PEP) (10) ADP A TP

Trong iu kin c hoc khng c O2, glucose u c chuyn a. Pyruvic thnh pyruvate qua 10 phn ng, trong 3 phn ng 1, 3 v 10 l phn Hnh 5.1: Con ng EMP ng mt chiu, cn cc phn ng khc l phn ng thun nghch. Tr cht

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u v cht cui, tt c cc cht trung gian trong chu trnh u dng phosphoryl ha. Gc phosphoryl mang 3 chc nng l gip cho cht trung gian mang in tch m cao khng th thot ra ngoi, l v tr gn enzyme v l v tr cung cp lin kt cao nng. Phng trnh chung: Glucose 2 pyruvate + 2ATP + 2 NADH2 ATP c to thnh t 2 phn ng 7 v 10. S to thnh ATP 2 phn ng trn gi l phosphoryl ha mc c cht v phosphate cao nng trc l mt phn ca phn t cht trao i (1,3 di - P - glycerate v PEP), khi c mt ca ADP th cht trao i s chuyn gc P cho ADP to thnh ATP. C th chia con ng EMP thnh 4 giai on: 1. Hot ha glucose to fructose - 1,6 diphosphate (c th xy ra nhiu monosaccharide khc). 2. B i phn t fructose - 1,6 diphosphate to AlPG v DAP. 3. xy ha AlPG thnh A2PG. 4. Chuyn ha A2PG to acid pyruvic (hnh 5.1). 1.2. ngha ca con ng EMP - Qu trnh ng phn cung cp cho t bo 6 trong s 12 tin cht dng tng hp cc n v kin trc: glucose - 6-P, fructose - 6-P, 3-Pglyceraldehyd, 3P - glycerate, PEP, pyruvate. - Cung cp nng lng cho hot ng sng ca t bo tuy hiu sut khng cao (khong 10%), nhng y l con ng nguyn thy nht gp hu ht sinh vt. 2. Con ng pentosophosphate xy ho (PP, HMP) Do mt t bin no (thiu enzyme) m mt s vi sinh vt c th chuyn ha glucose thnh pyruvate theo con ng PP (hnh 5.2). T glucose qua qu trnh decarboxyl ha to ribuloso - 5P v CO2. Cc phn ng tip theo l s chuyn ha gia pentoso- 5P v hexoso - 6P. ngha ca chu trnh: Xt v mt nng lng, t 1 phn t glucose khi phn gii to 35 ATP nhng qua con ng ny to riboso - 5P tham gia vo qu trnh sinh tng hp acid nucleic, to cc loi ng C4, C7... tham gia vo qu trnh sinh tng hp acid amin. Con ng ny to NADPH2 cn thit cho cc phn ng sinh tng hp trong t bo. y l con ng phn gii ng thng gp vi khun thuc ging Pseudomonas.

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Phng trnh tng qut: Glucose + 6O2


Glucose

6CO2 + 6H2O + 35ATP

Glucose Ribulose

Glucose Frutose Erytrose Xilulose Ribose Xilulose Xilulose Erytros Fructos Fructos Ribose Xilulose

Fructos

Hnh 5.2 Con ng pentosophosphate

3. Con ng KDPG (2keto - 3deoxy - 6Pgluconate) Phng trnh tng qut nh sau: Glucose 2 Pyruvate + ATP + NADH2 + NADPH2 Con ng KDPG gip cho nhiu vi khun s dng gluconate v ch gp mt s vi khun. E. coli v nhiu loi ca Clostridium chuyn ha glucose qua con ng EMP nhng phn gii gluconate qua con ng KDPG. Qua cc con ng phn gii glucose, pyruvate c to thnh. Nu trong iu kin hiu kh cc vi sinh vt s xy ho pyruvate to CO2 v nc. Nu iu kin k kh cc vi sinh vt s tin hnh qu trnh ln men hoc h hp k kh (hnh 5.3).

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Glucose

Glucose -6h h

6 - phosphoglucono-18-lactose

6 - phosphogluconate

2-keto-3 deoxy-6-phosphogluconate

Glyceraldehyd

pyruvate

1,3

Phosphoenol-pyruvate

3-phosphoglycerat

2-phosphoglycerate

Hnh 5.3 Con ng KDPG

4. xy ho pyruvate Pyruvate chim v tr trung tm trong trao i cht trung gian v l tin cht ca nhiu sn phm. Nhiu vi sinh vt xy ha pyruvate thnh acetyl - CoA ch yu qua 3 phn ng sau (hnh 5.4):

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Pyruvate +CoA+NAD+ Pyruvate + CoA + 2Fd Pyruvate + CoA + NAD+

Acetyl - CoA + NADH2 + CO2 (1) Acetyl - CoA + 2FdH + CO2 (2) Acetyl - CoA + formate (3) (Formate - Fd - ferredoxin)

Pyruvatecarboxylase

Vng tiasolium ca thiamin pyrophosphate

Pyruvate Enzyme Acid lipoic

Dihydrolipoidtransacetylase Enzyme Dihydrolipoid dehydrogenas

Enzyme Acid acetyl-lipoic

Enzyme Acid dihydrolipoic

Hnh 5.4 Qu trnh xy ha pyruvate

Phn ng (1) do phc h pyruvate - dehydrogenase (PDH) xc tc. PDH gp hu ht vi sinh vt hiu kh (khng gp vi khun k kh bt buc). PDH gm 3 enzyme, ngoi CoA v NAD+ cn cn TPP (thiamine pyrophosphate) v acid lipoic. Phn ng (2) xc tc bi pyruvate - Fd oxidoreductase, enzyme ny gp nhiu vi khun k kh nh Clostridium. Phn ng (3) do pyruvate - formate - liase xc tc, enzyme ny gp nhiu vi khun k kh tit acid formic (thuc h Enterobacteriaceae) v vi khun quang nng.

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II. Chu trnh tricarboxylic (Krebs)


Trong qu trnh xy ha hon ton, pyruvate s c xy ho trit thng qua chu trnh Krebs (chu trnh acid tricarboxylic = ATC). C ch c trnh by hnh 5.5.

Hnh 5.5 Chu trnh Krebs (theo Lehninger)

Trong chui phn ng vng ca chu trnh ATC, acid pyruvic b phn hu trit to CO2, coezyme dng kh v cc hp cht trung gian. Cc coezyme thng qua chui h hp vn chuyn H v electron n cho O2 phn t to nc ng thi gii phng nng lng ATP.

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ngha ca chu trnh: Ngoi chc nng xy ha tn cng, chu trnh ATC cn cung cp cho t bo 3 tin cht l oxalacetate (tng hp aspactate), - ketoglutarate v succinyl - CoA (tng hp nhn hem). V nng lng, chu trnh cung cp 1ATP mc phosphoryl ha c cht nhng sn sinh NADH2 i vo chui vn chuyn electron to ATP. Trong iu kin k kh chu trnh TCA khng c chc nng sinh nng lng nhng cng phi cung cp cho t bo 3 tin cht trn. T bo phi s dng phn ln pyruvate ln men thu nng lng, cn phn nh chuyn thnh acetyl - CoA nh pyruvate - ferredoxin - oxidoreductase. Phn ln cc vi sinh vt hiu kh xy ha cht dinh dng hu c thnh CO2 v H2O (v carbon trong phn t CO2 t mc xy ha cao nht nn ngi ta gi l qu trnh xy ha hon ton). Mt s vi sinh vt c kh nng xy ha c cht trong iu kin hiu kh nhng li sinh ra cc sn phm cha c xy ha hon ton (ketoacid, acid gluconic, acid malic, acid fumaric...). Nhiu qu trnh xy ha khng hon ton c ngha quan trng trong cng nghip ln men v chng sn sinh ra nhiu sn phm c gi tr.

III. Chui h hp (respiratory chain) v phosphororyl ha xy ha (oxidative phosphorylation)


Khc vi vi khun k kh, vi khun hiu kh c th tng hp ATP mnh m hn nhiu nh c chui h hp v ATP - synthetase. Hai h thng ny nm mng t bo Prokaryota hoc mng trong ti th ca t bo Eukaryota. Nng lng thot ra c chuyn thnh ATP, mt phn nh gii phng ra dng nhit. Cc thnh phn ca chui nm trong lp lipid kp, gm nhiu enzyme vn chuyn electron v hydro, cc coenzyme v nhm thm, cc dehydrogenase v h thng vn chuyn. Cc thnh phn quan trng nht tham gia vo xy ha hydro l : - Flavoprotein l cc enzyme cha nhm thm l FMN hoc FAD - vn chuyn hydro. - Protein Fe-S vn chuyn electron, cha nguyn t Fe, va lin kt vi S ca cysteine va lin kt vi S ca H2S. Cysteine l mt phn ca chui polypeptide, c th coi trung tm Fe - S l nhm thm ca chui polypeptide. Cc protein Fe - S cng tham gia vo s c nh N, kh sulphite, kh nitrite, quang hp... - Quinol: nm mng trong ca ti th, vi khun G- l ubiquinol (CoQ), vi khun G+ l naphtoquinol v lc lp l plastoquinol. Quinol, c bit l CoQ c c tnh a lipid do lp vo lp lipid kp ca mng, vn chuyn hydro hoc enzyme.

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- Cytochrome: vn chuyn electron, khng vn chuyn hydrogen. Cytochrome nhn electron t quinol, trong qu trnh vn chuyn mt s lng H+ tng ng vi s lng electron b tch vo mi trng (hnh 5.6).

Cyt

Cyt

Cyt

Cyt

Hnh 5.6 S chui h hp

Trong qu trnh vn chuyn 2[H] t NADH2 n O2 c 3 bc chuyn electron lin quan n to thnh ATP v c biu th bng h s P/O (mol ATP/mol nguyn t O). H s P/O = 3 khi 2[H] c chuyn qua NAD+ v P/O = 2 khi 2[H] t succinate qua FAD n O2. Do 1mol glucose khi phn gii qua chu trnh EMP v TCA vi tt c [H] tch ra c chuyn vo chui h hp to thnh nc s to 38 ATP. S tnh ton trn ng vi ti th v mt s vi khun. Tuy nhin, mt s vi khun do ch c 2 v tr phosphoryl ha nn s h hp hiu kh glucose ch cho 26 ATP (nh E. coli khng c NAD, Proteus vulgaris khng c cytochrome C). C ch phosphoryl ho xy ho (s to thnh ATP trong chui h hp) c gii thch theo thuyt ho thm thu ca Mitchell (1979). Theo thuyt ny, mng t bo vi khun v mng trong ti th khng thm cc ion k c H+ v OH- v km dn in. S sp xp cc protein ca mng (cc thnh phn vn chuyn electron, permease, ATP - sinthetase...) khin mng mang tnh cht khng i xng. S nh hng khng gian ca cc phn t enzyme dn n trao i cht c c tnh vect. Chui h hp bao gm mt dy cc cht vn chuyn hidro v electron sp xp lun phin trong mng. Do s xy ho c cht m pha trong mng c s tiu th H+ v pha mng ngoi gii phng H+. Trong s xy ho NADH2, 6H+

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c vn chuyn ra pha ngoi qua 3 nm. Vic vn chuyn proton do h hp lm xut hin mt gradien in ho gia 2 pha ca mng. Th proton ny s l ng lc ca s phosphoryl ho ngha l s to thnh ATP. Qu trnh c xc tc bi ATP - sinthetase, enzyme ny chuyn nng lng pht ra t dng electron thnh lin kt esterphosphate cao nng ca ATP. ATP - sinthetase gp cc mng tham gia vo s chuyn ho nng lng l mng ti th, lc lp v mng t bo vi khun. C l, dng H+ t ngoi i qua rnh ca ATP - sinthetase tr vo bn trong ti th hoc vi khun, nng lng thot ra c dng cho phn ng phosphoryl ho to ATP.

IV. S vn chuyn cht dinh dng vo t bo


tn ti, sinh trng v pht trin, t bo vi sinh vt phi thng xuyn trao i vt cht v nng lng vi mi trng bn ngoi. Mt mt chng nhn cc cht dinh dng cn thit t mi trng, mt khc chng thi ra cc sn phm trao i cht. Gia mi trng xung quanh v mi trng bn trong t bo tn ti mt hng ro thm thu, hng ro ny chnh l mng t bo cht. T bo nhn v thi cc cht mt cch chn lc. S xm nhp ca nc v cc cht ha tan qua mng t bo l mt qu trnh ng hc; t bo sng khng bao gi trng thi cn bng vi cc cht ca mi trng chung quanh. S vn chuyn cc cht qua mng t bo vi sinh vt tun theo mt trong hai c ch: khuch tn n gin (hay cn gi l vn chuyn th ng hay vn chuyn xui dng) v vn chuyn ch ng (vn chuyn ngc dng). Theo c ch khuch tn th ng cc phn t i qua mng nh s chnh lch nng (trong trng hp cc cht khng in phn) hay chnh lch in th (trong trng hp cc ion) hai pha ca mng. Hng lot nghin cu khng nh, tr nc ra rt t hp cht c th c vn chuyn qua mng theo c ch ni trn. Tri li, a s cc cht ha tan qua mng do c ch vn chuyn c bit. Nhng phn t vn chuyn sp xp trong mng lin kt vi cc phn t cht ha tan v chuyn chng vo b mt bn trong ca mng, t y cc phn t cht ha tan c chuyn vo t bo cht. Kiu khuch tn ny c gi l khuch tn xc tin. Cc phn t protein vn chuyn (carrier protein) ni trn gi l protein thm permease. S vn chuyn cc cht nh permease c th l th ng (khng cn nng lng ca t bo) hoc ch ng (cn nng lng). Theo c ch vn chuyn th ng, cht ha tan lin kt thun nghch vo mt v tr c bit trn phn t permease nm bn trong mng (c th cc l ca mng). Phc hp cht ha tan - permease c vn chuyn theo hai pha ca

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mng nh s chnh lch nng mt cht no , ngha l s vn chuyn din ra theo kiu xui dng. S vn chuyn th ng nh permease c chng minh mt s vi sinh vt. Tuy nhin, vi sinh vt c kh nng tch ly mt s cht vi nng cao hn nhiu so vi nng bn ngoi. Chng hn, nng K+ bn trong mt s t bo vi sinh vt c th ln hn nng bn ngoi hng ngn ln. m bo trung ha in, t bo cng ng thi thi ra bn ngoi cc ion H+ hoc Na+. Thm vo , ngi ta pht hin thy cc mng vi khun c hot tnh ca ATP - ase l enzyme c lin quan n vic vn chuyn cc cht. R rng, trong t bo vi sinh vt ngoi c ch vn chuyn th ng cn tn ti c ch vn chuyn ch ng nh permease. S vn chuyn ny c tin hnh ngc vi gradien nng ngha l theo kiu ngc dng, nng lng tiu th c th do ATP cung cp. Cho ti nay ngi ta phn lp c hng lot protein vn chuyn trong cc loi vi sinh vt. Ging nh enzyme, chng c tnh c hiu c cht khc nhau. Mt s c tnh c hiu gn nh tuyt i. Chng hn, permease ca galactose E.coli ch vn chuyn galactose. Cc permease ca cc ng v acid amin khc th hin tnh c hiu yu hn i vi cc cht ha tan. iu ng ch l trong vi khun s vn chuyn ch ng ca hu ht cc loi ng ph thuc vo qu trnh phosphoryl ha v h thng enzyme phosphotransferase. H thng ny bao gm hai enzyme EI, EII v mt protein vn chuyn bn nhit (Hpr: heat stable carried protein) c khi lng phn t thp. Cc thnh phn protein ca h thng ny c thun khit v phn ng din ra theo hai bc. Trc ht EI chuyn phosphate t phosphoenolpyruvate (PEP) n Hpr: Hpr + PEP Hpr - P + Pyruvate Sau EII chuyn phosphate t Hpr - P n C6 ca ng n. EI l chung cho nhiu loi ng nhng EII li c trng cho mi loi ng. Ngha l mt t bin no nh hng n vic tng hp EI s dn n mt kh nng vn chuyn nhiu loi ng. Tri li vi t bin nh th i vi EII ch nh hng n s vn chuyn ca mt loi ng (hnh 5.7).

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Ngoi
Glucose

Mng EII Hpr

Trong Glucose- 6P PEP

EII glucose

Hpr-P

pyruvic

Hnh 5.7 S vn chuyn ng qua mng t bo vi sinh vt

V. Trao i cht v nng lng


1. Cc ngun nng lng vi sinh vt Ty theo bn cht ca ngun nng lng s cp, c hai con ng c bn cung cp cho vi sinh vt: con ng quang dng v ha dng. Trong c hai trng hp, s thu nhn nng lng ca t bo c lin h vi s vn chuyn electron qua mng, do to ra mt gradient electron proton t phn ny i vi phn kia ca mng t bo cht. Chnh gradien nng ion l ngun gc ca lc bm proton (s khc bit in tch) c th c dng vo nhng mc ch khc nhau nh vn chuyn tch cc, sn xut cc cht giu nng lng, chuyn ng v.v.. . Trong th gii vi sinh vt c s khc bit rt ln v kh nng s dng cc ngun nng lng. Mt s vi khun hiu kh cng ging nh ng vt c kh nng tng hp ATP cho mnh t ng b phn gii theo con ng ng phn (glycolyse) v chu trnh Krebs nh chui h hp trn mng t bo cht (tng t nh chui h hp mng trong ca ty th). Cn nhng vi sinh vt k kh li thu nng lng cho hot ng sng ca mnh nh qu trnh ln men ca cc loi ng hoc nh mt loi chui vn chuyn electron trong c s dng cc hp cht khng phi l xy phn t lm cht nhn electron cui cng trong chui vn chuyn. y, chui vn chuyn cng khu tr trn mng t bo cht tng t nh chui vn chuyn nm trong cc ty th. i vi cc c th quang dng th li khc, chng c th thu nhn nng lng ca nh sng.

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ng v quan im nng lng sinh hc, th ty th, lc lp v t bo vi khun c nhiu nt tng ng. Tt c cc vi khun c trn mng t bo cht mt h enzyme ATP - synthetase tng t nh ATP - synthetase ca cc ty th v lc lp. Phc hp protein vn chuyn qua mng m bo bin i dng proton thnh nng lng trong mi lin kt phosphate dng ATP. 1.1.Cc c th quang dng (phototroph) v quang tng hp (photosynthesis) Hnh thi ca c th quang dng v quang tng hp gia vi khun, to, thc vt c s khc bit r rng. Mt khc, cu to cc c quan quang hp, sc t quang tng hp, cng khc bit (xem chng II). i vi thc vt, cht cho electron l H2O v qu trnh quang hp gii phng xy phn t. i vi vi khun quang hp khng thi xy v cht cho electron l nhng hp cht v c (nh H2S, S) hoc hp cht hu c (isopropanol) (xem thm chng VII). 1.2. Cc c th ha dng v xy ha sinh hc Phn ln vi khun khng c c quan v sc t quang hp. Chng ly nng lng hu ch cho cc qu trnh tng hp ca t bo nh gii phng nng lng trong cc phn ng ha hc. S trao i cht ca cc c th ho dng ni chung bao gm 3 giai on: - Phn ct cc i phn t ngoi t bo. - Phn gii cc phn t b to ra cc cht trao i trung gian (pyruvate, acetyl - CoA) v to nng lng hu ch (ATP). - Phn gii trit cc cht trao i trung gian thnh CO2 v H2O vi vic to ra lng ln ATP. l nhng phn ng xy ha c cht hu c v v c. Qu trnh xy ha mt c cht A c th xem l mt chui phn ng m trong c cht A mt i cc eletron ca mnh. C cht A gi l cht cho electron s bin thnh mt sn phm c xy ha. Tng t, mt hp cht B khc gi l cht nhn electron s bin thnh sn phm kh. Trong phn ln trng hp, mt hp cht A l mt cht hu c c th xem l cht c hydro ha, nh vy th qu trnh xy ha v thc cht l mt qu trnh kh hydro (dehydrogenation), trong khi qu trnh kh l qu trnh hydro ha (hydrogenation), t hp hai qu trnh ny l qu trnh xy ha kh (oxidoreduction). Qu trnh xy ho:

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- 2H+ AH2 (cht cho hydro) - 2e Qu trnh kh: + 2H+ B (cht nhn hydro) + 2e Kt qu: A + BH2 + Nng lng AH2 + B Trong chui phn ng, vic vn chuyn hydro hay electron t c cht sang cht nhn c thc hin nh hng lot enzyme, cc enzyme ny to thnh chui vn chuyn in t. Cc cht xc tc thch ng cao vi qu trnh xy ho c cht l nhng dehydrogenase. Nhng enzyme ny c trng cho mt loi c cht v t hp vi cc coenzyme m n ng vai tr l cht nhn hydro t c cht. l cc dn xut ca flavin (FMN, FAD) hoc ca pyridine (NAD, NADP) v nhiu hp cht khc. cc c th hiu kh, s vn chuyn cc electron gia coenzyme ca dehydrogenase v xy phn t c thc hin nh cc hp cht trung gian ca cytochrome. 2. Cc kiu h hp Thng thng khi cht nhn electron cui cng l xy phn t th gi l qu trnh h hp hiu kh v vi sinh vt thuc dng ny gi l vi sinh vt hiu kh (aerobes). Khi cht nhn electron cui cng l mt c cht khc khng phi l xy t do, th ngi ta gi l qu trnh ln men hoc c th l qu trnh h hp k kh v vi sinh vt thc hin cc qu trnh ny gi l cc c th k kh (anaerobes). Trong s cc c th k kh, cht nhn electron c th l cht hu c hoc cht v c. Nu cht nhn electron l cht hu c ngi ta gi l ln men, nu cht nhn l electron l cht v c ngi ta gi l h hp k kh (anaerobic respiration). H hp ca mt c th hiu kh l mt qu trnh vn chuyn electron v proton qua mng vi xy phn t lm cht nhn electron cui cng, cn h hp k kh dng ch mt qu trnh vn chuyn qua mng ca cc electron v proton n cht nhn electron cui cng l mt cht khc xy phn t, nh nitrate trong trng hp h hp nitrate (nitrate respiration), fumarate nh h hp fumarate (fumarate respiration ) v.v... BH2 (sn phm kh) A (sn phm xy ho) + Nng lng

115

Rt nhiu vi sinh vt c nhiu chui vn chuyn electron c th cng hot ng, hoc c hot ng trong nhng iu kin nui cy nht nh. V d nh E. coli, vi khun ng rut ny c chui vn chuyn electron vi xy phn t l cht nhn electron cui cng (chui hot ng nh c th hiu kh ) v chui h hp nitrate hot ng k kh trong mi trng nitrate, mt chui h hp fumarate, hot ng k kh trn mi trng cha c cht hu c m vi khun ny c th ln men. H hp c c trng trc ht l ni khu tr ca chui vn chuyn electron mng t bo cht i vi cc c th nhn s v mng trong ca ty th i vi cc c th nhn chun. H hp cn c trng bn cht ca cht nhn electron cui cng l xy phn t. S xy ho c cht v ng thi s kh ca xy c thc hin nh mt chui phn ng enzyme, trong c s tham gia ca dehydrogenase v cc coenzyme lin kt vi n. C ch thng thy h hp l con ng ca cc cytochrome gin tip. Ngy nay ngi ta hon ton bit r thnh phn v c ch hot ng ca cc cytochrome ny trong t bo nhn chun v trong mt s c th nhn s. Bn cnh qu trnh phosphoryl xy ho c cht, cn tn ti nhng con ng khc, c bit l con ng xy ho trc tip nh cc enzyme vn chuyn cc electron t c cht n xy vi s to thnh H2O2 . FADH2 + O2 FAD- oxydase FAD + H2O2 Hp cht ny rt c v cn phi c phn gii nh catalase, peroxydase hoc superoxyd dismutase trnh cho t bo khi b cht. H2O2 catalase H2O + 1/2 O2 + H2O2 + 2H + 2e peroxydase 2H2O Nhng con ng ny to rt t hoc khng to nng lng hu ch. Mt vi khun khng c enzyme trn, nhng con ng phn gii trn buc chng phi sng k kh bt buc, bi v xy phn t l tc nhn gy c vi chng. C mt s vi khun chu c hiu kh nh Streptococcus, chng khng c catalase, nhng c cc enzyme flavin (NAD - oxydase v NAD - peroxydase) cho php chng thc hin s cn bng trong h hp mng t bo cht. Tu thuc vo s lng v lng cc enzyme c th phn gii H2O2 m quyt nh tnh hiu kh hay k kh cc vi khun. Ln men ch s c mt ca chui vn chuyn electron nm trong t bo cht, khng c s t ng i qua ca dng electron hoc proton phn bn ny v phn bn kia ca mng t bo (mt dng vn chuyn th

116

cp c thit lp nu cn thit, nh nng lng ca ATP). V phng din trao i nng lng, y cng l mt qu trnh xy ha kh, nhng cht nhn electron cui cng l cc hp cht hu c, ch khng phi l cc phn t xy. Qu trnh ny cng gii phng nng lng nhng t hn nhiu so vi qu trnh h hp. Cc hp cht cho electron (AH2) v cht nhn electron (B) u l cc hp cht hu c. Vic vn chuyn electron c thc hin nh NAD. y c s khc bit r rt so vi h hp v bn cht ca cht vn chuyn electron v sn phm cui cng... C ch vn chuyn electron trong qu trnh ln men c tm tt nh sau: AH2 NAD+ BH2

B A NADH2 3. Nghin cu s trao i nng lng S khc bit v cc phn ng xy ha kh cc nhm vi sinh vt khc nhau l mt tiu chun dng nh loi chng, mt s nh nghin cu xut nhng phng php v mi trng c th nh loi nhanh chng v n gin cc nhm vi sinh vt. 3.1 Nghin cu kiu h hp xc nh kiu h hp ca mt loi vi khun, trong phng th nghim ngi ta s dng mi trng VF (nc tht - gan) ch c v a vo ng nghim, ri cy ging vo su, da vo kh nng pht trin trong cc ng nghim ta bit c l loi h hp g. 3.2 Dng phn ng oxydase Phn ng ny cho php s c mt ca chui h hp ang hot ng vi cytochrome C mng nh TMPD (tetra - methyl - paraphenylene diamine). Phn ng ny c bit hiu qu i vi vi khun G-, phn ng dng tnh vi cc vi khun hiu kh bt buc (tr Pseudomonas maltophyla v Acinatobacter), phn ng m tnh i vi cc vi khun k kh khng bt buc tr cc Vibrio v Aeromonas. c bit i vi cc vi khun ng rut l c phn ng m tnh mc d vi khun ny c chui h hp vi cc cytochrome. 3.3 Th catalase Enzyme catalase thng c cc vi khun G-, tr cc ging Streptococcus v Lactobacilus. Khi c mt H2O2, mt vi git huyn ph vi khun c catalase dng s lm si bt gii phng xy phn t. Nu:

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- Si nhiu bt: l huyn ph vi khun hiu kh - Si t bt: vi khun hiu - k kh - Khng c bt: vi khun k kh 3.4 Phn ng nitrate reductase S kh nitrate c th c nghin cu trn mi trng c nitrate (mi trng lng hoc c) pht hin s c mt ca nitrite (nh thuc th Griess), c th dn n 2 kt qu theo 2 c ch khc nhau: - Kh nitrate ng ha c xc tc bi nitrate reductase B to nitrite, cht ny c th c chuyn ha tip thnh mui ammon l ngun nit d c c th s dng. - Kh nitrate d ha hay h hp nitrate di nh hng ca nitrate reductase A v cc enzyme khc, cc nitrate s c kh hon ton thnh N2O v N2 s c gii phng ngoi t bo. H hp nitrate l loi h hp k kh. 3.5 Nghin cu kiu trao i cht Nghin cu ny c thc hin nh s dng glucose lm c cht, loi ng c dng ph bin nht nhiu loi vi khun. Th nghim cn nui cy 2 iu kin hiu kh v k kh. Sau khi nui t m vi nhit thch hp, c th c 4 trng hp xy ra: - Vi khun ln men khi c ging pht trin trong canh trng v lm acid ha trong 2 ng nghim. - Vi khun xy ha khi n ch lm acid ha trong ng nghim iu kin hiu kh. - Vi khun bt hot khi khng thy acid ha trong c 2 ng nghim. - Vi khun kim ha khi thy mi trng b kim ho trong iu kin hiu kh, iu ny c th thy khi dng peptone. 3.6 Kh cc sn phm lu hunh Th nghim kh cc sn phm xy ha ca lu hunh (sulfate, sulfite, thiosulfate v.v...) c th thy r nh to ra hydro c lu hunh ha (H2S...) pht hin di dng sulfua kim loi b en (sulfua st hay sulfua ch...). 3.7 o mc h hp H hp k (Respirometre) Warburg l dng c u tin dng xc nh mc s dng xy ca t bo sng. N bao gm mt bnh cch thy chu nhit mu cn nghin cu. Mu ny c t trong mt bnh cha ni vi mt p k, bnh cha mu v p k to thnh mt nhnh c

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c nh chc chn trn ng c mc nc di ng, s di chuyn y l do s giao ng ca mu nghin cu gy nn. Bnh cha mu c ni vi mt nhnh ca bnh cong ca p k v c iu chnh c mt dung tch kh nht nh phn trn ca mu nghin cu. loi b nh hng ca CO2 sinh ra trong qu trnh h hp, ngi ta t mt ng nh gia bnh ng mu nghin cu, trong , cha KOH 10% hp th CO2 do qu trnh h hp sinh ra. S thay i p sut trong p k s c tnh chuyn nh dung tch xy tiu th. Vi dng c h hp k ny khng nhng dng o s tiu th xy phn t m cn c th nghin cu cc phn ng enzyme khc na, trong s thay i kh xut hin do kh carbonic, kh amin ha. 4. S tch tr v s dng nng lng 4.1. Cc mi lin kt giu nng lng T bo sng tch tr nng lng vo cc hp cht v chng c th c gii phng d dng khi t bo cn. Phn ng ha hc ph bin l thy phn mi lin kt esterphosphate. Hp cht c t bo s dng thun li v thng xuyn nht l ATP. Ngoi ra cn c cc hp cht giu nng lng khc nh cc nucleotide UTP, GTP, CTP, TTP; cc acylphosphate, v d nh 1-3 diphosphoglycerate; cc enolphosphate nh PEP; cc acyl - thio - ester nh CoA - SH. CH2 R- C-OP V d: PEP (G = - 53,5 Kj.mole -1) + Cc acyl - thioester: O R-C-SP V d: Coenzyme A 4.2. Ngun gc hp cht cao nng ATP c th c tng hp bng hai c ch: - Qu trnh phosphoryl ha c cht xy ra trong t bo cht. - Qu trnh phosphoryl ha lin h vi gradien electron v proton xy ra mng t bo cht, y ngi ta thy c qu trnh phosphoryl xy ha v phosphoryl quang ha (photophosphorylation). Trong c hai trng hp qu trnh tng hp ATP ph thuc vo lng ADP v phosphate v c.

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+ Qu trnh phosphoryl ha c cht: trong qu trnh ny, mt phosphate v c c lin kt (nh xc tc bi enzyme) vi cc sn phm ca s phn gii. Hp cht xy ha l cht mang mt mi lin kt phosphate giu nng lng v c th c chuyn n ADP. Kiu hnh thnh ATP ny thng thy s phn gii glucose theo con ng EMP (giai on xy ha 3 - phosphoglyceraldehyd thnh acid 1,3diphosphoglyceric v s bin i hp cht ny thnh acid pyruvic). + Qu trnh phosphoryl ha cng lin h vi gradien proton. C khi vn chuyn electron trong qu trnh h hp (hiu kh hoc k kh) cng nh trong quang hp to ra mt gradien proton quan trng v n l nng lng d tr (c th c so vi mt t in c np sn), nng lng d tr ny c th c thu hi bng cch li cc proton mt bn mng m ngi ta thng so snh nh mt ng c sn sinh ATP. 5. S trao i carbohydrate 5.1. S phn gii carbohydrate Carbohydrate nu dng hp cht c trng lng phn t ln phi c thu phn thnh cc dng ng n c th i vo cc con ng phn gii ng cung cp nng lng cn thit cho hot ng sng ca c th hoc to ra cc hp cht trung gian cn cho s chuyn ho trao i cht. 5.2. S tng hp carbohydrate Tng t nh cc thc vt bc cao, vi sinh vt c kh nng tng hp cc oligo - v polysaccharide. Lng cc oligo - v polysaccharide ni bo t ti 60% khi lng kh ca t bo, cn polysaccharide ngoi bo c th vt nhiu ln khi lng ca vi sinh vt. Thnh t bo cng cha mt lng ln polysaccharide. Tt c cc oligo v polysaccharide c tng hp bng cch ko di chui saccharide c trc nh vic thm n v monosaccharide (X). n v monosaccharide ny tham gia vo phn ng dng nucleotide monosaccharide c hot ha, thng l dn xut ca uridine diphosphate (UDP.X) nhng i khi cng vi cc nucleotide purine v pirimidine khc. S tng hp din ra theo phn ng chung nh sau: ...X.X.X.X.X.X + UDP - X = ...X.X.X.X.X.X.X. + UDP (n - nhnh) (n + 1 - nhnh) Trong trng hp polysaccharide bao gm hai loi monosaccharide lin tip (X v Y) phn ng chung xy ra qua hai bc: Bc 1: ...X.Y.X.Y.X.Y + UDP - X = ... X.Y.X.Y.X.Y.X. + UDP

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Bc 2: ...X.Y.X.Y.X.Y.X + UDP - Y = ... X.Y.X.Y.X.Y.X.Y + UDP Con ng tng hp polysaccharide phn nhnh ta cn bit rt t. Trong dch chit t bo ca mt s loi nm si c cha enzyme kitine sinthetase xc tc phn ng chuyn cc nhnh N - acetylglucosamine t UDP - acetylglucosamine n phn t nhn. Mt cch tng t vic tng hp mannan ca thnh t bo nm men cng bao gm vic chuyn cc nhnh mannose t GDP - mannose n cht nhn. Trong vic tng hp tinh bt Chlorella v glycogen Arthrobacter c s tham gia ca ADP glucose. - Tng hp glycogen: Khi c iu kin ngoi cnh hn ch s pht trin ca vi khun th chng c th tch ly glycogen nh h enzyme trng hp cc glucose. Glycogen tch ly di dng cc ht trong cht ngyn sinh m chng ta c th pht hin bng cch nhum mu vi iode (hnh 5.8).

Hnh 5.8 S tng hp glycogen

- Tng hp levan v dextran: Cc levan (poly - 2,6-fructose) v cc dextran (poly -1,6 glucose) vi s phn nhnh - 1,3 hoc -1,4 c tng hp t saccharose theo phn ng sau: n saccharose n glucose + (fructose)n n saccharose (glucose)n + n fructose S hnh thnh nhng hp cht ny chnh l nguyn nhn ca hin tng nhy nht khi nui cy mt s loi vi khun trn mi trng giu saccharose. Cc hp cht polysaccharide cng vi hp cht nit s hnh thnh nn glycopeptide ca thnh t bo. 6. S trao i protein 6.1. S phn gii protein Khc vi ln men, c cht ca qu trnh thi ra l protein. y l mt trong nhng thnh phn quan trng ca xc b ng vt, thc vt v vi sinh vt. S phn gii cc hp cht hu c cha nit c ngha to ln

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i vi nng nghip v i vi vng tun hon vt cht trong t nhin. Ngi ta gi qu trnh phn gii ny l qu trnh ammon ha (ammonification). Mun phn gii protein, cng nh cc hp cht cao phn t khc, trc ht vi sinh vt phi tit ra protease ngoi bo v chuyn ha protein thnh cc hp cht c phn t nh hn (polypeptide, olygopeptide). Cc cht ny tip tc c phn hy thnh acid amin nh cc peptidase ngoi bo, hoc c xm nhp ngay vo t bo vi sinh vt sau mi chuyn ha thnh acid amin. Mt phn cc acid amin ny c vi sinh vt s dng trong tng hp protein, mt phn khc c phn gii theo nhiu con ng khc nhau to NH3, CO2 v nhiu sn phm trung gian khc. Nhng vi sinh vt khng c kh nng sn sinh cc enzyme phn gii protein ngoi bo s khng ng ha c cc loi protein thin nhin. Chng ch c th s dng c cc sn phm thy phn ca protein. Rt nhiu loi vi sinh vt khc nhau tham gia vo qu trnh ammon ha trong t nhin. ng ch l cc vi sinh vt sau: - Vi khun: Bacillus mycoides, B. subtilis, Proteus vulgais, Pseudomonas aeruginosa, E. coli... - X khun v nm: Streptomyces griseus, S. fradiae, Aspergillus niger, A. awamori, Penicillium camemberti, Mucor spp., ... Trong c th vi sinh vt, cc acid amin thng c chuyn ha nh qu trnh kh amin, qu trnh kh carboxyl hoc ng thi va kh amin va kh carboxyl. Khi phn gii cc acid amin ty theo tng loi phn ng s to cc sn phm trung gian, y l tin cht cho cc phn ng sinh tng hp hoc cn thit cho mt s qu trnh trao i cht, nh s to thnh acid fumaric t acid aspactic: HOOC- CH2- CH (NH2) - COOH HOOC-CH=CH-COOH+ NH3 hay to - ketoglutaric t acid glutamic, acid acetic t glycine, acid succinic t aspactic...). Trong qu trnh kh amin thy phn c s lin kt vi cc ion H+ v OH- nh s to thnh acid lactic t alanine: CH3-CH(NH2)-COOH + H2O CH3-CHOH-COOH + NH3

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Protein
Enzyme phn gii protein ngoi bo

Polypeptide Olygopeptide

Amino acid Peptidase

Cc Amino acid ni bo

Kh amin v Kh amin phn gii mch carbon

Chuyn amin v phn gii carbon

Trc tip dng trong qu trnh sinh tng hp protein

Nhiu sn phm sinh ra trong qu trnh phn gii acid amin (ru, acid hu c ...) s c li cun vo qu trnh phn gii to nng lng. Nhiu loi amin do vi khun sn sinh ra trong c th ngi v ng vt c tnh c, nh s tch ly histamine c th gy chng co git mch mu, cc hp cht diamine nh acmatine, putrecine gp nhiu trong tht c i thiu u c tnh c. Mt s vi khun c kh nng chuyn ha tryptophan thnh 2 cht c mi hi l indol v skatol. Trong qu trnh phn gii cc acid amin cha lu hunh (methionine, cysteine, cystine) s to ra H2S lm cho phn hoc tht c c mi hi.
NH2 HOOC- CH- (NH2)-CH2-SH + 2H2O H2S + NH3 + CH3COOH + HCOOH

(cysteine)

Khi qu trnh thi ra xy ra trong iu kin thong kh th qu trnh xy ha c tin hnh n cng, khi hu ht carbon c chuyn thnh CO2. Nu qu trnh ny din ra trong iu kin k kh th c s tch ly nhiu sn phm trung gian ni trn. Ngoi kh nng phn gii protein, vi sinh vt cn c kh nng phn gii mt s cht m khc nh urea, acid uric... S phn gii cc cht ny cng to ra cc sn phm trung gian nh acid formic, acid acetic, CO2,

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NH3..., cc cht ny c th tham gia trc tip vo qu trnh trao i cht v trao i nng lng ca t bo. Urea l cht hu c ch yu trong nc tiu ca ngi v ng. Trong urea c cha 47% nit nhng nu khng c vi sinh vt phn gii thnh NH3 th ngun nit ln lao ny cng tr thnh v ch i vi mi thc vt. Rt nhiu vi khun, x khun, nm si sng trong t c kh nng phn gii mnh m urea. ng ch l cc loi nh Planosarcina urea, Micrococcus urea, Bacillus pasteurii, Proteus vulgaris... Qu trnh phn gii urea xy ra rt n gin vi s xc tc ca urease: H2N- CO- NH2 + 2H2O (NH4)2CO3 (NH4)2 CO3 t bn vng do d dng phn gii tip: (NH4)2CO3 2NH3 + CO2 + H2O NH3 sinh ra do s phn gii hp cht hu c thng c xy ha thnh NO2- v tip tc xy ha n NO3-. Phn m ((NH4)2SO4 hoc NH4Cl) sau khi bn rung cng nhanh chng chuyn thnh NO3-. Qu trnh chuyn ho ny gi l qu trnh nitrate ha. Qu trnh ny bao gm 2 giai on: - Qu trnh nitrite ha c biu th bng phng trnh phn ng sau: 2NH3 + 3O2 2HNO2 + 2H2O + Q Nng lng sinh ra trong phn ng ny cng c vi khun nitrite s dng ng ha CO2 ca khng kh. Thc ra qu trnh ny tri qua nhiu phn ng trung gian v to nhiu sn phm trung gian, trong sn phm trung gian quan trng nht l hydroxilamin (NH2OH). Enzyme xc tc cho vic chuyn hydro l cc enzyme ca qu trnh h hp hiu kh. Vi khun nitrite l bn t dng bt buc vi cc chi i din l Nitrosomonas, Nitrosococcus, Nitrosospira v Nitrosocystis. - Qu trnh nitrate ha c biu th bng phng trnh sau: NO3- + Q NO2- + O2 Cng nh cc vi khun t dng ha nng khc, nng lng sinh trong phn ng xy ho NO2- c s dng ng ha CO2 khng kh. Vi khun nitrate ha in hnh l cc loi thuc ging Nitrobacter. y l nhng t bo hnh bu dc, kch thc khong 0,8-1,0m, thuc loi G-, khng to bo t, khun lc nh b v trong sut. Ngoi ra cn c mt s ging khc nh Nitrococcus, Nitrospira...

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6.2. S tng hp protein Ba qu trnh ch yu trong vic bin i v truyn t thng tin di truyn trong c th l: - S nhn i DNA to ra 2 phn DNA ging nhau v ging vi phn t DNA m. - Qu trnh sao m theo thng tin di truyn t DNA c sao sang RNA, sau c chuyn n ribosome. - Qu trnh dch m cho php chuyn dch thng tin di truyn thnh cc axit amin cu trc nn protein. Cng ngh di truyn cho php gii m trt t cc nucleotide thnh trt t cc axit amin c xc lp (h. 5.9).

Hnh 5.9 S sinh tng hp axit amin (theo Lehninger)

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Phn ln cc vi sinh vt c kh nng tng hp 20 loi axit amin. Con ng tng hp axit amin vi sinh vt c nghin cu kh t m. Nguyn liu dng cho qu trnh tng hp l nhng sn phm trung gian nh pyruvate, - ketoglutarate, oxaloacetate, fumarate... Trong phn ln trng hp, nhm amin c a vo giai on cui cng ca sinh tng hp nh s chuyn amin, mt s acid amin c hnh thnh nh hng lot bin i ca cc acid amin khc, trong trng hp khng cn s chuyn amin. Nhiu loi L - acid amin c sinh tng hp bng con ng ln men nh vi sinh vt nh lysine, glutamic, alanine, glycine... Hin nay nhiu loi acid amin t nhin l sn phm ca cng ngh sinh hc bng cch thy phn protein, tng hp ha hc hoc sinh tng hp nh vi sinh vt. Trong nhiu trng hp ngi ta phi hp gia tng hp ha hc v sinh tng hp, phng php ny c nhiu u im l cho cc dng L - acid amin l dng thng gp trong c th. 7. S trao i lipid 7.1. S phn gii lipid Lipid (ester phc tp ca glycerine v acid bo) v cc cht sp (ester phc tp ca acid bo v ru bc mt) c nhiu vi sinh vt dng lm ngun thc n v nng lng. So vi cc c cht khc th y l loi c cht c ng ha vi tc chm, qu trnh ny c thc hin bi nhiu nhm vi sinh vt khc nhau nh vi khun, x khun, vi nm Bc u tin ca qu trnh ng ha lipid l s phn gii thnh glycerine (hoc ru n nguyn t) v cc acid bo. Qu trnh ny c xc tc nh lipase ni bo hoc ngoi bo. Sau khi c phosphoryl ha, glycerine s c chuyn ha theo con ng EMP, cn cc acid bo i vo chu trnh - xy ha (hnh 5.10). Nh vy, c qua mt vng xy ha han ton chui carbon ca phn t acid bo li mt i 2C. C tip tc nh vy th ton b chui carbon s c chuyn ha thnh acetyl - CoA, cht ny c chuyn ha nh chu trnh TCA hoc chu trnh acid glyoxilic. Mt s nm mc thuc cc chi nh Penicillium v Aspergillus c th xy ha acid bo mt cch khng trit v tch ly li trong mi trng metylketone to mi kh chu (gp trong du m b nhim mc).

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Hnh 5.10: Chu trnh - xy ha axit bo

Cu hi n tp ca chng 5
1. Cc hnh thc vn chuyn cc cht qua mng? 2. C ch ca qu trnh sinh nng lng ca h hp hiu kh v k kh? 3. Cc kiu h hp vi sinh vt? 4. Cc con ng phn gii hexose? 5. Cc kiu trao i cht vi sinh vt? 6. C ch v ngha ca chu trnh acid tricarboxylic (ATC)? 7. S phn gii cc hp cht protein? 8. Vai tr ca vi sinh vt trong vng tun hon nit? 9. Vai tr ca vi sinh vt trong tun hon carbon?

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Chng 6

H hp k kh
I.Khi nim chung
Nhiu vi khun hiu kh c th sinh trng c trong iu kin khng c oxy, lc chng dng nitrate (hoc sulfate) lm cht nhn in t cui cng, v vy gi l h hp nitrate (hoc h hp sulfate). Kh nng chuyn in t cho nitrate v sulfate, gip cho vi khun thc hin tng i y qu trnh oxy ha hon ton c cht, m khng cn c s tham gia ca oxy phn t, nh vi sinh vt thu c nhiu nng lng hn so vi qu trnh ln enzyme (hnh 6.1) Ngoi ra, c mt s t vi khun (Clostridium aceticum) li c th dng CO2 lm cht nhn hydro trong h hp k kh.

Hnh 6.1: S qu trnh h hp hiu kh, k kh v ln enzyme

II. H hp nitrate, ammonium ha nitrate v kh nitrogen


H hp nitrate hay l kh d ho nitrate khc vi qu trnh kh ng ho nitrate ch, cc sn phm kh trong trng hp ny khng c t bo s dng tip, m thng c tit ra ngoi mi trng.

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Rt nhiu vi khun hiu kh khi sng trong iu kin k kh c kh nng dng nitrate lm cht nhn hydro cui cng, v d mt s loi thuc cc chi Bacillus, Aerobacter v E. coli kh nitrate thnh amoniac (qu trnh amon ho nitrate), trong khi mt s khc li kh nitrate gii phng nit phn t (N2) hoc (N2O), ngi ta cho rng qu trnh amon ho nitrate v phn nitrate ho c chung giai on u (hnh 6.2)

Hnh 6.2: S qu trnh h hp nitrate

Vi khun nitrate ha thuc chi Nitrobacter dng nitrite lm ngun nng lng. C th biu th chui h hp ca vi khun nitrate nh sau:

Hnh 6.3: Chui h hp ca vi khun nitrate ha

Nhiu loi vi sinh vt c kh nng dng nitrate lm ngun thc n nit, kh ng ho nitrate qua nitrit thnh amoniac, hp cht ny s tham gia tng hp trong t bo ca chng:
NO 3 NO x NH2OH NH + 2 4

Khi ammonium ho nitrate ta c:

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8[H] + H+ + NO 3

NH + + OH- + 2H2O 4 N2 + 6H2O

Cn khi kh nitrate ho:


10[H] + 2H+ + NO 3

Khi h hp nitrate, c bit l khi phn nitrate ho, c cht hu c c oxy ho hon ton n CO2 v H2O. Nng lng sinh ra vi nitrate l cht nhn hydro ch thp hn trong trng hp cht nhn l oxy phn t khong 10%. ATP cng c to ra nh kt qu ca qu trnh phosphoryl ha trong chui h hp, v vy qu trnh phn nitrate ha m bo hot tnh sinh trng ca vi sinh vt mc cao. nhiu loi vi sinh vt c kh nng h hp nitrate nh E.coli th chui vn chuyn in t tng t nh trong trng hp hiu kh, ch c citocromo-oxydase c thay bng nitratereductase. Giai on u ca qu trnh kh nitrate c xc tc nh nitratereductase. Nhiu vi khun phn nitrate ha c kh nng dng khng nhng nitrate m c nitrite lm cht nhn hydrogen, M. denitrificans cn kh c c N2O. Ty theo loi vi khun m sn phm ca kh nitrate d ha v nitrite d ha l N2, N2O hay NO. Do vi khun phn nitrate ha c kh nng dng nitrate ch yu lm + cht nhn in t, m khng c kh nng kh n thnh NH 4 , cho nn mun nui cy chng phi cn b sung thm ngun m vo mi trng + (pepton hoc NH 4 ). Vi khun phn nitrate ha phn b rt rng trong t nhin, nhng loi thng gp nh: P. denitrificans, P. aeruginosa, P. fluorescens, Micrococcus denitrificans, Hydrogenomonas agilis... Vi khun phn nitrate ha l tc nhn sinh hc lm ngho nitrogen ca t (cn phn nitrate ha hc xy ra mnh khi t qu nhiu acid) qu trnh ny xy ra mnh khi t b k kh (t ngp nc, khng ti...)hoc khi dng phn m (nitrate) cng vi phn chung trn nhng rung la ngp nc, y phn nitrate dng bn cho la t hiu qu rt t, v nitrate c th mt ht rt nhanh, ch khong mi gi sau khi bn. Khi t thong kh qu trnh phn nitrate b c ch, v oxy phn t c ch t bo vi khun tng hp enzyme nitratereductase v nitritereductase.

III. H hp sulfate
Phn ln vi sinh vt v thc vt c th dng sulfate lm ngun dinh dng lu hunh tng hp cchp cht ca c th cha S (acid amin cha S, enzyme...). l qu trnh kh ng ha sulfate. Ch c mt

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s t vi sinh vt thuc 2 chi Desulfovibrio v Desulfotomaculum li dng sulfate lm cht nhn hydro cui cng trong h hp k kh. Qu trnh h hp sulfate cng tng t nh qu trnh h hp nitrate, nhng cc vi sinh vt thc hin qu trnh ny u thuc loi k kh bt buc, chng ch c th dng sulfate lm cht nhn hydro cui cng trong qu trnh oxy ha c cht. H2S l sn phm ca qu trnh c sinh ra theo kh ng:
2 8[H] + SO 4 H2S + 2H2O + 2OH-

Vi khun thc hin qu trnh h hp sulfate trc khi b kh thnh H2S phi tri qua nhiu giai on trung gian m c nhng giai on cho n nay ngi ta cha bit c mt cch chc chn:
2 SO 4 APS SO 2 S23

(Adenosin-5-phosphosulfate) Nng lng sinh ra trong qu trnh h hp sulfate c vi sinh vt s dng ng ha cc hp cht hu c (acid hu c, amino acid) mt s vi khun kh sulfate ha c kh nng t dng cacbon, chng dng hydro phn t kh sulfate v s dng nng lng sinh ra trong qu trnh h hp sulfate ng ha CO2 trong khng kh. 4H2 + H2SO4 H2S + 2H2O + Q Vi khun kh sulfate ha c th phn gii pyruvate hnh thnh H2S: 4CH3COCOOH + H2SO4 CH3COOH +4CO2 +H2S Vi khun kh sulfate ha thng gp nhng vng bn ly c kh H2S, tc l nhng ni c qu trnh phn gii k kh cc hp cht hu c. Trong 1ml nc bn cha 104 106 vi khun kh sulfate, cn trong bn c H2S s lng ca chng ln ti 107 t bo. Hng triu nm nay, chng tham gia tch cc vo qu trnh hnh thnh qung lu hunh, m du ha. Desulfotomaculum ruminis tham gia tch cc vo qu trnh kh sulfate v to thnh H2S trong nhng d dy c ca ng vt nhai li (tru, b...). Bng cch kh sulfate nhng vi sinh vt ca nhm ny tham gia tch cc vo chu trnh chuyn ha chuyn ha lu hunh trong t nhin. Mt khc, do sinh ra H2S c th lm c cht hng lot, H2S lm n mn cc b phn kim loi ca cc cng trnh chn su di t hoc di nc.

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Thiobacillus denitrificans Thiobacillus thiooxidans Hnh 6.4: Mt s loi vi khun lu hunh

IV. H hp carbonate to thnh methane Nhng vi sinh vt ny c kh nng hnh thnh methane t carbonate e. Chng ly nng lng cho hot ng sng ca mnh t qu trnh oxy ha k kh cc hp cht khong hay cc hp cht hu c n gin nh acid formic hay mt s acid bo bc cao. Nhng vi sinh vt sinh methane bao gm 4 nhm ph khc nhau v hnh thi: -Cc vi sinh vt sinh methane khng sinh bo t (Methanobacterium) -Cc vi sinh vt sinh methane sinh bo t (Methaneobacillus) -Cc vi sinh vt hnh cu sinh methane (Methanococcus) -Cc 8 cu khun sinh methane (Methanosarcina)

Hnh 6.5: Khun lc Methanosarcina acetivorans.

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Methanobacterium formocicum

Methanospirillum sp.strain TM20-1

Hnh 6.6: Hnh dng mt s loi vi khun methane

Ngi ta thng gp nhng vi sinh vt sinh methane y nhng ao h, m v y bin, ni m iu kin k kh rt thun li cho chng pht trin. Methaneobacterium barkeri. C kh nng chuyn ho CO thnh CH4. Sn phm trung gian ca qu trnh chuyn ho l CO2 v H2. 4CO + 4H2O 4 CO2 + 4H CO2 + 4H2 CH4 + H2O 4CO + 2H2O CH4 + 3CO2 Vi khun sinh methane c chng minh l loi vi sinh vt c kh nng sinh tng hp mnh vitamin B12. S gi thuyt v s hnh thnh CH4 t CO2 hoc acetate vi khun sinh methane c th c trnh by nh sau:

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Hnh 6.7: S gi thit sinh tng hp VTM B12 vi khun sinh methane

V. H hp carbonate to thnh acetate Qu trnh h hp carbonate to thnh acetate mi c pht hin gn y vi khun methane, kh sulfate.

Hnh 6.8: Hnh dng Vi khun Methanobacterium thermoautotrophicum

Kt qu nghin cu vi khun Methanobacterium thermoautotrophicum cho thy nh sau:

Theo con ng ny, 1phn t CO2 b [H] c nng lc kh thnh CH3-X, 1phn t CO2 khc b CO dehydrogenase kh thnh CO. Qua qu trnh cacbocyl ha CH3X s sinh ra Acetyl-X, sau thnh Acetyl-CoA. Di s xc tc ca piruvate siterase, Acetyl-CoA s tip thu 3 phn t CO2 cacbocyl ha thnh acid pyruvic. Acid pyruvic thng qua cc con ng trao i cht quen thuc tng hp ra cc cht hu c cn tht cho t bo.

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Cu hi n tp chng 6
1. So snh nhng im khc nhau gia h hp hiu kh v h hp k kh ? 2. Nhng im ging nhau v khc nhau gia h hp nitrate v h hp sulfate. 3. V tr vi khun, x khun, nm mc, nm men trong cc h thng phn loi hin nay ? 4. Vai tr ca vi khun methane trong thin nhin v trong i sng con ngi ?

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Chng 8

Cc qu trnh ln men
I. Con ng Embden-Meyerhof chung cho cc qu trnh ln men (EM) : Bc chuyn ha
Ln men l mt qu trnh oxi ha-kh cn bng ni sinh, trong mt s nguyn t ca ngun nng lng (cht cho in t) tr nn c tnh kh hn, trong khi mt s nguyn t khc tr nn oxi ha hn, cn nng lng th c sinh ra nh s phosphorin ha c cht. Con ng trao i cht chung i vi s ln men glucose l con ng ng phn, cng cn c gi l con ng Embden-Meyerhof. ng phn c chia thnh ba bc chnh, mi bc gm mt s phn ng ring bit do enzyme xc tc. Bc mt gm cc phn ng chun b, khng c cc phn ng oxi ha v cng khng gii phng nng lng, song li dn n vic to ra hai phn t trung gian quan trng, glixeraldehit-3-phosphate (GAP). Trong bc hai, s oxi ha-kh xy ra, nng lng c d tr di dng ATP v hai phn t piruvat c to thnh. Trong bc ba, mt phn ng oxi ha-kh th hai xy ra v cc sn phm ln men (chng hn ethanol v CO2, hoc acid lactic), s c to thnh. Trong s to thnh hai phn t acid 1,3 iphosphoglixeric c hai phn t NAD+ b kh thnh NADH. Tuy nhin, mt t bo ch cha mt lng nh NAD+, v nu nh ton b NAD+ b chuyn thnh NADH th phn ng oxi ha glucose s b nh ch; s oxi ha tip tc ch c th xy ra khi mt phn t NAD+ c mt tip nhn cc in t va c gii phng ra. S "tc ng" ny c th c khc phc trong ln men bng cch oxi ha NADH tr li thnh NAD+ nh cc phn ng kh piruvat thnh mt trong hng lot cc sn phm ln men khc nhau. Trong trng hp ca nm men, piruvat b kh thnh ethanol vi s gii phng CO2. cc vi khun lactic, piruvat b kh thnh acid lactic. Nhiu con ng kh piruvat cng gp cc vi sinh vt nhn s khc, song kt qu tht s u ging nhau, tc l NADH phi c hi phc li thnh dng oxi ha, NAD+, cho cc phn ng thu nhn nng lng ca ln men c th tip tc. L mt coenzyme d khuch tn, NADH c th tch khi GAP-dedihydrogenase, gn vo mt enzyme khc, (chng hn lactat-dedihydrogenase, enzyme kh piruvat thnh acid lactic), ri li lp tc khuch tn tr li sau khi NADH c oxi ha thnh NAD+ lp li chu trnh mt ln na.

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Trong bt k mt phn ng thu nng lng no, s oxi ha bao gi cng phi c cn bng vi s kh v phi c mt cht nhn no ng ch sn nhn mt in t va c tch ra. Trong trng hp ny, s kh NAD+ ti mt bc trong con ng ng phn s c cn bng bi s oxi ha n ti mt bc khc. (Cc) sn phm cui cng cng phi nm trong trng thi cn bng oxi ha-kh vi c cht ban u, tc l glucose.
Bng 6.1: Cc qu trnh ln men ph bin vi sinh vt Kiu ln men Ln men ru Ln men lactic ng hnh Ln men lactic d hnh Acid propionic Acid hn hp Phn ng tng th Hexose 2 Ethanol + 2 CO2 Hexose 2 Lactat + 2 H+ Hexose Lactat + Ethanol + CO2 + H+ Lactat Propionat + Acetate + CO2 Hexose Ethanol + 2,3Butandiol + Xucxinat + Lactat + Acetate + Focmat + H2 + CO2 Hexose Butirat + Acetate + H2 + CO2 Hexose Butanol + Acetate + Aceton + Ethanol + H2 + CO2 Ethanol + Acetate + CO2 Caproat + Butirat + H2 Fructose 3 Acetate + 3 H+ 4 H2+ 2 CO2 + H+ Acetate + 2 H2O Acetate + H2O CH4 + HCO 3 Vi sinh vt thc hin Nm men Zymomonas Streptococcus Mt s Lactobacillus Leuconostoc Mt s Lactobacillus Propionibacterium Clostridium propionicum Cc vi khun ng rut Escherichia, Salmonella, Shigella, Klebsiella, Enterobacter Clostridium butyricum Clostridium acetobutylicum Clostridium kluyveri Clostridium aceticum Acetobacterium Methanothrix Methanosarcina

Acid butiric Butanol Caproat Homoacetic

Sinh methane

II. Tnh a dng ca ln men


Bng 6.1 trnh by mt s kiu ln men chnh c phn loi da trn cc sn phm c to thnh. l cc qu trnh ln men ru, acid lactic, acid propionic, acid hn hp, acid butiric v acid homoacetic. C

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nhiu qu trnh ln men c phn loi da trn c cht c ln men thay v cc sn phm. Chng hn, nhiu vi khun k kh to thnh bo t (chi Clostridium) ln men cc amino acidvi s sn sinh acetate, amoniac v H2. Cc loi Clostridium khc, nh C. acidiurici v C. purinolyticum ln men cc purin nh xantin hoc aenin vi s to thnh acetate, focmat, CO2, v amoniac. Cn nhng bn k kh khc th ln men cc hp cht thm. Chng hn, vi khun Pelobacter acidigallici ln men hp cht thm phloroglucinol (1,3,5-benzenetriol, C6H6O3) theo con ng sau : Phloroglucinol (C6H6O3) +3 H2O 3 acetate- + 3H+, Go' = -142,5 kJ/phn ng Nhiu qu trnh ln men khng thng thng ch c thc hin bi mt nhm rt hn ch cc vi khun k kh, v trong mt s trng hp ch bi mt vi khun duy nht. Mt s v d c nu trn bng 6.2. Nhiu trong s cc vi khun ny c th c xem nh cc chuyn gia v trao i cht v chng c kh nng phn gii mt hoc nhiu c cht m cc vi khun khc khng phn gii c.
Bng 6. 2: Mt s qu trnh ln men khng thng thng Kiu ln men Acetylen Glycerin Phn ng cn bng Vi sinh vt thc hin tng th 2 C2H2 + 3 H2O Ethanol + Pelobacter acetylenicus Acetate + H+ Acetobacterium spp. 4 Glycerin + 2 HCO
3

7 Acetate + 5 H+ + 4 H2O Resocxinol 2 C6H4(OH)2 + 6 H2O 4 (mt hp cht Acetate + Butirat + 5 H+ thm) Xinamat (mt 2C9H7O2 + 2 H2O C9H9O2 + hp cht thm) Benseat + Acetate + H+ Phlorogluxinol C6H6O3 + 3 H2O (mt hp cht 3 Acetate + 3 H+ thm) Putresxin 10 C4H12N2 + 26 H2O 6 Acetate + 7 Butirat + 20 NH4+ + 16 H2 + 13 H+ Xitrat Xitrat + 2 H2O Focmat + 2 Acetate + HCO 3 + H+ Aconitat

Clostridium spp. Acetovibrio multivorans Pelobacter masiliensis Pelobacter acidigallici Cc vi khun k kh gram dng khng sinh bo t, cha phn loi Bacteroides sp.

Aconitat + H++ 2 H2O 2 CO2 Acidaminococcus fermentans + 2 Acetate + H2

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Glioxilat Xucxinat Oxalat Malonat

4 Glioxilat +3 H+ + 3 H2O 6 CO2 + 5 H2 + Glicolat Xucxinat + H2O Propionat + HCO 3

Vi khun gram m cha phn loi Propionigenium modestum

Oxalat + H2O Focmat + Oxalobacter formigenes HCO 3 Malonat + H2O Acetate + Malonomonas rubra Sporomusa malonica HCO 3

III. Ln men khng c s phosphorin ha c cht Vi mt s c cht, nng lng sinh ra khng ghp i vi s tng hp ATP trc tip nh phosphorin ha c cht, tuy nhin cc hp cht ny vn tr gip sinh trng ln men ca mt c th no . Trong nhng trng hp ny s phn gii c cht c gn lin vi nhng bm ion c dng to nn mt graien proton hoc natri qua mng. V d v kiu ny c cc qu trnh ln men ca Propionigenium modestum hoc Oxalobacter formigenes, c hai u gn s ln men cc acid icacboxilic vi cc bm ion sinh nng lng lin kt vi mng. Propionigenium modestum thc hin phn ng sau y : Xucxinat2- + H2O Propionat- + HCO3- , Go' = -20,5 kJ/phn ng Phng trnh tng th ny thu khng nng lng t do ghp i vi s tng hp ATP trc tip nh phosphorin ha c cht, tuy nhin n li c dng lm phn ng thu nng lng duy nht cho sinh trng ca vi khun. S d nh vy l v s loi CO2 khi xucxinat

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Hnh 6.1: Ln men lin kt vi bm ion vi khun (nng lng thu c khng qua phosphorin ha c cht)

(qua metilmalonil-CoA v ecacboxilase lin kt vi mng bi Propionigenium modestum c ghp i vi s xut Na+ qua mng t bo cht. Mt ATPse chuyn v Na+ nm trn mng ca P. modestum s dng graien ny hot ha s tng hp ATP. Oxalobacter formigenes thc hin s ln men oxalat : Oxalat2- + H2O Focmat- + HCO3- , Go' = -26,7 kJ/phn ng pH trung tnh, oxalat tn ti di dng ion ha oxalat-, v s loi cacboxil thnh focmat- tiu th mt proton. Sau s xut focmat- khi t bo s to nn mt ng lc nh proton (proton motive force = PMF) c kh nng ghp i vi s tng hp ATP nh mt ATPase chuyn v proton nm trn mng.

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iu th v v c o trong trao i cht ca c Propionigenium modestum ln Oxalobacter formigenes nm ch s tng hp ATP xy ra khng cn c phosphoril ha c cht ln chui vn chuyn in t; tuy nhin, s to thnh ATP theo c ch ha thm vn xy ra da vo mt bm Na+/H+ kt hp vi s loi CO2 khi cc acid hu c. iu cn rt ra t nhng s ln men ny l : cc phn ng, d ch thu c di 31,8 kJ, con s cn thit to thnh mt ATP, hoc khng c kh nng ghp i vi s phosphorin ha c cht, cng khng th lp tc b coi l khng c kh nng tr gip sinh trng cho mt vi khun. Nu nh phn ng c th ghp i c vi mt graien ion th s sn sinh ATP vn c kh nng xy ra v do vy vn c sinh trng (Hnh 6.1). IV. Hin tng cng dng (syntrophy) Trong vi sinh vt hc c nhiu v d v hin tng cng dng, mt hin tng m hai sinh vt khc nhau c th cng nhau phn gii mt cht no - v bo ton nng lng ca qu trnh phn gii n mc khng c th no trong hai c th mt mnh c th lm c. Trong a s trng hp, bn cht ca mt phn ng cng dng c lin quan ti kh dihydro (H2) sinh ra bi mt trong hai i tc v c tiu th bi i tc kia. Do vy, cho n nay, cng dng cng cn c gi l s chuyn H2 lin loi. K tiu th H2 c th l bt k mt c th no trong s cc vi khun kh sunfat, ln men homoacetate, v vi khun sinh methane. Hy xem xt trng hp cng dng lin quan ti qu trnh ln men ethanol thnh acetate v s sn sinh methane sau . Nh c th thy trn hnh 2, bn ln men ethanol thc hin mt phn ng khng thun li v mt nng lng t do (Go' dng). Tuy nhin, H2 c sinh ra bi i tc ln men ru li l mt cht cho in t y gi tr i vi qu trnh hnh thnh methane do mt vi khun sinh methane thc hin. V khi cng hai phn ng li th phn ng tng th s l mt phn ng to nhit v c th tr gip sinh trng ca c hai i tc trong hn hp cng dng. Ln men ethanol 2CH3CH2OH + 2H2O 4H2 + 2CH3COO- +2H+ +19,4 kJ /phn ng Sinh methane 4H2+ CO2 - 130,7 kJ/phn ng CH4 + 2H2O Phn ng cng dng 2CH3CH2OH + CO2 - 111,3 kJ/phn ng CH4 + 2CH3COO

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Mt v d khc ca hin tng cng dng l s oxi ha butirat thnh acetate cng vi H2 bi vi khun cng dng oxi ha acid bo Syntrophomonas: Butirat- + 2H2O 2acetate- + H+ + 2H2 , Go' = +48,2 kJ/phn ng S bin i nng lng t do ca phn ng ny rt khng thun li v trong ng ging thun khit, Syntrophomonas khng th mc c trn butirat. Song nu H2 va sinh ra trong phn ng c tiu th ngay bi mt c th i tc (nh mt vi khun sinh methane) th Syntrophomonas li sinh trng rt tt trong ng ging cy chung vi bn tiu th H2.

V. Ln men ru nh nm men v vi khun


Ethanol l mt trong s cc sn phm ln men ph bin nht gp vi sinh vt. Bn thn thc vt v nhiu loi nm cng tch ly ethanol di cc iu kin k kh. Vi sinh vt sn sinh ethanol ch yu l nm men, c bit l cc chng thuc loi Saccharomyces cerevisiae. Ging nh a s nm, nm men l nhng c th h hp hiu kh, nhng khi vng mt khng kh chng s ln men hirat cacbon thnh ethanol v CO2. Nhiu vi khun k kh v hiu kh cng to thnh ethanol nh cc sn phm chnh hoc sn phm ph khi ln men cc hexose hoc pentose. 1. S to thnh ethanol nh nm men S ln men glucose thnh ethanol v CO2 nh Saccharomyces cerevisiae din ra theo con ng Embden-Meyerhof. S chuyn ha piruvat thnh ethanol gm hai bc. Trong bc mt, piruvat c loi CO2 thnh acetaldehit nh piruvat-ecacboxilase vi s tham gia ca tiaminpirophosphate. Trong bc sau, acetaldehit b kh thnh ethanol nh alcohol-edihydrogenase cha NADH.
Glucose 2 Piruvat

2NAD

2NADH 2 Acetaldehyt

2CO2

2Ethanol

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Bng s chuyn H ny, dihydro tch ra do triosephosphatedehydrogenase xc tc s c tiu th, nh vy trng thi oxi ha-kh s c cn bng. 2. Cc dng phng trnh Neuberg Cc phng php v pht hin ca Carl Neuberg v ln men ru khng ch c ngha lch s. ng khm ph ra rng, khng ch glucose m c piruvat cng c nm men ln men v acetaldehit xut hin nh mt sn phm trung gian c th b bt gi bi dihydrosulfit. Dihydrosulfit vn khng c i vi nm men. Nu b sung dihydrosulfit vo mt mi trng cha glucose ang c nm men ln men th acetaldehit s b kt ta do to thnh phc cht : CH3-CHO + NaHSO3 CH3- CHOH -SO3Na Lc glycerin s xut hin nh mt sn phm ln men mi, ng thi hiu sut ethanol v CO2 s gim xung. Ln men vi s c mt ca dihydrosulfit c s dng trong cng nghip sn xut glycerin. Nguyn l ca n l, acetaldehit b bt gi, v do vy khng th hot ng nh mt cht nhn in t c na. Lc ny, thay cho acetaldehit, dihydroxiacetonphosphate c s dng lm cht nhn in t v b kh thnh glycerin-3-phosphate ri tip tc b loi phosphate tr thnh glycerin. Phng trnh phn ng din ra nh sau : Glucose + Dihydrosulfit Glycerin + Acetaldehit-sulfit + CO2 Phng trnh ln men ci bin ny c gi l dng ln men Neuberg 2. Khi b sung cc cht kim (NaHCO3, Na2HPO4) th glycerin cng c to thnh v acetaldehit b chuyn thnh ethanol v acetate qua phn ng ha hai, v do vy khng cn hon thnh c chc nng ca mt cht nhn in t. Phng trnh ln men c tn l dng ln men Neuberg 3 v c vit nh sau : Glucose + 2H2O Ethanol + Acetate + 2Glycerin + 2CO2 Dng ln men thng thng c Neuberg gi l dng ln men 1.

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3. Hiu ng Pasteur Hiu ng Pasteur l s c ch ln men ru (hay qu trnh ng phn) bi oxi (tc l bi h hp hiu kh). S chuyn sang h hp khng nhng lm gim s to thnh ethanol v CO2 m cn lm gim s hp th ng. C hai c ch chu trch nhim i vi hin tng ny. Mt mt, l s cnh tranh ADP v phosphate v c gia hai qu trnh phosphorin chui h hp v phosphorin ha c cht. S loi dihydro xy ra trong phn ng oxi ha glyceraldehyt-3-phosphate cn ADP v phosphate v c (Pvc) : Glyceraldehyt-3-phosphate + NAD + Pvc NAD + ATP
H

3-phosphoglicerate +

Do vy, s chuyn ha c cht (glucose) qua con ng EM ph thuc vo s c mt ca ADP v phosphate. Tuy nhin, trong iu kin hiu kh, s phosphorin ha chui h hp cng cnh tranh ADP v phosphate pht sinh ATP. Lc nng ni bo ca ADP v phosphate gim, v hu qu l, s chuyn ha glucose v do vy, s to thnh ethanol cng s gim. Khi dng initrophenol (DNP) lm bt hot phosphorin ha chui h hp, ADP v phosphate li tr nn c sn i vi phn ng loi dihydro ca glyceraldehyt-3-phosphate, bi vy ngi ta thy ngay trong iu kin thang kh, s tiu th glucose (cho ln men) vn tng. C ch th hai chu trch nhim i vi hiu ng Pasteur l c ch iu ho bng enzyme d lp th. Hnh 3 tm tt nhng quan h iu ha ch cht trong hiu ng ny. Trong iu kin thong kh, s ln men ru ch to thnh mt vi ATP tnh trn mi phn t glucose. duy tr s cung cp nng lng, t bo phi s dng nhiu glucose. T l ca ATP/AMP trong trng hp ny rt thp. Tri li, khi oxi xm nhp vo mi trng, ATP c to thm rt nhiu nh s qu trnh phosphorin ha chui h hp. Enzyme cha kha d lp th iu ha s s dng glucose trong nm men ln men ru l phosphofructokinase. l enzyme khng thun nghch u tin ca con ng EM. N xc tc cho s phosphorin ha fructose-6-phosphate thnh fructose-1,6-iphophat nh ATP v b km hm d lp th bi ATP. S km hm theo c ch lin kt ngc ny lm tng nng glucose-6-phosphate trong t bo do tnh thun nghch ca phn ng enzyme trc . Glucose-6-phosphate km hm d lp th hexokinase, km hm c s hp thu glucose, do lm gim s tiu th ng ny.

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Trong khi ATP km hm d lp th phosphofructokinase th AMP li hot ha n. Bi vy mt t l ATP/AMP thp nh trong ln men li kch thch s hp thu v s trao i glucose. Hot tnh ca phosphofructokinase cng b km hm bi xitrat. Nh cht hiu ng m tnh th hai ny m tc dng km hm ca ATP c tng cng, tri li cc ion amn li kch thch enzyme ny. S iu ha m t trn c th c chng minh d dng khi ngi ta o nng ni bo ca glucose, glucose-6-phosphate, fructose-6phosphate, fructose-1,6-diphosphate v cc triose-phosphate trc v sau khi chuyn t iu kin nui k kh sang iu kin nui hiu kh. Ngay sau khi c thng kh, nng glucose-6-phosphate v fructose-6-P ca t bo tng nhanh, cn nng ca fructose-1,6-diphosphate v cc triosephosphate li gim mnh. y, phosphofructosekinase hot ng nh mt ci van c iu chnh bi cc aenilat cng nh cc cht trao i trung gian khc. Nu van b ng, cc cht trao i nm pha trn van s b ng, cn cc cht nm bn di van c tip tc chuyn ha v do vy, nng gim i. Hiu ng Pasteur cng gp vi khun (chng hn, E. coli) v m ng vt. Nguyn tc iu ha l ging nhau, ch c nhng khc bit v loi cht hiu ng. Chng hn, E. coli, khng phi AMP m ADP mi l cht hiu ng dng. Nhng c ch c bn ca trao i cht th rt ging nhau, cn cc c ch iu ha th thng c tnh c hiu loi v c hiu chng. 4.K thut sn xut ethanol nh nm men Cc loi nm men dng trong sn xut ethanol l cc nm n bo mang nhiu c im phong ph v sinh trng v pht trin. Cc loi c quan tm trc ht trong sn xut cng nghip l Saccharomyces cerevisiae, Saccharomyces uvarum (carslbergensis), Schizosaccharomyces pombe v Kluyveromyces sp. Ngoi ra, nhiu bin chng bt ngun t cc nm men ny c to ra nh k thut di truyn. Cc tnh cht mong mun ca mt qu trnh sn xut ethanol cng nghip ph thuc nhiu vo vic la chn chng vi sinh vt dng trong ln men. Cc chng ny phi c mt nng sut sn phm cao trn mt n v c cht c s dng, mt tc ln men cao, tnh chu cn cn thit, kh nng sng st nhit cao, tnh bn trong cc iu kin ln men v tnh chu pH thp.

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Kh nng s dng cc c cht khc nhau ph thuc vo loi. Ni chung, cc c th ny c kh nng sinh trng v ln men ethanol c hiu qu cc gi tr pH gia 3,5-6,0 v nhit t 28-35oC. Hin nay ngi ta quan tm nhiu n vic to ra cc th t bin khuyt h hp t nm men nh vic s dng cc tc nhn ha hc nh cc thuc nhum hoc cc hp cht lm bin tnh protein. Cc bin chng ny cha ADN ti th b bin i theo hng c ch s to ra cc enzyme cn thit cho s trao i cht hiu kh v do vy thch hp vi s tng hp ru. 5. Hiu sut ln men Nm men, di iu kin k kh chuyn ho glucose thnh ethanol trc ht bng con ng Embden-Meyerhof. Phn ng tht s bao gm s to thnh 2 mol ethanol, 2 mol CO2 v 2 mol ATP trn mi mol glucose c ln men. Do vy, v mt trng lng mi gam glucose v l thuyt c th to ra 0,51 g ru. Tuy nhin sn lng t c trong ln men thc t thng khng vt qu 90-95% con s l thuyt. S d nh vy l v mt phn cht dinh dng c s dng tng hp sinh khi mi v cho cc phn ng lin quan ti s duy tr t bo. Cng cn c cc phn ng ph xy ra (thng dn ti Glycerin v sucxinat) tiu th ti 4-5% c cht tng s. Nu loi tr c cc phn ng ny th sn lng ethanol c th tng thm c 2,7%. Mt nng nh oxi cn phi c cung cp cho nm men ln men v y l thnh phn cn thit trong s sinh tng hp cc cht bo cha bo ha v cc lipit. Thng thng oxi c gi trong dch ln men nng 0,05-0,1 mm Hg tnh theo p sut oxi. Bt c nng no nm trn gii hn u s kch thch sinh trng, vi phm n s to thnh ethanol (hiu ng Pasteur). Cc nhu cu tng i ca cc cht dinh dng c dng trong s tng hp ethanol t l thun vi cc thnh phn chnh ca t bo nm men. Chng bao gm C, O, N, v H. Thp hn mt cht l P, S, K v Mg dng tng hp cc thnh phn th yu. Cc mui v c (nh Mn, Co, Cu, Zn) v cc nhn t sinh trng hu c (acid amin, acid nucleic, v cc vitamin) c yu cu di dng vt. Nhiu loi nguyn liu dng sn xut ru quy m ln (nh r ng ma hay actiso Jerusalem) ngoi hirat C cn cung cp ton b cc cht dinh dng cn thit cho sinh trng ca nm men. Trong mt s trng hp, ngi ta cng a thm vo mt s cht dinh dng b sung nh cc mui amn, phosphate K hay ngun cao ng r tin.

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Hnh 6.3: Hiu ng Pasteur C ch iu ha nh enzyme d lp th Phosphofructosekinase

Nm men rt mn cm vi s c ch bi ethanol. Nng 1-2% (w/v) lm chm sinh trng ca vi sinh vt v nng cn 10% (w/v) tc sinh trng ca nm men gn nh ngng hn. Bn cht ca hin tng km hm ny l rt phc tp. Vic thm cn vo pha log lm gim nhanh tc sinh trng ca nm men (c l do tc ng ln s tng hp protein), gim kh nng sng st ca t bo (qua s bin tnh khng thun nghch ca cc enzyme), v mt mc thp hn nhiu,

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ethanol lm gim tc tng hp bn thn n. Cc dn liu v mc chu cn ca mt s chng nm men ph thuc rt nhiu vo thnh phn acid bo trong mng t bo cht cho thy rng thnh phn acid bo kch thch hoc km hm s tit ethanol khi bo tng. Cc kt qu khc cung chng minh c rng ethanol c to ra trong ln men (ethanol t sinh) c ch ln sinh trng ca t bo mnh hn l ethanol c a vo mt cch nhn to t bn ngoi. 6. S to thnh ethanol nh vi khun Trong s vi khun, kiu ln men ru ph bin nm men (EM, piruvat-ecacboxilase) ch gp Sarcina ventriculi. y l loi vi khun chu kh yu, d dng phn lp c t t, nhng cng gp c trong dch tiu ha ca nhng ngi mc bnh d dy. Cc vi khun ny c coi l khng bnh thng v c kch thc rt ln (ng knh 4 m), to nn cc dng hp bao gi (cha trn 64 t bo mi hp , c lin kt vi nhau nh xenlulose), chu pH (sinh trng c pH t 0,9 n 9,8) v to thnh ni bo t. T dch p ln men ca cy da di (Agave americana) vng Mexico ngi ta phn lp c mt loi vi khun hnh que, di ng, c lng roi hai cc, c kh nng to thnh ethanol. Vi khun ny c tn l Zymomonas mobilis, phn gii glucose theo con ng 2-keto-3deoxi-6phosphogluconat (KDPG) ct piruvat thnh acetaldehit v ioxit cacbon. Acetaldehit c kh thnh ethanol. Ethanol, ioxit cacbon v mt lng nh acid lactic l nhng sn phm ln men duy nht. Ngi ta rng trong ru mnh ch t da di, cc nguyn t cacbon s 2 v s 3 cng nh s 5 v s 6 thay th cc nguyn t cacbon s 1 v s 2 cng nh s 5 v s 6 trong ru ch t nm men.

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Trong qu trnh ln men ca mt s Enterobacteriaceae v Clostridium, ethanol xut hin nh mt sn phm ph. Tuy nhin, tin cht ca ethanol, acetaldehit, khng c gii phng trc tip t piruvat nh piruvatecacboxilase m c to thnh nh s kh acetil-CoA. Cc vi khun ln men lactic d hnh (v d Leuconostoc mesenteroides) to ra alcohol theo mt con ng hon ton khc. Qua cc bc u tin ca con ng pentosephosphate (PP), glucose c phn gii thnh pentosephosphate. Phosphoketolase tn cng vo xilulose -5- phosphate : Xilulose-5-phosphate+Pvc Acetilphosphate + glyceraldehyt-3-phosphate Acetilphosphate va to thnh nh acetaldehit-edihydrogenase v alcohol-edihydrogenase s c kh thnh ethanol. Sn phm phn gii cn li l glyceraldehyt-3-phosphate, s c kh thnh lactat qua piruvat. VI. Ln men lactic v h Lactobacteriaceae Cc vi khun lactic c xp chung vo h Lactobacteriaceae. Mc d nhm ny khng c s ging nhau v hnh thi v, bao gm c vi khun hnh que di, hnh que ngn ln cc vi khun hnh cu, song v mt sinh l chng c chung cc c im kh c trng. Tt c cc i din u l cc vi khun gram dng, khng to thnh bo t (tr Sporolactobacillus) v khng di ng. thu nng lng chng hon ton ph thuc vo cc hirat cacbon v tit ra acid lactic (lactat). i lp vi cc Enterobacteriaceae l bn cng sn sinh acid lactic, chng thuc bn ln men bt buc. Chng khc cha cc hemin (xitocrom, catalase). Mc d vy, cc vi khun thuc h Lactobacteriaceae c kh nng sinh trng khi c mt oxi khng kh, chng thuc bn k kh song ng thi cng l bn chu kh. Mt vi khun sinh trng hiu kh m khng cha catalase th gn nh chc chn l mt vi khun lactic (tr ngoi l Acetobacter peroxydans v Shigella dysenteriae). 1. Nhu cu v cc cht b sung v nhn t sinh trng Mt c im na ca cc vi khun lactic l c nhu cu v cc cht b sung. Khng c i din no c th mc c trn mi trng v c thun khit cha glucose v cc mui amn. a s trong chng cn hng lot cc vitamin (lactoflavin, tiamin, acid pantotenic, acid nicotinic, acid folic, biotin), v cc acid amin, cc baz purin v pirimiin. V vy ngi ta thng nui chng trn cc mi trng dinh dng phc tp cha

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nhng s lng kh ln cao nm men, dch c chua, dch ng sa hoc thm ch c mu na. iu ngc nhin l mt s vi khun lactic (v c cc vi khun ln men khc) khi sinh trng trn mi trng dinh dng cha mu c th to thnh cc xitocrom v thm ch c th tin hnh qu trnh phosphorin ha chui h hp. V vi khun lactic thiu nng lc tng hp cc pocphirin nn khi cc pocphirin c b sung vo mi trng th mt s vi khun lactic c th to thnh cc sc t hem tng ng. Vi khun lactic c coi nh nhng k qu qut v trao i cht. C th rng do hu qu ca s chuyn ha sinh trng trong sa hoc trn nhng mi trng giu cht dinh dng v cht sinh trng, chng mt i kh nng tng hp nhiu cht trao i. Trong khi chng li c kh nng m a s cc vi khun khc khng c, l kh nng s dng lactose. Kh nng ny nhiu vi khun ng rut (v d Escherichia coli) cng c. Lactose khng c mt trong gii thc vt, n c cc ng vt c v to thnh v tit ra cng vi sa hoc c hp thu vo cng vi sa. Vic s dng c lactose cng c th c xem nh mt s thch nghi vi cc iu kin tn ti trong ng rut ca ng vt c v. Lactose l mt isaccarit, trc khi i vo cc con ng phn gii hexose n phi c phn ct : Lactose + H2O
- Galactozidase

D-glucose + D-galactose

Sau khi c phosphorin ha galactose c chuyn thnh glucosephosphate. Do c s sn sinh mt lng ln lactat nn mi trng dinh dng cn phi c trung ha. Thng ngi ta dng cacbonat canxi. Trn mt mi trng thch dinh dng c b sung cacbonat canxi c th nhn ra s to thnh acid qua cc vng trong sut bao quanh cc khun lc. Ni sng ch yu ca cc vi khun lactic l sa v nhng ni sn xut v ch bin sa, thc vt nguyn vn hoc ang t phn hy, cng nh ng rut v nim mc ca ngi v ng vt. Do to thnh mt lng ln lactat v do tnh chu chua m di cc iu kin mi trng thch hp cc vi khun lactic pht trin nhanh, v vy c th d dng phn lp cc vi khun lactic trn cc mi trng dinh dng chn lc.

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2. Ln men lactic ng hnh Vi khun lactic ln men ng hnh to thnh ton b hoc gn nh ton b l lactat. Chng phn gii glucose qua con ng EM, cha cc enzyme cn thit, k c alolase v chuyn dihydro tch ra trong qu trnh loi dihydro ca glyceraldehyt-3-phosphate sang piruvat : Glucose
2 Piruvat

2 NAD

2 NADH

2 Lactat

Vic xut hin D-, L+ hay DL- lactat l ph thuc vo tnh chuyn ha khng gian ca enzyme vn chuyn lactat-edihydrogenase v vo s c mt ca mt lactat-racemase. Ch mt phn nh piruvat c loi cacboxil v chuyn thnh acetate, ethanol, CO2 v acetoin. Vic to thnh cc sn phm ph ph thuc vo s c mt hay vng mt ca oxi. 3. Ln men lactic d hnh Vi khun ln men lactic d hnh thiu cc enzyme ch yu ca con ng EM l alolase v triosephosphate-isemerase. on phn gii glucose u tin ch xy ra qua con ng pentosephosphate, tc l qua glucose-6-phosphate, 6-phosphogluconat v ribulose-5-phosphate (hnh). Cht ny nh mt epimerase c chuyn thnh xilulose-5-phosphate v nh phosphoketolase c phn gii trong mt phn ng ph thuc taminpirophosphate lm xut hin glyceraldehyt-3-phosphate v acetilphosphate. Cc t bo Leuconostoc mesenteroides khng sinh trng v c ra sch ln men glucose thnh lactat, ethanol v CO2 theo mt t l gn nh tng ng: C6H12O6 CH3 - CHOH - COOH + CH3 - CH2 - OH + CO2 cc c th ny acetilphosphate b kh qua acetil-CoA v acetaldehit thnh ethanol. Cc vi khun ln men d hnh khc chuyn mt phn hoc

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ton b acetilphosphate thnh acetate, trong lin kt phosphate giu nng lng c chuyn sang ADP v c s dng di dng ATP. Dihydro tha trong trng hp ny c truyn cho glucose lm xut hin manit. Glyceraldehyt-3-phosphate c chuyn qua piruvat thnh lactat. Ribose c L. mesenteroides ln men thnh lactat v acetate. *Ln men lactic d hnh Bifidobacterium Vi khun lactic d hnh Bifidobacterium bifidum c dng ch V hay ch Y (bifidus ting Hi Lp c ngha l b chia i, b phn ct), l thnh vin trong khu h ng rut ca tr s sinh, trc ht l cc tr nui bng sa m. Vi khun ny cn N-acetilglucosemin ch c trong sa ngi m khng c trong sa b. Cc i din thuc chi Bifidobacterium l nhng vi khun k kh nghim ngt, khng chu kh v cn mt kh quyn cha CO2 (10% CO2) sinh trng. Bifidobacterium chuyn ha glucose theo phng trnh : 2 C6H12O6 2 CH3 - CHOH - COOH + 3 CH3 - COOH Bifidobacterium ln men glucose qua con ng ph phosphoketolase, khng cha aldolase cng nh glucose-6-phosphatdehydrogenase, song li cha cc phosphoketolase hot ng, phn gii fructose-6-phosphat v xilulose-6-phosphat thnh acetilphosphate v eritro-4-phosphate hoc glixeraldehyt-3-phosphate. Hexose c chuyn ha theo con ng sau y :

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Hnh 6.4: Ln men lactic d hnh :1. Glucose-6-phosphat-dehydrogenase

2. Phosphogluconat-dedihydrogenase 3. Epimerase ; 4. Phosphoketolase 4. ng dng ca ln men lactic Nu dung dch ng khng kh trng cha c cc ngun nit phc tp v cc cht sinh trng mt cch t nhin trong trong iu kin k kh hoc di dng lp cao th cc vi khun lactic s nhanh chng pht trin pha bn trong. Chng lm gim pH xung di 5 v qua c ch sinh trng ca cc vi khun k kh khc l bn khng c kh nng sinh trng chua ny. Trong cc mi trng lm giu ny nhm vi khun no pht trin c l tu thuc vo cc iu kin c bit. Nh c tnh bo qun v kh trng da trn nguyn tc acid ha m cc vi khun lactic

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c s dng, trong nng nghip, trong ni tr, trong ngnh sn xut v ch bin sa. 4.1.Thc n chua. Vi khun lactic phn b trn cc nguyn liu thc vt gi mt vai tr to ln trong vic bo qun thc n gia sc. to iu kin cho ln men lactic xy ra, l cc loi cy ly c hoc c ... c nn cht trong cc hm , thm r ng nng cao t l C/N v to chua ban u bng cch thm acid formic hay acid v c. 4.2.Da l mt sn phm ca qu trnh ln men lactic. Bp ci c ct nh, trn v bp vi mui n 2-3%. Trong iu kin ym kh, ln men lactic s xy ra mt cch t nhin, lc u bi Leuconostoc - vi s to thnh CO2, v v sau c s tham gia ca Lactobacillus plantarum. 4.3.Cc sn phm sa. Sa c mt thnh phn dinh dng cao, cha nhiu nc v giu cc mui khong, protein (ch yu l cazein), m b, ng (c bit l lactose) v cc vitamin. trong bu v ca ng vt th n l v trng, nhng khi i ra khi nm v th sa b nhim bi khu h bnh thng ca ng vt, sau c b sung thm bi cc vi sinh vt khc t ngi vt sa, my hoc bnh cha. L mt mi trng gn nh hon ho nn sa rt mn cm vi s sinh trng ca vi sinh vt. Khi sa trong phng nhiu loi vi khun s ln men lactose to thnh acid v lm thay i tnh cht v kin trc ca sa. Qu trnh ny c th xy ra t nhin hoc c con ngi bt chc nh trong qn trnh sn xut phomat hoc iagua. Trong cc giai on sm ca ln men sa lactose b tn cng nhanh bi Streptococcus lactis v Lactobacillus spp. S tit acid lactic v s gim pH lm protein ca sa b ng li thnh cc vn (curd). S ng cng lm tch ring phn dch ng sa (whey) trn b mt. Cc vn cng c th c to nn nh tc dng ca vi sinh vt hoc nh mt enzyme (rennin) ly t d dy b. Mt lt sa nng 1032g bao gm 902g nc v 130g cht kh trong cha 49g lactose; 39g cht bo (l nhng git m ng knh 1-8 m gm glixerit v cc cht tan trong lipit, nh colesterol v cc vitamin A, D, E, K; 32,7g protein m ch yu l cazein; 9g cht khong (gm phosphate, xitrat, KCl, NaCl v MgCl2). *Sa chua (sa b). Sa chua l sn phm ca s ln men sa lng bng s chua ngu nhin di tc ng ca nhiu vi khun sinh acid

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lactic khc nhau (nh Streptococcus lactis hay S. cremoris), hoc ca mt s lng t hn nhiu cc vi khun gy thm (nh Streptococcus diacetylacis, Betacoccus citrovorus) hoc bng vic cy vo sa un nng cc ging vi khun thun khit. Khi ln men din ra, lactose c chuyn ho thnh acid lactic, acid ny s ngng kt cazein pH 4-5. Sa ng, c, c v chua, ty theo vng c tn gi khc nhau. Sa chua c lm t sa y (cha t nht 3% m ca sa) thng c trn vi bt sa vt vng nhm lm tng hm lng cht rn ton phn v ci thin cu trc gel protein. mt s nc, sa chua cng c sn xut t sa cu, sa tru, sa tun lc hoc sa nga. Kem chua c sn xut bi mt qu trnh rt ging quy trnh dng sn xut sa chua ch tr vic kem c ph c s dng lm nguyn liu th. *Iagua. Iagua c lm t cc loi sa cha cc hm lng m khc nhau bng cch ln men nh cc ging c bit. N c mt ng c mn ging nh gel, mt chua r rt v hng v d chu, khc bit r rng vi loi sa chua bnh thng. Iagua c sn xut bng cch cy vo sa kh trng Pasteur ng nht khong 1,5-3% cc vi khun lactic a nhit (ging hn hp ca mt hoc nhiu chng nh Streptococcus thermophilus hay Lactobacillus bulgaricus) v sau gi 42-45oC trong khong 3 gi. qunh mn c hnh thnh trong thi gian ny. Sn phm cui cng c v chua ( chua 40-60oSH). Phn quan trng trong hng thm c bit ca iagua l cc hp cht cacbonyl vi ixetyl v acetaldehit chim u th. Mt loi igua c bit c tn l sa aciophilus c sn xut nh Lactobacillus acidophil. Vic thm ng v hoa qu vo iagua (iagua hoa qu) lm tng ng k sc tiu th sn phm ny. *Kefia v Kumis. Kefia v Kumis l cc loi ung cha ru giu CO2, nhiu bt. Khu h vi sinh vt bao gm nm men Torula (chu trch nhim i vi qu trnh ln men ru) v cc vi khun Streptococcus lactis v Lactobacillus caucaticus (chu trch nhim i vi ln men lactic). Cc ht kefia dng sn xut kefia cha cc cc sa ng cng vi cc vi khun kefia. Kumis c lm t sa nga hay sa d do cc ging kumis thun khit (Thermobacterrium bulgaricus v nm men Candida) . C hai ung lm t sa ny u l cc sn phm a phng ca vng Caucasus v cc vng tho nguyn Turkestan. Kefia cha acid lactic (0,5- 1%), mt s lng ru ng k (0,5-2%), CO2 v mt s sn phm phn gii cazein sinh ra t hot ng phn gii protein ca nm men. *Phomat. Phomat l sn phm thu c t sa ng bng cch loi b dch ng sa v bng cch lm chn cc vn khi c mt mt khu h

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vi sinh vt c bit. C nhiu loi phomat vi hnh dng v kch thc khc nhau. c xp thnh nhm da vo loi sa s dng (b, d, cu), s to thnh cc vn (dng acid, dng rennin hay dng c hai), kin trc hay c, v hm lng cht bo. Trong tng nhm, cc loi phomat ring bit c c trng bi hng thm. S sn xut phomat v c bn bao gm hai cng on : s to thnh cc vn v qu trnh lm chn. *S to thnh cc vn Hm lng cht bo ca sa c iu chnh ti mc mong mnn, v khi cn, hm lng protein cng c iu chnh. Cc cht ph gia bao gm mui canxi nhm ci thin s ngng kt protein v kin trc ca phomat, nitrat km hm khu h vi khun k kh hnh thnh bo t v cc sc t to mu. Sa th hoc sa kh trng c trn trong mt b nhit 15-59oC vi mt loi ging khi ng (vi khun lactic hay vi khun propionic, nm si, nh Penicillium camemberti, P. candidum, P. roqueforti , cc vi khun mang sc t mu vng hoc mu nh Bacterium linens, cng cc cu khun v nm men) v sa s ng li thnh mt khi bn lng mm, ccvn, sau ln men lactic, c ni tip bng s b sung rennin hoc mt t hp no m thng thng nht l t hp gia acid v rennin. Gel protein ny s c ct thnh cc hnh khi nh trong lc ang c un nng ri sau c khuy nh. Dch ng sa c loi b cn phn cc vn cha m th c chuyn sang qu trnh lm rn. Khi rn mong mun t c, cc vn c chuyn sang qu trnh lm chn. *Lm chn Qu trnh lm chn ph thuc vo thnh phn khi phomat, c bit l hm lng nc, khu h vi sinh vt v cc iu kin bn ngoi nh nhit v m ca cc phng lm chn. S chn ca phomat mm xy ra t ngoi vo trong, v th trong giai on u s c mt phn v chn v mt phn li cha chn. S chn khng ng u xy ra l do c s chnh lch v pH. bn trong khi phomat, pH thp v c mt acid lactic. ngoi v, cc loi nm si c trng pht trin thch hp hn cc pH cao nng pH ln nh phn ng loi CO2 t cc amino acid. S chn phomat cng xy ra ng u trn ton b khi phomat. S to thnh v ch l kt qu ca s kh b mt, iu ny c th trnh c nh vic bao gi khi phomat trong cc mng plastic thch hp trc khi qu trnh lm chn bt u. Thi gian lm chn thay i ty trng hp, ko di t vi ngy i vi phomat mm ti vi thng thm ch vi

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nm i vi cc loi phomat cng. Sn lng trn 100 kg sa nc l 8 kg phomat cng v ti 12 kg phomat mm. Mi thnh phn ca phomat u chu s bin i v mt sinh ho hc cc mc khc nhau trong qu trnh lm chn. Lactose c phn gii thnh acid lactic nh ln men lactic ng hnh. Trong loi phomat chea chng hn, pH gim t 6,55 xung 5,15 tnh t lc b sung ging khi ng cho ti khi c thnh khun. Khi c mt vi khun propionic (nh trong trng hp ca phomat Emmental), acid lactic c chuyn ha tip tc thnh acid propionic, acid acetic, v CO2 theo phn ng : 3 CH3CHOHCOOH 2 CH3CH2COOH + CH3COOH + CO2 + H2O T l gia acid propionic v acid acetic b nh hng bi th oxi ha kh ca phomat v t l ny s thp khi c mt mui nitrat chng hn. Phng thc v mc phn gii m ca sa ph thuc vo khu h vi sinh vt tham gia vo qu trnh lm chn phomat. S gii phng cc acid bo, c bit l cc acid nh hng n hng thm ca phomat ph thuc vo tnh c hiu ca cc lipase. Ngoi cc acid bo t do, 2-alkanon v 2-alkanol cng c to thnh nh cc sn phm ph ca s -oxi ho cc acid bo. Nm si, c bit l Penicillium roqueorti, s dng -ketoaxyl-CoA eaxylase (tiodihydroase) v -ketoacid ecacboxylase cung cp cc hp cht in hnh cho hng thm ca cc phomat bn mm. S phn gii protein thnh cc amino acid xy ra qua cc peptit nh cc sn phm trung gian. Ty thuc vo loi sa m 20-40% cazein c chuyn ho thnh cc dn xut protein ha tan trong 5-15% l cc acid amin. Phm vi pH 3-6 l ti u i vi hot tnh proteinase ca Penicillium roqueorti. S phn gii protein b nh hng mnh bi hm lng mui cha trong phomat. Hm lng amino acid l 2,6-9% phn cht rn ca phomat v l phn quan trng trong hng thm ca phomat. Nhng li trong qu trnh lm chn c th to nn nhng peptit c v ng. Cc amino acid c chuyn ha tip tc. Trong giai on sm ca s lm chn, vi pH thp, chng b loi CO2 thnh cc amino. Trong giai on mun, vi pH cao, cc phn ng oxi ho chim u th. S phn gii protein khng ch ng gp vo s to thnh hng thm, n cn nh hng n kin trc ca phomat. Khi phomat mm b lm chn qu mc, s phn gii protein c th dn n vic dch ha gn nh ton b khi phomat.

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Cu hi n tp chng 8 1. Trong t bo, qu trnh ng phn din ra u ? Phn ng ny khc nhau nh th no khi khng c mt oxy v khi c mt oxy ? 2. Phn t no m u con ng ng phn ? Khi ng phn hon thnh, phn t no cn li. Bao nhiu ATP c to thnh mc phosphoril ha c cht ? Con s ATP c to thnh l bao nhiu, v v sao con s ny li khc vi tng s ATP c to thnh ? Bao nhiu ATP s c to thnh b sung nu oxy c mt ? 3. Khi ngung glucose l khng hn ch th ci g s l nhn t gii hn trong phn ng ng phn ? 4. Hai c ch ti to NAD+ l nhng c ch no ? Chng khc nhau ch no ? u l mt qu trnh hiu kh v u l mt qu trnh k kh ? 5. Ln men khc h hp oxy ha ch no khi xt v phng din cc cht nhn in t v s sn sinh nng lng ? 6. S trao i in t to nn ATP khc nhau nh th no trong cc trng hp NADH c to thnh trong ng phn, NADH c to thnh trong chu trnh acid citric, v FADH2 c to thnh trong chu trnh acid citric ? 7. Hiu qu ca vic phn gii hiu kh hon ton mt phn t glucose vi khun l bao nhiu ? So snh vi hiu qu ca mt mnh ng phn? 8. Hiu ng Pasteur c gp cc vi khun ln men lactic ng hnh khng ? V sao ? 9. Gii thch ti sao mt s nm men, nh Rhodotorula hay Geotrichum, khi tha ngun C, ng thi thiu N v P th lipit li c tch ly ? 10. Cho v d v nhng trng hp ln men vi khun trong c s to thnh nhiu hn 2 mol ATP trn mt mol glucose b phn hy.

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Chng 8

Cc qu trnh ln men
I. Con ng Embden-Meyerhof chung cho cc qu trnh ln men (EM) : Bc chuyn ha
Ln men l mt qu trnh oxi ha-kh cn bng ni sinh, trong mt s nguyn t ca ngun nng lng (cht cho in t) tr nn c tnh kh hn, trong khi mt s nguyn t khc tr nn oxi ha hn, cn nng lng th c sinh ra nh s phosphorin ha c cht. Con ng trao i cht chung i vi s ln men glucose l con ng ng phn, cng cn c gi l con ng Embden-Meyerhof. ng phn c chia thnh ba bc chnh, mi bc gm mt s phn ng ring bit do enzyme xc tc. Bc mt gm cc phn ng chun b, khng c cc phn ng oxi ha v cng khng gii phng nng lng, song li dn n vic to ra hai phn t trung gian quan trng, glixeraldehit-3-phosphate (GAP). Trong bc hai, s oxi ha-kh xy ra, nng lng c d tr di dng ATP v hai phn t piruvat c to thnh. Trong bc ba, mt phn ng oxi ha-kh th hai xy ra v cc sn phm ln men (chng hn ethanol v CO2, hoc acid lactic), s c to thnh. Trong s to thnh hai phn t acid 1,3 iphosphoglixeric c hai phn t NAD+ b kh thnh NADH. Tuy nhin, mt t bo ch cha mt lng nh NAD+, v nu nh ton b NAD+ b chuyn thnh NADH th phn ng oxi ha glucose s b nh ch; s oxi ha tip tc ch c th xy ra khi mt phn t NAD+ c mt tip nhn cc in t va c gii phng ra. S "tc ng" ny c th c khc phc trong ln men bng cch oxi ha NADH tr li thnh NAD+ nh cc phn ng kh piruvat thnh mt trong hng lot cc sn phm ln men khc nhau. Trong trng hp ca nm men, piruvat b kh thnh ethanol vi s gii phng CO2. cc vi khun lactic, piruvat b kh thnh acid lactic. Nhiu con ng kh piruvat cng gp cc vi sinh vt nhn s khc, song kt qu tht s u ging nhau, tc l NADH phi c hi phc li thnh dng oxi ha, NAD+, cho cc phn ng thu nhn nng lng ca ln men c th tip tc. L mt coenzyme d khuch tn, NADH c th tch khi GAP-dedihydrogenase, gn vo mt enzyme khc, (chng hn lactat-dedihydrogenase, enzyme kh piruvat thnh acid lactic), ri li lp tc khuch tn tr li sau khi NADH c oxi ha thnh NAD+ lp li chu trnh mt ln na.

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Trong bt k mt phn ng thu nng lng no, s oxi ha bao gi cng phi c cn bng vi s kh v phi c mt cht nhn no ng ch sn nhn mt in t va c tch ra. Trong trng hp ny, s kh NAD+ ti mt bc trong con ng ng phn s c cn bng bi s oxi ha n ti mt bc khc. (Cc) sn phm cui cng cng phi nm trong trng thi cn bng oxi ha-kh vi c cht ban u, tc l glucose.
Bng 6.1: Cc qu trnh ln men ph bin vi sinh vt Kiu ln men Ln men ru Ln men lactic ng hnh Ln men lactic d hnh Acid propionic Acid hn hp Phn ng tng th Hexose 2 Ethanol + 2 CO2 Hexose 2 Lactat + 2 H+ Hexose Lactat + Ethanol + CO2 + H+ Lactat Propionat + Acetate + CO2 Hexose Ethanol + 2,3Butandiol + Xucxinat + Lactat + Acetate + Focmat + H2 + CO2 Hexose Butirat + Acetate + H2 + CO2 Hexose Butanol + Acetate + Aceton + Ethanol + H2 + CO2 Ethanol + Acetate + CO2 Caproat + Butirat + H2 Fructose 3 Acetate + 3 H+ 4 H2+ 2 CO2 + H+ Acetate + 2 H2O Acetate + H2O CH4 + HCO 3 Vi sinh vt thc hin Nm men Zymomonas Streptococcus Mt s Lactobacillus Leuconostoc Mt s Lactobacillus Propionibacterium Clostridium propionicum Cc vi khun ng rut Escherichia, Salmonella, Shigella, Klebsiella, Enterobacter Clostridium butyricum Clostridium acetobutylicum Clostridium kluyveri Clostridium aceticum Acetobacterium Methanothrix Methanosarcina

Acid butiric Butanol Caproat Homoacetic

Sinh methane

II. Tnh a dng ca ln men


Bng 6.1 trnh by mt s kiu ln men chnh c phn loi da trn cc sn phm c to thnh. l cc qu trnh ln men ru, acid lactic, acid propionic, acid hn hp, acid butiric v acid homoacetic. C nhiu qu trnh ln men c phn loi da trn c cht c ln men

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thay v cc sn phm. Chng hn, nhiu vi khun k kh to thnh bo t (chi Clostridium) ln men cc amino acidvi s sn sinh acetate, amoniac v H2. Cc loi Clostridium khc, nh C. acidiurici v C. purinolyticum ln men cc purin nh xantin hoc aenin vi s to thnh acetate, focmat, CO2, v amoniac. Cn nhng bn k kh khc th ln men cc hp cht thm. Chng hn, vi khun Pelobacter acidigallici ln men hp cht thm phloroglucinol (1,3,5-benzenetriol, C6H6O3) theo con ng sau : Phloroglucinol (C6H6O3) +3 H2O 3 acetate- + 3H+, Go' = -142,5 kJ/phn ng Nhiu qu trnh ln men khng thng thng ch c thc hin bi mt nhm rt hn ch cc vi khun k kh, v trong mt s trng hp ch bi mt vi khun duy nht. Mt s v d c nu trn bng 6.2. Nhiu trong s cc vi khun ny c th c xem nh cc chuyn gia v trao i cht v chng c kh nng phn gii mt hoc nhiu c cht m cc vi khun khc khng phn gii c.
Bng 6. 2: Mt s qu trnh ln men khng thng thng Phn ng cn bng Vi sinh vt thc hin tng th Acetylen 2 C2H2 + 3 H2O Ethanol + Pelobacter acetylenicus Acetate + H+ Acetobacterium spp. Glycerin 4 Glycerin + 2 HCO 3 + 7 Acetate + 5 H + 4 H2O Resocxinol 2 C6H4(OH)2 + 6 H2O 4 Clostridium spp. (mt hp cht Acetate + Butirat + 5 H+ thm) Xinamat (mt 2C9H7O2 + 2 H2O C9H9O2 + Acetovibrio multivorans hp cht thm) Benseat + Acetate + H+ Phlorogluxinol Pelobacter masiliensis C6H6O3 + 3 H2O (mt hp cht 3 Acetate + 3 H+ Pelobacter acidigallici thm) Putresxin 10 C4H12N2 + 26 H2O 6 Cc vi khun k kh gram Acetate + 7 Butirat + 20 NH4+ + dng khng sinh bo t, cha phn loi 16 H2 + 13 H+ Xitrat Xitrat + 2 H2O Focmat + 2 Bacteroides sp. Acetate + HCO 3 + H+ Aconitat Glioxilat Aconitat + H++ 2 H2O 2 CO2 Acidaminococcus fermentans + 2 Acetate + H2 + 4 Glioxilat +3 H + 3 H2O 6 Vi khun gram m cha Kiu ln men

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Xucxinat Oxalat Malonat

CO2 + 5 H2 + Glicolat phn loi Xucxinat + H2O Propionat + Propionigenium modestum HCO 3 Oxalat + H2O Focmat + Oxalobacter formigenes HCO 3 Malonat + H2O Acetate + Malonomonas rubra Sporomusa malonica HCO 3

III. Ln men khng c s phosphorin ha c cht Vi mt s c cht, nng lng sinh ra khng ghp i vi s tng hp ATP trc tip nh phosphorin ha c cht, tuy nhin cc hp cht ny vn tr gip sinh trng ln men ca mt c th no . Trong nhng trng hp ny s phn gii c cht c gn lin vi nhng bm ion c dng to nn mt graien proton hoc natri qua mng. V d v kiu ny c cc qu trnh ln men ca Propionigenium modestum hoc Oxalobacter formigenes, c hai u gn s ln men cc acid icacboxilic vi cc bm ion sinh nng lng lin kt vi mng. Propionigenium modestum thc hin phn ng sau y : Xucxinat2- + H2O Propionat- + HCO3- , Go' = -20,5 kJ/phn ng Phng trnh tng th ny thu khng nng lng t do ghp i vi s tng hp ATP trc tip nh phosphorin ha c cht, tuy nhin n li c dng lm phn ng thu nng lng duy nht cho sinh trng ca vi khun. S d nh vy l v s loi CO2 khi xucxinat

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Hnh 6.1: Ln men lin kt vi bm ion vi khun (nng lng thu c khng qua phosphorin ha c cht)

(qua metilmalonil-CoA v ecacboxilase lin kt vi mng bi Propionigenium modestum c ghp i vi s xut Na+ qua mng t bo cht. Mt ATPse chuyn v Na+ nm trn mng ca P. modestum s dng graien ny hot ha s tng hp ATP. Oxalobacter formigenes thc hin s ln men oxalat : Oxalat2- + H2O Focmat- + HCO3- , Go' = -26,7 kJ/phn ng pH trung tnh, oxalat tn ti di dng ion ha oxalat-, v s loi cacboxil thnh focmat- tiu th mt proton. Sau s xut focmat- khi t bo s to nn mt ng lc nh proton (proton motive force = PMF) c kh nng ghp i vi s tng hp ATP nh mt ATPase chuyn v proton nm trn mng.

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iu th v v c o trong trao i cht ca c Propionigenium modestum ln Oxalobacter formigenes nm ch s tng hp ATP xy ra khng cn c phosphoril ha c cht ln chui vn chuyn in t; tuy nhin, s to thnh ATP theo c ch ha thm vn xy ra da vo mt bm Na+/H+ kt hp vi s loi CO2 khi cc acid hu c. iu cn rt ra t nhng s ln men ny l : cc phn ng, d ch thu c di 31,8 kJ, con s cn thit to thnh mt ATP, hoc khng c kh nng ghp i vi s phosphorin ha c cht, cng khng th lp tc b coi l khng c kh nng tr gip sinh trng cho mt vi khun. Nu nh phn ng c th ghp i c vi mt graien ion th s sn sinh ATP vn c kh nng xy ra v do vy vn c sinh trng (Hnh 6.1). IV. Hin tng cng dng (syntrophy) Trong vi sinh vt hc c nhiu v d v hin tng cng dng, mt hin tng m hai sinh vt khc nhau c th cng nhau phn gii mt cht no - v bo ton nng lng ca qu trnh phn gii n mc khng c th no trong hai c th mt mnh c th lm c. Trong a s trng hp, bn cht ca mt phn ng cng dng c lin quan ti kh dihydro (H2) sinh ra bi mt trong hai i tc v c tiu th bi i tc kia. Do vy, cho n nay, cng dng cng cn c gi l s chuyn H2 lin loi. K tiu th H2 c th l bt k mt c th no trong s cc vi khun kh sunfat, ln men homoacetate, v vi khun sinh methane. Hy xem xt trng hp cng dng lin quan ti qu trnh ln men ethanol thnh acetate v s sn sinh methane sau . Nh c th thy trn hnh 2, bn ln men ethanol thc hin mt phn ng khng thun li v mt nng lng t do (Go' dng). Tuy nhin, H2 c sinh ra bi i tc ln men ru li l mt cht cho in t y gi tr i vi qu trnh hnh thnh methane do mt vi khun sinh methane thc hin. V khi cng hai phn ng li th phn ng tng th s l mt phn ng to nhit v c th tr gip sinh trng ca c hai i tc trong hn hp cng dng. Ln men ethanol 2CH3CH2OH + 2H2O 4H2 + 2CH3COO- +2H+ +19,4 kJ /phn ng Sinh methane 4H2+ CO2 - 130,7 kJ/phn ng CH4 + 2H2O Phn ng cng dng 2CH3CH2OH + CO2 - 111,3 kJ/phn ng CH4 + 2CH3COO

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Mt v d khc ca hin tng cng dng l s oxi ha butirat thnh acetate cng vi H2 bi vi khun cng dng oxi ha acid bo Syntrophomonas: Butirat- + 2H2O 2acetate- + H+ + 2H2 , Go' = +48,2 kJ/phn ng S bin i nng lng t do ca phn ng ny rt khng thun li v trong ng ging thun khit, Syntrophomonas khng th mc c trn butirat. Song nu H2 va sinh ra trong phn ng c tiu th ngay bi mt c th i tc (nh mt vi khun sinh methane) th Syntrophomonas li sinh trng rt tt trong ng ging cy chung vi bn tiu th H2.

V. Ln men ru nh nm men v vi khun


Ethanol l mt trong s cc sn phm ln men ph bin nht gp vi sinh vt. Bn thn thc vt v nhiu loi nm cng tch ly ethanol di cc iu kin k kh. Vi sinh vt sn sinh ethanol ch yu l nm men, c bit l cc chng thuc loi Saccharomyces cerevisiae. Ging nh a s nm, nm men l nhng c th h hp hiu kh, nhng khi vng mt khng kh chng s ln men hirat cacbon thnh ethanol v CO2. Nhiu vi khun k kh v hiu kh cng to thnh ethanol nh cc sn phm chnh hoc sn phm ph khi ln men cc hexose hoc pentose. 1. S to thnh ethanol nh nm men S ln men glucose thnh ethanol v CO2 nh Saccharomyces cerevisiae din ra theo con ng Embden-Meyerhof. S chuyn ha piruvat thnh ethanol gm hai bc. Trong bc mt, piruvat c loi CO2 thnh acetaldehit nh piruvat-ecacboxilase vi s tham gia ca tiaminpirophosphate. Trong bc sau, acetaldehit b kh thnh ethanol nh alcohol-edihydrogenase cha NADH.
Glucose 2 Piruvat

2NAD

2NADH 2 Acetaldehyt

2CO2

2Ethanol

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Bng s chuyn H ny, dihydro tch ra do triosephosphatedehydrogenase xc tc s c tiu th, nh vy trng thi oxi ha-kh s c cn bng. 2. Cc dng phng trnh Neuberg Cc phng php v pht hin ca Carl Neuberg v ln men ru khng ch c ngha lch s. ng khm ph ra rng, khng ch glucose m c piruvat cng c nm men ln men v acetaldehit xut hin nh mt sn phm trung gian c th b bt gi bi dihydrosulfit. Dihydrosulfit vn khng c i vi nm men. Nu b sung dihydrosulfit vo mt mi trng cha glucose ang c nm men ln men th acetaldehit s b kt ta do to thnh phc cht : CH3-CHO + NaHSO3 CH3- CHOH -SO3Na Lc glycerin s xut hin nh mt sn phm ln men mi, ng thi hiu sut ethanol v CO2 s gim xung. Ln men vi s c mt ca dihydrosulfit c s dng trong cng nghip sn xut glycerin. Nguyn l ca n l, acetaldehit b bt gi, v do vy khng th hot ng nh mt cht nhn in t c na. Lc ny, thay cho acetaldehit, dihydroxiacetonphosphate c s dng lm cht nhn in t v b kh thnh glycerin-3-phosphate ri tip tc b loi phosphate tr thnh glycerin. Phng trnh phn ng din ra nh sau : Glucose + Dihydrosulfit Glycerin + Acetaldehit-sulfit + CO2 Phng trnh ln men ci bin ny c gi l dng ln men Neuberg 2. Khi b sung cc cht kim (NaHCO3, Na2HPO4) th glycerin cng c to thnh v acetaldehit b chuyn thnh ethanol v acetate qua phn ng ha hai, v do vy khng cn hon thnh c chc nng ca mt cht nhn in t. Phng trnh ln men c tn l dng ln men Neuberg 3 v c vit nh sau : Glucose + 2H2O Ethanol + Acetate + 2Glycerin + 2CO2 Dng ln men thng thng c Neuberg gi l dng ln men 1.

152

3. Hiu ng Pasteur Hiu ng Pasteur l s c ch ln men ru (hay qu trnh ng phn) bi oxi (tc l bi h hp hiu kh). S chuyn sang h hp khng nhng lm gim s to thnh ethanol v CO2 m cn lm gim s hp th ng. C hai c ch chu trch nhim i vi hin tng ny. Mt mt, l s cnh tranh ADP v phosphate v c gia hai qu trnh phosphorin chui h hp v phosphorin ha c cht. S loi dihydro xy ra trong phn ng oxi ha glyceraldehyt-3-phosphate cn ADP v phosphate v c (Pvc) : Glyceraldehyt-3-phosphate + NAD + Pvc NAD + ATP
H

3-phosphoglicerate +

Do vy, s chuyn ha c cht (glucose) qua con ng EM ph thuc vo s c mt ca ADP v phosphate. Tuy nhin, trong iu kin hiu kh, s phosphorin ha chui h hp cng cnh tranh ADP v phosphate pht sinh ATP. Lc nng ni bo ca ADP v phosphate gim, v hu qu l, s chuyn ha glucose v do vy, s to thnh ethanol cng s gim. Khi dng initrophenol (DNP) lm bt hot phosphorin ha chui h hp, ADP v phosphate li tr nn c sn i vi phn ng loi dihydro ca glyceraldehyt-3-phosphate, bi vy ngi ta thy ngay trong iu kin thang kh, s tiu th glucose (cho ln men) vn tng. C ch th hai chu trch nhim i vi hiu ng Pasteur l c ch iu ho bng enzyme d lp th. Hnh 3 tm tt nhng quan h iu ha ch cht trong hiu ng ny. Trong iu kin thong kh, s ln men ru ch to thnh mt vi ATP tnh trn mi phn t glucose. duy tr s cung cp nng lng, t bo phi s dng nhiu glucose. T l ca ATP/AMP trong trng hp ny rt thp. Tri li, khi oxi xm nhp vo mi trng, ATP c to thm rt nhiu nh s qu trnh phosphorin ha chui h hp. Enzyme cha kha d lp th iu ha s s dng glucose trong nm men ln men ru l phosphofructokinase. l enzyme khng thun nghch u tin ca con ng EM. N xc tc cho s phosphorin ha fructose-6-phosphate thnh fructose-1,6-iphophat nh ATP v b km hm d lp th bi ATP. S km hm theo c ch lin kt ngc ny lm tng nng glucose-6-phosphate trong t bo do tnh thun nghch ca phn ng enzyme trc . Glucose-6-phosphate km hm d lp th hexokinase, km hm c s hp thu glucose, do lm gim s tiu th ng ny.

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Trong khi ATP km hm d lp th phosphofructokinase th AMP li hot ha n. Bi vy mt t l ATP/AMP thp nh trong ln men li kch thch s hp thu v s trao i glucose. Hot tnh ca phosphofructokinase cng b km hm bi xitrat. Nh cht hiu ng m tnh th hai ny m tc dng km hm ca ATP c tng cng, tri li cc ion amn li kch thch enzyme ny. S iu ha m t trn c th c chng minh d dng khi ngi ta o nng ni bo ca glucose, glucose-6-phosphate, fructose-6phosphate, fructose-1,6-diphosphate v cc triose-phosphate trc v sau khi chuyn t iu kin nui k kh sang iu kin nui hiu kh. Ngay sau khi c thng kh, nng glucose-6-phosphate v fructose-6-P ca t bo tng nhanh, cn nng ca fructose-1,6-diphosphate v cc triosephosphate li gim mnh. y, phosphofructosekinase hot ng nh mt ci van c iu chnh bi cc aenilat cng nh cc cht trao i trung gian khc. Nu van b ng, cc cht trao i nm pha trn van s b ng, cn cc cht nm bn di van c tip tc chuyn ha v do vy, nng gim i. Hiu ng Pasteur cng gp vi khun (chng hn, E. coli) v m ng vt. Nguyn tc iu ha l ging nhau, ch c nhng khc bit v loi cht hiu ng. Chng hn, E. coli, khng phi AMP m ADP mi l cht hiu ng dng. Nhng c ch c bn ca trao i cht th rt ging nhau, cn cc c ch iu ha th thng c tnh c hiu loi v c hiu chng. 4.K thut sn xut ethanol nh nm men Cc loi nm men dng trong sn xut ethanol l cc nm n bo mang nhiu c im phong ph v sinh trng v pht trin. Cc loi c quan tm trc ht trong sn xut cng nghip l Saccharomyces cerevisiae, Saccharomyces uvarum (carslbergensis), Schizosaccharomyces pombe v Kluyveromyces sp. Ngoi ra, nhiu bin chng bt ngun t cc nm men ny c to ra nh k thut di truyn. Cc tnh cht mong mun ca mt qu trnh sn xut ethanol cng nghip ph thuc nhiu vo vic la chn chng vi sinh vt dng trong ln men. Cc chng ny phi c mt nng sut sn phm cao trn mt n v c cht c s dng, mt tc ln men cao, tnh chu cn cn thit, kh nng sng st nhit cao, tnh bn trong cc iu kin ln men v tnh chu pH thp.

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Kh nng s dng cc c cht khc nhau ph thuc vo loi. Ni chung, cc c th ny c kh nng sinh trng v ln men ethanol c hiu qu cc gi tr pH gia 3,5-6,0 v nhit t 28-35oC. Hin nay ngi ta quan tm nhiu n vic to ra cc th t bin khuyt h hp t nm men nh vic s dng cc tc nhn ha hc nh cc thuc nhum hoc cc hp cht lm bin tnh protein. Cc bin chng ny cha ADN ti th b bin i theo hng c ch s to ra cc enzyme cn thit cho s trao i cht hiu kh v do vy thch hp vi s tng hp ru. 5. Hiu sut ln men Nm men, di iu kin k kh chuyn ho glucose thnh ethanol trc ht bng con ng Embden-Meyerhof. Phn ng tht s bao gm s to thnh 2 mol ethanol, 2 mol CO2 v 2 mol ATP trn mi mol glucose c ln men. Do vy, v mt trng lng mi gam glucose v l thuyt c th to ra 0,51 g ru. Tuy nhin sn lng t c trong ln men thc t thng khng vt qu 90-95% con s l thuyt. S d nh vy l v mt phn cht dinh dng c s dng tng hp sinh khi mi v cho cc phn ng lin quan ti s duy tr t bo. Cng cn c cc phn ng ph xy ra (thng dn ti Glycerin v sucxinat) tiu th ti 4-5% c cht tng s. Nu loi tr c cc phn ng ny th sn lng ethanol c th tng thm c 2,7%. Mt nng nh oxi cn phi c cung cp cho nm men ln men v y l thnh phn cn thit trong s sinh tng hp cc cht bo cha bo ha v cc lipit. Thng thng oxi c gi trong dch ln men nng 0,05-0,1 mm Hg tnh theo p sut oxi. Bt c nng no nm trn gii hn u s kch thch sinh trng, vi phm n s to thnh ethanol (hiu ng Pasteur). Cc nhu cu tng i ca cc cht dinh dng c dng trong s tng hp ethanol t l thun vi cc thnh phn chnh ca t bo nm men. Chng bao gm C, O, N, v H. Thp hn mt cht l P, S, K v Mg dng tng hp cc thnh phn th yu. Cc mui v c (nh Mn, Co, Cu, Zn) v cc nhn t sinh trng hu c (acid amin, acid nucleic, v cc vitamin) c yu cu di dng vt. Nhiu loi nguyn liu dng sn xut ru quy m ln (nh r ng ma hay actiso Jerusalem) ngoi hirat C cn cung cp ton b cc cht dinh dng cn thit cho sinh trng ca nm men. Trong mt s trng hp, ngi ta cng a thm vo mt s cht dinh dng b sung nh cc mui amn, phosphate K hay ngun cao ng r tin.

155

Hnh 6.3: Hiu ng Pasteur C ch iu ha nh enzyme d lp th Phosphofructosekinase

Nm men rt mn cm vi s c ch bi ethanol. Nng 1-2% (w/v) lm chm sinh trng ca vi sinh vt v nng cn 10% (w/v) tc sinh trng ca nm men gn nh ngng hn. Bn cht ca hin tng km hm ny l rt phc tp. Vic thm cn vo pha log lm gim nhanh tc sinh trng ca nm men (c l do tc ng ln s tng hp protein), gim kh nng sng st ca t bo (qua s bin tnh khng thun nghch ca cc enzyme), v mt mc thp hn nhiu,

156

ethanol lm gim tc tng hp bn thn n. Cc dn liu v mc chu cn ca mt s chng nm men ph thuc rt nhiu vo thnh phn acid bo trong mng t bo cht cho thy rng thnh phn acid bo kch thch hoc km hm s tit ethanol khi bo tng. Cc kt qu khc cung chng minh c rng ethanol c to ra trong ln men (ethanol t sinh) c ch ln sinh trng ca t bo mnh hn l ethanol c a vo mt cch nhn to t bn ngoi. 6. S to thnh ethanol nh vi khun Trong s vi khun, kiu ln men ru ph bin nm men (EM, piruvat-ecacboxilase) ch gp Sarcina ventriculi. y l loi vi khun chu kh yu, d dng phn lp c t t, nhng cng gp c trong dch tiu ha ca nhng ngi mc bnh d dy. Cc vi khun ny c coi l khng bnh thng v c kch thc rt ln (ng knh 4 m), to nn cc dng hp bao gi (cha trn 64 t bo mi hp , c lin kt vi nhau nh xenlulose), chu pH (sinh trng c pH t 0,9 n 9,8) v to thnh ni bo t. T dch p ln men ca cy da di (Agave americana) vng Mexico ngi ta phn lp c mt loi vi khun hnh que, di ng, c lng roi hai cc, c kh nng to thnh ethanol. Vi khun ny c tn l Zymomonas mobilis, phn gii glucose theo con ng 2-keto-3deoxi-6phosphogluconat (KDPG) ct piruvat thnh acetaldehit v ioxit cacbon. Acetaldehit c kh thnh ethanol. Ethanol, ioxit cacbon v mt lng nh acid lactic l nhng sn phm ln men duy nht. Ngi ta rng trong ru mnh ch t da di, cc nguyn t cacbon s 2 v s 3 cng nh s 5 v s 6 thay th cc nguyn t cacbon s 1 v s 2 cng nh s 5 v s 6 trong ru ch t nm men.

157

Trong qu trnh ln men ca mt s Enterobacteriaceae v Clostridium, ethanol xut hin nh mt sn phm ph. Tuy nhin, tin cht ca ethanol, acetaldehit, khng c gii phng trc tip t piruvat nh piruvatecacboxilase m c to thnh nh s kh acetil-CoA. Cc vi khun ln men lactic d hnh (v d Leuconostoc mesenteroides) to ra alcohol theo mt con ng hon ton khc. Qua cc bc u tin ca con ng pentosephosphate (PP), glucose c phn gii thnh pentosephosphate. Phosphoketolase tn cng vo xilulose -5- phosphate : Xilulose-5-phosphate+Pvc Acetilphosphate + glyceraldehyt-3-phosphate Acetilphosphate va to thnh nh acetaldehit-edihydrogenase v alcohol-edihydrogenase s c kh thnh ethanol. Sn phm phn gii cn li l glyceraldehyt-3-phosphate, s c kh thnh lactat qua piruvat. VI. Ln men lactic v h Lactobacteriaceae Cc vi khun lactic c xp chung vo h Lactobacteriaceae. Mc d nhm ny khng c s ging nhau v hnh thi v, bao gm c vi khun hnh que di, hnh que ngn ln cc vi khun hnh cu, song v mt sinh l chng c chung cc c im kh c trng. Tt c cc i din u l cc vi khun gram dng, khng to thnh bo t (tr Sporolactobacillus) v khng di ng. thu nng lng chng hon ton ph thuc vo cc hirat cacbon v tit ra acid lactic (lactat). i lp vi cc Enterobacteriaceae l bn cng sn sinh acid lactic, chng thuc bn ln men bt buc. Chng khc cha cc hemin (xitocrom, catalase). Mc d vy, cc vi khun thuc h Lactobacteriaceae c kh nng sinh trng khi c mt oxi khng kh, chng thuc bn k kh song ng thi cng l bn chu kh. Mt vi khun sinh trng hiu kh m khng cha catalase th gn nh chc chn l mt vi khun lactic (tr ngoi l Acetobacter peroxydans v Shigella dysenteriae). 1. Nhu cu v cc cht b sung v nhn t sinh trng Mt c im na ca cc vi khun lactic l c nhu cu v cc cht b sung. Khng c i din no c th mc c trn mi trng v c thun khit cha glucose v cc mui amn. a s trong chng cn hng lot cc vitamin (lactoflavin, tiamin, acid pantotenic, acid nicotinic, acid folic, biotin), v cc acid amin, cc baz purin v pirimiin. V vy ngi ta thng nui chng trn cc mi trng dinh dng phc tp cha

158

nhng s lng kh ln cao nm men, dch c chua, dch ng sa hoc thm ch c mu na. iu ngc nhin l mt s vi khun lactic (v c cc vi khun ln men khc) khi sinh trng trn mi trng dinh dng cha mu c th to thnh cc xitocrom v thm ch c th tin hnh qu trnh phosphorin ha chui h hp. V vi khun lactic thiu nng lc tng hp cc pocphirin nn khi cc pocphirin c b sung vo mi trng th mt s vi khun lactic c th to thnh cc sc t hem tng ng. Vi khun lactic c coi nh nhng k qu qut v trao i cht. C th rng do hu qu ca s chuyn ha sinh trng trong sa hoc trn nhng mi trng giu cht dinh dng v cht sinh trng, chng mt i kh nng tng hp nhiu cht trao i. Trong khi chng li c kh nng m a s cc vi khun khc khng c, l kh nng s dng lactose. Kh nng ny nhiu vi khun ng rut (v d Escherichia coli) cng c. Lactose khng c mt trong gii thc vt, n c cc ng vt c v to thnh v tit ra cng vi sa hoc c hp thu vo cng vi sa. Vic s dng c lactose cng c th c xem nh mt s thch nghi vi cc iu kin tn ti trong ng rut ca ng vt c v. Lactose l mt isaccarit, trc khi i vo cc con ng phn gii hexose n phi c phn ct : Lactose + H2O
- Galactozidase

D-glucose + D-galactose

Sau khi c phosphorin ha galactose c chuyn thnh glucosephosphate. Do c s sn sinh mt lng ln lactat nn mi trng dinh dng cn phi c trung ha. Thng ngi ta dng cacbonat canxi. Trn mt mi trng thch dinh dng c b sung cacbonat canxi c th nhn ra s to thnh acid qua cc vng trong sut bao quanh cc khun lc. Ni sng ch yu ca cc vi khun lactic l sa v nhng ni sn xut v ch bin sa, thc vt nguyn vn hoc ang t phn hy, cng nh ng rut v nim mc ca ngi v ng vt. Do to thnh mt lng ln lactat v do tnh chu chua m di cc iu kin mi trng thch hp cc vi khun lactic pht trin nhanh, v vy c th d dng phn lp cc vi khun lactic trn cc mi trng dinh dng chn lc.

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2. Ln men lactic ng hnh Vi khun lactic ln men ng hnh to thnh ton b hoc gn nh ton b l lactat. Chng phn gii glucose qua con ng EM, cha cc enzyme cn thit, k c alolase v chuyn dihydro tch ra trong qu trnh loi dihydro ca glyceraldehyt-3-phosphate sang piruvat : Glucose
2 Piruvat

2 NAD

2 NADH

2 Lactat

Vic xut hin D-, L+ hay DL- lactat l ph thuc vo tnh chuyn ha khng gian ca enzyme vn chuyn lactat-edihydrogenase v vo s c mt ca mt lactat-racemase. Ch mt phn nh piruvat c loi cacboxil v chuyn thnh acetate, ethanol, CO2 v acetoin. Vic to thnh cc sn phm ph ph thuc vo s c mt hay vng mt ca oxi. 3. Ln men lactic d hnh Vi khun ln men lactic d hnh thiu cc enzyme ch yu ca con ng EM l alolase v triosephosphate-isemerase. on phn gii glucose u tin ch xy ra qua con ng pentosephosphate, tc l qua glucose-6-phosphate, 6-phosphogluconat v ribulose-5-phosphate (hnh). Cht ny nh mt epimerase c chuyn thnh xilulose-5-phosphate v nh phosphoketolase c phn gii trong mt phn ng ph thuc taminpirophosphate lm xut hin glyceraldehyt-3-phosphate v acetilphosphate. Cc t bo Leuconostoc mesenteroides khng sinh trng v c ra sch ln men glucose thnh lactat, ethanol v CO2 theo mt t l gn nh tng ng: C6H12O6 CH3 - CHOH - COOH + CH3 - CH2 - OH + CO2 cc c th ny acetilphosphate b kh qua acetil-CoA v acetaldehit thnh ethanol. Cc vi khun ln men d hnh khc chuyn mt phn hoc

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ton b acetilphosphate thnh acetate, trong lin kt phosphate giu nng lng c chuyn sang ADP v c s dng di dng ATP. Dihydro tha trong trng hp ny c truyn cho glucose lm xut hin manit. Glyceraldehyt-3-phosphate c chuyn qua piruvat thnh lactat. Ribose c L. mesenteroides ln men thnh lactat v acetate. *Ln men lactic d hnh Bifidobacterium Vi khun lactic d hnh Bifidobacterium bifidum c dng ch V hay ch Y (bifidus ting Hi Lp c ngha l b chia i, b phn ct), l thnh vin trong khu h ng rut ca tr s sinh, trc ht l cc tr nui bng sa m. Vi khun ny cn N-acetilglucosemin ch c trong sa ngi m khng c trong sa b. Cc i din thuc chi Bifidobacterium l nhng vi khun k kh nghim ngt, khng chu kh v cn mt kh quyn cha CO2 (10% CO2) sinh trng. Bifidobacterium chuyn ha glucose theo phng trnh : 2 C6H12O6 2 CH3 - CHOH - COOH + 3 CH3 - COOH Bifidobacterium ln men glucose qua con ng ph phosphoketolase, khng cha aldolase cng nh glucose-6-phosphatdehydrogenase, song li cha cc phosphoketolase hot ng, phn gii fructose-6-phosphat v xilulose-6-phosphat thnh acetilphosphate v eritro-4-phosphate hoc glixeraldehyt-3-phosphate. Hexose c chuyn ha theo con ng sau y :

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Hnh 6.4: Ln men lactic d hnh :1. Glucose-6-phosphat-dehydrogenase

2. Phosphogluconat-dedihydrogenase 3. Epimerase ; 4. Phosphoketolase 4. ng dng ca ln men lactic Nu dung dch ng khng kh trng cha c cc ngun nit phc tp v cc cht sinh trng mt cch t nhin trong trong iu kin k kh hoc di dng lp cao th cc vi khun lactic s nhanh chng pht trin pha bn trong. Chng lm gim pH xung di 5 v qua c ch sinh trng ca cc vi khun k kh khc l bn khng c kh nng sinh trng chua ny. Trong cc mi trng lm giu ny nhm vi khun no pht trin c l tu thuc vo cc iu kin c bit. Nh c tnh bo qun v kh trng da trn nguyn tc acid ha m cc vi khun lactic

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c s dng, trong nng nghip, trong ni tr, trong ngnh sn xut v ch bin sa. 4.1.Thc n chua. Vi khun lactic phn b trn cc nguyn liu thc vt gi mt vai tr to ln trong vic bo qun thc n gia sc. to iu kin cho ln men lactic xy ra, l cc loi cy ly c hoc c ... c nn cht trong cc hm , thm r ng nng cao t l C/N v to chua ban u bng cch thm acid formic hay acid v c. 4.2.Da l mt sn phm ca qu trnh ln men lactic. Bp ci c ct nh, trn v bp vi mui n 2-3%. Trong iu kin ym kh, ln men lactic s xy ra mt cch t nhin, lc u bi Leuconostoc - vi s to thnh CO2, v v sau c s tham gia ca Lactobacillus plantarum. 4.3.Cc sn phm sa. Sa c mt thnh phn dinh dng cao, cha nhiu nc v giu cc mui khong, protein (ch yu l cazein), m b, ng (c bit l lactose) v cc vitamin. trong bu v ca ng vt th n l v trng, nhng khi i ra khi nm v th sa b nhim bi khu h bnh thng ca ng vt, sau c b sung thm bi cc vi sinh vt khc t ngi vt sa, my hoc bnh cha. L mt mi trng gn nh hon ho nn sa rt mn cm vi s sinh trng ca vi sinh vt. Khi sa trong phng nhiu loi vi khun s ln men lactose to thnh acid v lm thay i tnh cht v kin trc ca sa. Qu trnh ny c th xy ra t nhin hoc c con ngi bt chc nh trong qn trnh sn xut phomat hoc iagua. Trong cc giai on sm ca ln men sa lactose b tn cng nhanh bi Streptococcus lactis v Lactobacillus spp. S tit acid lactic v s gim pH lm protein ca sa b ng li thnh cc vn (curd). S ng cng lm tch ring phn dch ng sa (whey) trn b mt. Cc vn cng c th c to nn nh tc dng ca vi sinh vt hoc nh mt enzyme (rennin) ly t d dy b. Mt lt sa nng 1032g bao gm 902g nc v 130g cht kh trong cha 49g lactose; 39g cht bo (l nhng git m ng knh 1-8 m gm glixerit v cc cht tan trong lipit, nh colesterol v cc vitamin A, D, E, K; 32,7g protein m ch yu l cazein; 9g cht khong (gm phosphate, xitrat, KCl, NaCl v MgCl2). *Sa chua (sa b). Sa chua l sn phm ca s ln men sa lng bng s chua ngu nhin di tc ng ca nhiu vi khun sinh acid

163

lactic khc nhau (nh Streptococcus lactis hay S. cremoris), hoc ca mt s lng t hn nhiu cc vi khun gy thm (nh Streptococcus diacetylacis, Betacoccus citrovorus) hoc bng vic cy vo sa un nng cc ging vi khun thun khit. Khi ln men din ra, lactose c chuyn ho thnh acid lactic, acid ny s ngng kt cazein pH 4-5. Sa ng, c, c v chua, ty theo vng c tn gi khc nhau. Sa chua c lm t sa y (cha t nht 3% m ca sa) thng c trn vi bt sa vt vng nhm lm tng hm lng cht rn ton phn v ci thin cu trc gel protein. mt s nc, sa chua cng c sn xut t sa cu, sa tru, sa tun lc hoc sa nga. Kem chua c sn xut bi mt qu trnh rt ging quy trnh dng sn xut sa chua ch tr vic kem c ph c s dng lm nguyn liu th. *Iagua. Iagua c lm t cc loi sa cha cc hm lng m khc nhau bng cch ln men nh cc ging c bit. N c mt ng c mn ging nh gel, mt chua r rt v hng v d chu, khc bit r rng vi loi sa chua bnh thng. Iagua c sn xut bng cch cy vo sa kh trng Pasteur ng nht khong 1,5-3% cc vi khun lactic a nhit (ging hn hp ca mt hoc nhiu chng nh Streptococcus thermophilus hay Lactobacillus bulgaricus) v sau gi 42-45oC trong khong 3 gi. qunh mn c hnh thnh trong thi gian ny. Sn phm cui cng c v chua ( chua 40-60oSH). Phn quan trng trong hng thm c bit ca iagua l cc hp cht cacbonyl vi ixetyl v acetaldehit chim u th. Mt loi igua c bit c tn l sa aciophilus c sn xut nh Lactobacillus acidophil. Vic thm ng v hoa qu vo iagua (iagua hoa qu) lm tng ng k sc tiu th sn phm ny. *Kefia v Kumis. Kefia v Kumis l cc loi ung cha ru giu CO2, nhiu bt. Khu h vi sinh vt bao gm nm men Torula (chu trch nhim i vi qu trnh ln men ru) v cc vi khun Streptococcus lactis v Lactobacillus caucaticus (chu trch nhim i vi ln men lactic). Cc ht kefia dng sn xut kefia cha cc cc sa ng cng vi cc vi khun kefia. Kumis c lm t sa nga hay sa d do cc ging kumis thun khit (Thermobacterrium bulgaricus v nm men Candida) . C hai ung lm t sa ny u l cc sn phm a phng ca vng Caucasus v cc vng tho nguyn Turkestan. Kefia cha acid lactic (0,5- 1%), mt s lng ru ng k (0,5-2%), CO2 v mt s sn phm phn gii cazein sinh ra t hot ng phn gii protein ca nm men. *Phomat. Phomat l sn phm thu c t sa ng bng cch loi b dch ng sa v bng cch lm chn cc vn khi c mt mt khu h

164

vi sinh vt c bit. C nhiu loi phomat vi hnh dng v kch thc khc nhau. c xp thnh nhm da vo loi sa s dng (b, d, cu), s to thnh cc vn (dng acid, dng rennin hay dng c hai), kin trc hay c, v hm lng cht bo. Trong tng nhm, cc loi phomat ring bit c c trng bi hng thm. S sn xut phomat v c bn bao gm hai cng on : s to thnh cc vn v qu trnh lm chn. *S to thnh cc vn Hm lng cht bo ca sa c iu chnh ti mc mong mnn, v khi cn, hm lng protein cng c iu chnh. Cc cht ph gia bao gm mui canxi nhm ci thin s ngng kt protein v kin trc ca phomat, nitrat km hm khu h vi khun k kh hnh thnh bo t v cc sc t to mu. Sa th hoc sa kh trng c trn trong mt b nhit 15-59oC vi mt loi ging khi ng (vi khun lactic hay vi khun propionic, nm si, nh Penicillium camemberti, P. candidum, P. roqueforti , cc vi khun mang sc t mu vng hoc mu nh Bacterium linens, cng cc cu khun v nm men) v sa s ng li thnh mt khi bn lng mm, ccvn, sau ln men lactic, c ni tip bng s b sung rennin hoc mt t hp no m thng thng nht l t hp gia acid v rennin. Gel protein ny s c ct thnh cc hnh khi nh trong lc ang c un nng ri sau c khuy nh. Dch ng sa c loi b cn phn cc vn cha m th c chuyn sang qu trnh lm rn. Khi rn mong mun t c, cc vn c chuyn sang qu trnh lm chn. *Lm chn Qu trnh lm chn ph thuc vo thnh phn khi phomat, c bit l hm lng nc, khu h vi sinh vt v cc iu kin bn ngoi nh nhit v m ca cc phng lm chn. S chn ca phomat mm xy ra t ngoi vo trong, v th trong giai on u s c mt phn v chn v mt phn li cha chn. S chn khng ng u xy ra l do c s chnh lch v pH. bn trong khi phomat, pH thp v c mt acid lactic. ngoi v, cc loi nm si c trng pht trin thch hp hn cc pH cao nng pH ln nh phn ng loi CO2 t cc amino acid. S chn phomat cng xy ra ng u trn ton b khi phomat. S to thnh v ch l kt qu ca s kh b mt, iu ny c th trnh c nh vic bao gi khi phomat trong cc mng plastic thch hp trc khi qu trnh lm chn bt u. Thi gian lm chn thay i ty trng hp, ko di t vi ngy i vi phomat mm ti vi thng thm ch vi

165

nm i vi cc loi phomat cng. Sn lng trn 100 kg sa nc l 8 kg phomat cng v ti 12 kg phomat mm. Mi thnh phn ca phomat u chu s bin i v mt sinh ho hc cc mc khc nhau trong qu trnh lm chn. Lactose c phn gii thnh acid lactic nh ln men lactic ng hnh. Trong loi phomat chea chng hn, pH gim t 6,55 xung 5,15 tnh t lc b sung ging khi ng cho ti khi c thnh khun. Khi c mt vi khun propionic (nh trong trng hp ca phomat Emmental), acid lactic c chuyn ha tip tc thnh acid propionic, acid acetic, v CO2 theo phn ng : 3 CH3CHOHCOOH 2 CH3CH2COOH + CH3COOH + CO2 + H2O T l gia acid propionic v acid acetic b nh hng bi th oxi ha kh ca phomat v t l ny s thp khi c mt mui nitrat chng hn. Phng thc v mc phn gii m ca sa ph thuc vo khu h vi sinh vt tham gia vo qu trnh lm chn phomat. S gii phng cc acid bo, c bit l cc acid nh hng n hng thm ca phomat ph thuc vo tnh c hiu ca cc lipase. Ngoi cc acid bo t do, 2-alkanon v 2-alkanol cng c to thnh nh cc sn phm ph ca s -oxi ho cc acid bo. Nm si, c bit l Penicillium roqueorti, s dng -ketoaxyl-CoA eaxylase (tiodihydroase) v -ketoacid ecacboxylase cung cp cc hp cht in hnh cho hng thm ca cc phomat bn mm. S phn gii protein thnh cc amino acid xy ra qua cc peptit nh cc sn phm trung gian. Ty thuc vo loi sa m 20-40% cazein c chuyn ho thnh cc dn xut protein ha tan trong 5-15% l cc acid amin. Phm vi pH 3-6 l ti u i vi hot tnh proteinase ca Penicillium roqueorti. S phn gii protein b nh hng mnh bi hm lng mui cha trong phomat. Hm lng amino acid l 2,6-9% phn cht rn ca phomat v l phn quan trng trong hng thm ca phomat. Nhng li trong qu trnh lm chn c th to nn nhng peptit c v ng. Cc amino acid c chuyn ha tip tc. Trong giai on sm ca s lm chn, vi pH thp, chng b loi CO2 thnh cc amino. Trong giai on mun, vi pH cao, cc phn ng oxi ho chim u th. S phn gii protein khng ch ng gp vo s to thnh hng thm, n cn nh hng n kin trc ca phomat. Khi phomat mm b lm chn qu mc, s phn gii protein c th dn n vic dch ha gn nh ton b khi phomat.

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Cu hi n tp chng 8 1. Trong t bo, qu trnh ng phn din ra u ? Phn ng ny khc nhau nh th no khi khng c mt oxy v khi c mt oxy ? 2. Phn t no m u con ng ng phn ? Khi ng phn hon thnh, phn t no cn li. Bao nhiu ATP c to thnh mc phosphoril ha c cht ? Con s ATP c to thnh l bao nhiu, v v sao con s ny li khc vi tng s ATP c to thnh ? Bao nhiu ATP s c to thnh b sung nu oxy c mt ? 3. Khi ngung glucose l khng hn ch th ci g s l nhn t gii hn trong phn ng ng phn ? 4. Hai c ch ti to NAD+ l nhng c ch no ? Chng khc nhau ch no ? u l mt qu trnh hiu kh v u l mt qu trnh k kh ? 5. Ln men khc h hp oxy ha ch no khi xt v phng din cc cht nhn in t v s sn sinh nng lng ? 6. S trao i in t to nn ATP khc nhau nh th no trong cc trng hp NADH c to thnh trong ng phn, NADH c to thnh trong chu trnh acid citric, v FADH2 c to thnh trong chu trnh acid citric ? 7. Hiu qu ca vic phn gii hiu kh hon ton mt phn t glucose vi khun l bao nhiu ? So snh vi hiu qu ca mt mnh ng phn? 8. Hiu ng Pasteur c gp cc vi khun ln men lactic ng hnh khng ? V sao ? 9. Gii thch ti sao mt s nm men, nh Rhodotorula hay Geotrichum, khi tha ngun C, ng thi thiu N v P th lipit li c tch ly ? 10. Cho v d v nhng trng hp ln men vi khun trong c s to thnh nhiu hn 2 mol ATP trn mt mol glucose b phn hy.

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Chng 9

Vi khun quang hp v c nh m
I.Vi sinh vt quang hp
1.Cc vi khun quang quang hp (Phototrophic bacteria) 1.Vi khun lu hunh mu ta (Purple sulfur bacteria): a-HChromatiaceae: b-HEctothiorhodospiraceae: 2-Vi khun khng lu hunh mu ta (Nonsulfure purple bacteria) 3.Vi khun lu hunh mu lc (Green sulfure bacteria) 4.Vi khun khng lu hunh mu lc (Green nonsulfur bacteria) 5-Vi khunlam(Ngnh Cyanobacteria) a- Nhm I (c tc gi gi l b Chroococcales): b- Nhm II (c tc gi gi l b Pleurocapsales): c- Nhm III (c tc gi gi l b Oscillatorriales): d- Nhm IV (c tc gi gi l b Nostocales) : e- Nhm V (c tc gi gi l b Stigonematales) 1.1.Vi khun lu hunh mu ta (Purple sulfur bacteria) Thuc nhm ny l cc vi khun k kh bt buc, c kh nng quang t dng v c (photolithoautotroph), t bo c cha chlorophyll a hoc b , h thng quang hp cha cc mng hnh cu hay hnh phin (lamellar) gn vi mng sinh cht. dng lm ngun cho in t (electron donors) trong quang hp thng s dng H2, H2S hay S . C kh nng di ng vi tin mao mc cc, c loi chu mao, t l G+C l 45-70%. a- H Chromatiaceae: 1.1.Chi Thiospirium 1.2. Chi Chromatium 1.3. Chi Thiocapsa 1.4. Chi Thiocystis 1.5. Chi Thiospirillum 1.6. Chi Thiorhodovibrio 1.7. Chi Amoebobacter 1.8. Chi Lamprobacter 1.9. Chi Lamprocystis 1.10.Chi Thiodyction

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1.11.Chi Thiopedia 1.12. Chi Rhabdochromatium 1.13. Chi Thiorhodococcus

Chromatium

Thiocapsa

Thiocystis

Thiospirillum

Lamprocystis

Thiopedia

Hnh 9.1: Mt s i din vi khun lu hunh mu ta thuc H Chromatiaceae

169

b- H Ectothiorhodospiraceae: 1.1- Chi Ectothiorhodospirace 1.2- Chi Halorhodospira 1.2-Vi khun khng lu hunh mu ta (Nonsulfure purple bacteria) Vi khun khng lu hunh mu ta l nhm vi khun quang d dng hu c (photoorganoheterotrophs) thng k kh bt buc, mt s loi l quang t dng v c khng bt buc (trong ti l ho d dng hu c- chemoorganoheterotrophs). T bo cha chlorophyl a hoc b, h thng quang hp cha cc mng hnh cu hay hnh phin (lamellar) gn vi mng sinh cht. dng lm ngun cho in t (electron donors) trong quang hp thng s dng cht hu c, i khi s dng hp cht lu hunh dng kh hoc H2. C kh nng di ng vi tin mao mc cc, hoc khng di ng, mt s loi c ti kh (gas vesicles), t l G+C l 61-72%. 2.1- Chi Blastochloris 2.2- Chi Phaeospirillum 2.3- Chi Rhodobacter 2.4- Chi Rhodobium 2.5- Chi Rhodocista 2.6- Chi Rhodocyclus 2.7- Chi Rhooferax 2.8- Chi Rhodomicrobium 2.9- Chi Rhodoplanes 2.10-Chi Rhodopila 2.11- Chi Rhodopseudomonas 2.12- Chi Rhodospira 2.13- Chi Rhodospirillum 2.14- Chi Rhodothalassium 2.15- Chi Rhodovibrio 2.16-Chi Rhodovulum 2.17- Chi Rosespira 2.18- Chi Rubiviva

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Rhodospirillum

Rhodospirillum di KHV in t

Rhodopseudomonas

Rhodopseudomonas di KHV in t

Rhodobacter

Rhodopila

Rhodocyclus purpureus

Rhomicrobium

Hnh 9.2: Mt s i din vi khun khng lu hunh mu ta

171

1.3.Vi khun lu hunh mu lc (Green sulfure bacteria) Thuc nhm ny l cc vi khun k kh bt buc, c kh nng quang t dng v c (photolithoautotroph), t bo c cha chlorophyll a cng vi b , c hoc e, cha caroten nhm 5, h thng quang hp lin quan n cc lc th (chlorosom) v c lp i vi mng sinh cht. dng lm ngun cho in t (electron donors) trong quang hp thng s dng H2, H2S hay S . Ht lu hunh tch lu bn ngoi t bo Khng c kh nng di ng , mt s loi c ti kh; t l G+C l 48-58%. 3.1- Chi Chlorobium 3.2- Chi Prosthecochloris 3.3- Chi Pelodictyon 3.4- Chi Ancalichliris 3.5- Chi Chloroherpeton

Chlorobium

Pelodictyon

Prosthecochloris

Hnh 9.3: Mt s i din vi khun lu hunh mu lc

1.4.Vi khun khng lu hunh mu lc (Green nonsulfur bacteria) Thuc nhm ny l cc vi khun a bo, dng si,thng k kh khng bt buc ,thng l quang d dng (photoheterotroph), c loi quang t dng hoc ho d dng. T bo c cha chlorophyll a v c, trong iu kin k kh thy c chlorosom. dng lm ngun cho in t (electron donors) trong quang d dng l glucose, axit amin, axit hu c; trong quang t dng l H2, H2S. Di ng bng phng thc trn (gliding) , t l G+C l 53-55%. Chi in hnh l Chloroflexus., Chloronema

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Chloronema

Chloroflexus

Hnh 9.4: Mt s i din vi khun khng lu hunh mu lc

1.5- Vi khun lam (Ngnh Cyanobacteria) Theo NCBT (2005) th Vi khun lam bao gm nhng b sau y:

-Chlorococcales -Gloeobacteria -Nostocales -Oscillatoriales -Pleurocapsales -Prochlorales

Trc y thng nhm ln l To lam (Cyanophyta). Thc ra y l nhng c th nhn nguyn thu, khng lin quan g n to , ngoi kh nng quang hp hiu kh (quang t dng v c) v dng H2O lm cht cho in t trong qu trnh quang hp. Vi khun lam cha chlorophyll a v phycocyanin- phycobiliprotein. Mt s loi c sc t phycoerythrin. Chng phi hp vi sc t lc to nn mu nu. Mng lin kt vi phycobilisom. n bo hoc a bo dng si. Khng di ng hoc di ng bng cch trn (gliding), mt s loi c ti kh (gas vesicles).Nhiu loi c d t bo (heterocysts) v c kh nng c nh nit. Vi khun lam c mt khp mi ni, trong t, trn , trong sui nc nng, trong nc ngt v nc mn. Chng c nng lc chng chu cao hn so vi thc vt i vi cc iu kin bt li nh nhit cao, pH thp. Mt s loi c kh nng sng cng sinh vi cc c th khc nh Ru, Dng x, Tu...Nhiu loi cng sinh vi nm to ra a y (Lichen). Vi khun lam c th l sinh vt xut hin sm nht trn Tri t

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Vi khun lam c chia thnh 5 nhm (subsection) nh sau: a- Nhm I (c tc gi gi l b Chroococcales): Hnh que hoc hnh cu n bo, khng c dng si hay dng kt khi (aggregate); phn i hoc ny chi; khng c d t bo (heterocytes). Hu ht khng di ng. T l G+C l 31-71% . Cc chi tiu biu l: -Chamaesiphon -Chroococcus -Gloeothece -Gleocapsa -Prochloron

Chamaesiphon

Chroococcus

Prochloron Hnh 9.5: Mt s i din vi khun lam thuc b Chroococcales b-Nhm II (c tc gi gi l b Pleurocapsales): Hnh que hoc hnh cu n bo. c th to dng kt khi (aggregate); phn ct nhiu ln to ra cc baeocytes; khng c d t bo.Ch c cc baeocytes l c di ng. T l G+C l 40-46% . Cc chi tiu biu l: -Pleurocapsa -Dermocapsa -Chroococcidiopsis

Glooeothece

Gleocapsa

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Pleurocapsa

Dermocapsa

Chroococcidiopsis

Hnh 9.6: Mt s i din vi khun lam thuc b Pleurocapsales

c-Nhm III (c tc gi gi l b Oscillatorriales): Dng si (filamentous) ; dng lng (trichome) khng phn nhnh ch c cc t bo dinh dng; phn i trn mt phng, c kiu t on (fragmentation); khng c d t bo; thng di ng. T l G+C l 3467%. Cc chi tiu biu l: -Lyngbya -Osscillatoria -Prochlorothrix -Spirulina -Pseudanabaena

Lyngbya

Oscillatoria

Prochlorothrix

Spirulina

Pseudanabaena

Hnh 9.7: Mt s i din vi khun lam thuc b Oscillatorriales

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d-Nhm IV (c tc gi gi l b Nostocales) Dng si ; dng lng (trichome) khng phn nhnh c th cha cc t bo bit ho (specialized cell) ; phn i trn mt phng, c kiu t on to thnh on sinh sn (hormogonia) ; c t bo d hnh ; thng di ng c th sn sinh bo t mng dy (akinetes). T l G+C l 38-47%. Cc chi tiu biu l : -Anabaena -Cylindrospermum -Aphanizomenon -Nostoc -Scytonema -Calothrix

Anabaena

Anabaena trong Bo hoa du

Cylindrospermum

Calothrix

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Nostoc

Scytonema

Hnh 9.8: Mt s i din vi khun lam thuc b Nostocales

e-Nhm V (c tc gi gi l b Stigonematales) : Lng (trichome) dng si, phn nhnh hoc do cc t bo nhiu hn mt chui to thnh ; phn i theo nhiu mt phng, hnh thnh on sinh sn (hormogonia) ; c t bo d hnh ; c th sn sinh bo t mng dy ( alkinetes), c hnh thi phc tp v bit ha (differentiation). T l G+C l 42-44%. Cc chi tiu biu l : -Fischerella -Stigonema -Geitlerinema

Stigonema

Fischerella

Geitlerinema

Hnh 9.9: Mt s i din vi khun lam thuc b Stigonematales

2.Trao i cht cc vi sinh vt quang dng Tt c cc vi khun quang hp u cha sc t quang hp. Sc t quang hp vi khun c gi l bacteriochlorophyll. Chlorophyll v bacteriochlorophyll cn c gi l cht dip lc v cht khun lc. Cht dip lc, cht khun lc v huyt sc t c cu trc tng t nh nhau. l mt vng pocphiril do 4 nhn pirol lin kt vi nhau. Li ca cht dip lc v cht khun lc l Mg, cn li ca huyt sc t l Fe, cht dip lc a

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khc vi cht khun lc a,b,c,d,e 7 gc R (t R1 n R7) c th thy r s khc nhau ny trong bng sau y:
Bng 9.1: S sai khc gia cht dip lc a v cc loi cht khun lc

Ch thch: a gia C3 v C4 khng c ni i, khng thm H b gia C3 v C4 khng c ni i, c thm H C th thy r hn s phn bit ca hai loi chlorophyll, bacteriochlorophyll v hem...bng cch quan st hnh sau y:

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H. 9.10: Cu trc ha hc ca mt s sc t quang hp

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Ngoi cc loi chlorophyll vi khun t dng quang nng cn c cha mt s cc sc t thuc loi carotenoit. Carotenoit vi khun khng ging vi carotenoit to hoc thc vt. Di y l vi v d:

H. 9.11: Cu trc ha hc cc loi carotenoit

vi khun t dng quang nng c hai loi phosphoryl ha quang hp: phosphoryl ha quang hp tun hon v phosphoryl ha quang hp khng tun hon. Ch thch Bchl*: trng thi kch pht ca bacteriochlorophyll Ru-5-P: Ribulose-5-phosphate H2A: Cht v c cho hydrogen

H.9.12:Phosphoryl ha quang hp kiu tun hon

Trong iu kin k kh mt s vi khun quang hp c th s dng nng lng ca nh sng thc hin phn ng phosphoryl ha sn sinh ra ATP. Electron t bacteriochlorophyll c tch ra di tc dng ca nh sng, sau tham gia vo chui h hp tun hon v quay tr li Bchl. Trn ng i sn sinh ra ATP. Vic sinh ra ATP c thc hin ring r vi vic sinh ra [H] c nng lc kh. [H] c nng lc kh c sinh ra t cc cht v c cho hydrogen (nh H2S...).Qu trnh quang hp ny khng sn sinh ra oxi.

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H.9.12: Phosphoryl ha quang hp kiu khng tun hon

Ch thch: Chlb* v Chla* l trng thi kch pht. y l kiu phn ng phosphoryl ha quang hp gp vi khun lam v cng tng t nh to v cy xanh. y chui vn chuyn electron l khng tun hon v vy cn gi l dng chy electron khng tun hon. Quang hp xy ra trong iu kin c oxi v c 2 h thng quang. H thng quang I c cha Chl a (cht dip lc a) v c th s dng c tia c bc sng di. H thng quang II c cha Chl.b v c th s dng c tia sng lam (bc sng ngn). Qu trnh quang hp ny c sn sinh oxi v xy ra ng thi vic sinh ATP( h thng quang hp II) v vic sinh [H] c nng lc kh (trong NAD PH2) l H+ v e-, c sinh ra sau s quang gii nc. Sau quang gii nc sn sinh ra O2 +2H+ + 2e, electron s lin tip i qua chui electron (I v II), sau cng em electron n cho NADP+ sn sinh ra 2 phn ng phophoryl ha sinh ATP. h thng I c sinh NADPH2 v ATP, cn h thng II c sinh oxi v ATP. Mt s vi khun a mn t dng quang nng c qu trnh quang hp kh dc bit. i din cho nhm ny l cc vi khun hay gp trn cc hi sn p mui (nh vi khun Halobacterium halobium, H.eutirubrum...) Mng cht t bo ca chng phn thnh 2 phn: phn mu v phn mu ta. Phn mu c cha cytocrom, flavoprotein.. ca chui h hp dng oxi ha phosphoryl ha. Phn mu ta rt k l. Trn mng hin ln cc vt khoang (ng knh khong 0,5m), phn b c lp vi

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nhau, chim ti mt na tng din tch ca mng t bo vi khun. Mng ny c kh nng thc hin mt qu trnh quang hp c o. C mt loi protein gi l bacteriorodopsin, rt ging vi rodopsin, loi protein c trong t bo hnh tr trn vng mc ca mt. Bacteriorodopsin chim ti 75% mng mu ta. Nhng vi khun ny c th sinh trng c trong ti hoc ngoi sng nu c mt oxi, khi khng c mt oxi chng ch sinh trng c ngoi sng. Chng c 2 con ng thu nhn nng lng: mt ng oxi ha phosphoryl ha khi c oxi v mt ng oxi ha phosphoryl ha khi c chiu sng (quang hp). Tc to ra ATP l cao nht khi c chiu sng bng bc sng 550-600nm.

H.9.13: S minh ha 2 h thng quang

3. C ch quang hp
3.1. Pha sng quang hp Pha sng l giai on u ca quang hp, giai on ny nh sng l nhn t trc tip tham gia vo quang hp nn gi l pha sng. Pha sng xy ra qua 2 giai on: giai on quang l v giai on quang ho. * Giai on quang l: Nh tnh cht quang ho ca nh sng v kh nng hp th nng lng nh sng ca phn t chlorophyll m nng lng ca nh sng c chuyn sang nng lng ca in t trong cc phn t chlorophyll. Nng lng ny li chuyn n 2 tm quang hp l 700 v 680 thc hin giai on quang ho tip. * Giai on quang ho: quang ho l giai on chuyn ho nng lng ca cc in t trong 2 tm quang hp c lm giu bi nng lng nh sng thnh nng lng cha ng trong cc hp cht giu nng lng l ATP v NADPH2. Giai on quang ho xy ra ti 2 tm quang hp bi 2 phn ng

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quang ho m phn chnh l quang phn ly nc v phosphoryl ho. - Quang phn ly nc: Nh nng lng nh sng vi s tham gia ca sc t v h thng cc cht oxi ho trong lc lp m nc b phn hu. 2H2O ( 4H+ + 4 + O2 ) - Phosphoryl ho : y l qu trnh tng hp ATP nh nng lng ca qu trnh oxi ho xy ra trong chui vn chuyn quang hp. ADP + H3PO4 ATP + H2O C 3 hnh thc Phosphoryl ho xy ra: + Phosphoryl ho vng. + Phosphoryl ho khng vng. + Phosphoryl ho ho vng gi. Kt qu chung ca pha sng l: 12H2O +18ADP +18H3PO4+12NADP+ 18ATP + 12NADPH+H+ + 6O2 + 18H2O Pha sng to ra ATP v NADPH+H+ cung cp cho pha ti quang hp. 3.2. Pha ti quang hp Sau khi pha sng to ATP v NADPH+H+, giai on tip theo ca quang hp l s dng ATP v NADPH+H+ kh CO2 to sn phm s cp ca quang hp l C6H12O6. Qu trnh ny khng cn nh sng nn gi l pha ti. Pha ti din ra nhiu con ng khc nhau, mi con ng c trng cho mt nhm thc vt. C 3 con ng ng ho CO2 trong quang hp: chu trnh Calvin, chu trnh Hatch-Slack v chu trnh CAM. * Chu trnh Calvin (chu trnh C3) - y l con ng ng ho CO2 ph bin nht do Calvin tm ra. Chu trnh xy ra qua 3 giai on chnh: + Tip nhn CO2 : Ribulozo 1,5dP tip nhn CO2 sau bin i thnh 2 phn t APG. APG l sn phm u tin nn chu trnh cn c gi l chu trnh C3. + APG b kh thnh AlPG nh ATP v NADPH+H+ ca pha sng. + AlPG s ti to li Ribulozo 1,5dP ng thi to nn sn phm ca chu trnh l C6H12O6. Tm tt chu trnh Calvin:

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C5 + CO2 + ATP + NADPH ---> C6H12O6


H. 9.14: Chu trnh Calvin www.starsandseas.com/. ../cellcalvin.htm

II.C inh nitrogen phn t


Trong khng kh c rt nhiu N phn t, nhng tuyt i a s sinh vt khng s dng c ngun N ny. Ch c mt s VSV l c th hp th c N. Qua hot ng sng ca chng N s c chuyn thnh N hp cht (protein v cc sn phm thy phn protein). Hot ng ny c gi l s c nh N phn t. Qu trnh c nh N c tc dng rt ln n i sng cc sinh vt trn tri t. Trn c bn trong nham thch khng cha hp cht N. Cho nn ngun N trong t hu nh hon ton do tc dng c nh N ca VSV m hnh thnh. Hng nm cy trng li ly i mt lng N ng k. Hp cht N c hnh thnh trong qu trnh c nh ny li chnh l ngun N ch yu b sung cho t. Cc loi VSV c nh N khng nhiu lm, c th chia lm hai loi ln nh sau: 1.Vi sinh vt c nh N sng t do (khng cng sinh): bao gm mt s vi khun, x khun. Trong s ny quan trng nht l cc loi thuc chi Azotobacter v loi Clostridium Pasteurianum. 1. Azotobacter 1.1.S lc v Azotobacter Azotobacter l vi khun c nh N sng t do trong t, hiu kh, khng c bo t. Chng c phn lp v nui cy thun khit t nm

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1901 do nh VSV H Lan Beijerinck. i din in hnh ca chi ny l Azotobacter chroococcum. Do iu kin nui cy v la tui khc nhau m Azotobacter c hnh thi thay i. Lc cn non Azotobacter l nhng trc khun mp hnh que, c tin mao, c kh nng di ng, khi gi th tr thnh t bo hnh cu c gip mc xung quanh. Azotobacter c th s dng nhiu loi hp cht hu c lm ngun thc n C. Chng cng cn nhiu nguyn t khong, c bit l 2 nguyn t vi lng bor (B) v molipbden (Mo)(Mo cn cho tc dng c nh N). Khi sng trong iu kin khng c N hp cht Azotobacter s dng N ca khng kh bin thnh hp cht N ca c th sng. Khi sng trong mi trng thc n N hu c hoc v c th tc dng c nh N s rt thp hoc khng c. Azotobacter thch hp vi iu kin hiu kh va phi v pH trung tnh hoc hi kim. Trong iu kin thch hp c mi khi tiu th 1gam hydratcarbon Azotobacter c th c nh c 10- 15mg N. V Azotobacter c kh nng c nh N mnh m nh vy nn t cui th k 18 c nhiu nc nghin cu s dng chng lm phn vi khun (gi l phn Azotobacterin). Vic s dng Azotobacterin cng ang pht trin mnh nhiu nc v thu c kt qu. Tuy nhin s dng Azotobacterin khng phi l vn tip ging n gin, m cn phi to iu kin chng pht trin mnh c nh c nhiu N, ngha l s dng k thut lin hon. ch to Azotobacterin trc ht ta nui tht nhiu Azotobacter trn mi trng c hoc lng c bm khng kh. Sau em trn chng vi than bn hoc cc cht nui dng khc ch thnh bt Azotobacterin. 1.2. Mt s c im sinh hc ca vi khun Azotobacter a. Hnh thi khun lc Khi nui trong mi trng thch,vi khun Azotobacter c khun lc nhy, li hoc tan, lc u khng mu, sau bin thnh mu nu ti, thm ch n mu en nhng khng lm nhum mu mi trng khun lc. Ngoi ra mt s loi Azotobacter c dng nhn nheo, khun lc c mu vng lc, mu hng . V nhy ca vi khun Azotobacter cha khong 75% l cht hirit ca acid uronic v cha khong 0.023% N.

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H. 9.15: Hnh dng t bo Azotobacter sp. (a) di knh hin vi in t (b) tiu bn m (www-micro.msb.le.ac.uk/ video/Azotobacter.html) b. Hnh thi t bo vi khun Azotobacter Azotobacter l loi vi khun Gram m khng sinh bo t. Khi cn non t bo c dng que u trn ng ring l hay xp thnh tng i chng cht, cht t bo nhum mu ng u, c kh nng di ng nh tin mao, kch thc khong :2,0- 7,0 10- 2,5 m . Khi gi t bo Azotobacter mt kh nng di ng, kch thc thu nh li bin thnh dng hnh cu. Sinh cht xut hin nhiu ht ln nhn. l cc ht volutin, granulose, cc git m Lng ADN trong t bo Azotobacter thng thp hn so vi nhiu loi vi khun khc (0,70- 0,81%). Tm li hnh dng t bo Azotobacter v chu k bin i ca chng ph thuc vo tui ca ng ging v iu kin pht trin. 1.3. Cc c tnh sinh l, sinh ho. a. c im nui cy : Vi khun Azotobacter thuc loi vi khun hiu kh, sng theo phng thc d dng. Chng s dng nhiu loi hp cht hu c khc nhau: disacarit, dextrin, tinh bt, acid hu c, hp cht thm, tuy vy, nhiu tc gi cho bit c khng t cc chng Azotobacter khng c kh nng ng ho lactose, manitol hoc natribenzoat. b. Kh nng s dng ngun N: Trn cc mi trng khng cha N khun lc Azotobacter c dng nhy, li, i khi nhn nheo. Chng t vi khun Azotobacter c kh nng sinh trng trn mi trng khng c N. S d chng tn ti c l v c kh nng ng ho mui amonium, ur. Mt s chng Azotobacter c kh

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nng s dng nitrit, nitrat. Hai loi acid thch hp nht i vi nhu cu dinh dng ca Azotobacter l acid glutamic v axtit asparaginic. 1.4. c im phn loi ca vi khun Azotobacter Theo Becking (1974) th vi khun c nh m thuc chi Azotobacter c 4 loi: A. chroococcum; A. beijerinckii; A. vinelandii; A. agilis . 1.5. nh hng cc nhn t sinh thi n s sinh trng v pht trin ca cc vi khun Azotobacter Vi khun Azotobacter hiu kh, s pht trin v kh nng c nh N ca chng trong t chu nh hng ca cc iu kin sau: a. m ca t Azotobacter i hi m rt cao ca t. Nhu cu v m ca chng cao hn so vi cc vi khun khc, tng ng vi nhu cu ca cy trng. V vy t gp chng vng kh hn, s kh hn ca t ch c bo xc ca chng mi chu ng c. m thch hp:75- 80%. b. thong kh . thong kh ca t c lin quan n qu trnh c nh N ca Azotobacter. Tuy vy vi khun thuc loi hiu kh nhng c th pht trin c trong iu kin vi hiu kh. Qu trnh c nh N ca Azotobacter b gim khi th xi ho kh ca mi trng cao qu +200 mv hoc thp qu () 200 mv. Nh vy, khng kh qu mnh cng c ch qu trnh c nh N phn t, khi nng xi trong khng kh l 4% qu trnh c nh N vt qu ba ln so vi khi xi l 10- 20%. c . Nhit . Nhit nh hng n Azotobacter khng ging nhau cc vng a l khc nhau. vng nhit i Azotobacter thch hp vi nhit 35400C. nhit 70C Azotobacter c hot ng c nh N thp hn 5 ln so vi 450C. T bo sinh dng ca Azotobacter khng sng c khi x l 500C trong 30 pht, nhit 800C s cht rt nhanh. Nhit thch hp i vi s pht trin ca Azotobacter vo khong 20-300C. d. pH ca t. pH ca t nh hng khng ging nhau n s pht trin v kh nng c nh N ca Azotobacter. Azotobacter thch hp nht vi pH = 7,28,2, song c th pht trin c pH = 4,5- 9,0. Cc loi Azotobacter khc nhau mn cm mt cch khc nhau i vi pH ca mi trng. Chng hn: pH thp nht ca mi trng i vi Azotobacter

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chroococcum v A. beijerinck khong 5,5; i vi A. marocytoges l khong 4,6. Phn ln Azotobacter ch pht trin pH ln hn 6, v vy t gp chng t chua. Cng c th phn lp c mt s chng t t chua nhng cc chng ny thng mt kh nng c nh N phn t. e. Phn m Trong t Azotobacter c kh nng ng ho cc cht dinh dng t mi trng. Ngun N i vi Azotobacter khng phi ch l N phn t m cn l mui amon, nitrat, amino acid. Tu thuc vo nng ca cc hp cht cha N c trong mi trng m qu trnh c nh N s b c ch nhiu hay t . g. Phn ln. Pht pho rt cn cho qu trnh c nh N ca Azotobacter ch bt u xy ra khi nng PO43- t n 4 mg trong 100 ml mi trng. Ngc li khi nng PO43- t ti 800 mg trong 100 ml qu trnh c nh N s bt u ngng li. S mn cm mnh m ca Azotobacter vi pht pho cho php ngi ta s dng chng nh loi vi khun ch th xc nh nhu cu v phospho ca t. h. Phn kali. S pht trin ca Azotobacter rt cn n kali, nhng vi lng nh. Nu a vo mi trng mt lng mui kali qu d tha chng s lm c ch s pht trin ca Azotobacter, c th tc hi ny l do gc anion ca mui ny gy ra. i. Canxi. Canxi nh hng ln n s pht trin ca Azotobacter. Khi thiu canxi t bo Azotobacter s to thnh nhiu khng bo nh hng xu i vi vic tng hp ATP v s to thnh cc polyphotphat. Nhng iu tra Vit Nam cho thy trong t c lng cha CaO cao hn 0,4% lun c mt mt s lng ln cc t bo Azotobacter. Hu nh khng pht trin trong mu t c c lng cha CaO di 0,25% (Nguyn Ln Dng 1965) Ring i vi A.chroococcum nng CaCl2 ,thch hp nht l 0,01% ,nu cao hn s nh hng khng tt i vi hot ng c nh N ca chng. C tc gi dng A. chroococum nh l loi vi khun ch th xc nh nhu cu v vi ca t, nguyn t Mg c Azotobacter i hi vi s lng cao hn st khong 10 ln .

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Cng vi nguyn t i lng, cc nguyn t vi lng cng cn thit i vi Azotobacter, ng ch hn c l molybden, bo(Bo), mangan(Mn) k. Cc nhn t sinh hc S pht trin v c nh N ca Azotobacter trong t cn chu nh hng mt thit ca khu h cc sinh vt t. Bn cnh cc nhm vi sinh vt c nh hng tt cn c nhiu nhm c kh nng c ch s pht trin ca Azotobacter . c nhiu cng trnh nghin cu cp n mi quan h ga Azotobacter vi cy trng vi s lng cao hn nhiu so vi ngoi vng r . Mt s chng A.chroococum c kh nng sinh ra mt s cht chng nm c tc dng kh rng (c ch Aspergillus, Penicillium, Fusarium, Alternaria ) 1.6. Tnh hnh nghin cu ng dng Azotobacter. Trong nhiu nm, nhiu nc khc nhau ,ngi ta sn xut quy m cng nghip hoc th cng nghip nhng ch phm Azotobacter v gi l azotobacterin. l nhng dch nui cy Azotobacter c hp th vo than bn hoc cc loi t giu cht hu c trung ho v b sung thm mt t phn phospho, kali Khc vi nitragin, azotobacterin khi dng bn cho cy trng thng a n nhng hiu qu khng ln v khng n nh (nu c lm tng sn lng thng cng khng tng qu 10% so vi i chng ). a s cc tc gi cho rng hiu qu ca vic s dng azotobacterin ch tng i vi cc t giu cht hu c hoc trong trng hp c bn thm nhiu phn khong. C l tc dng ch yu ca chng khng phi kha cnh c nh N m l kha cnh tng hp cc cht hot ng sinh hc c tc dng kch thch sinh trng ca cy trng . Vit Nam, vic sn xut v s dng ch phm azotobacterin ch t ra trong nhng iu kin thm canh c kh nng di do v phn bn, cn bn vi v bn phn ln lm tng s lng Azotobacter trong t. Azotobacterin ch c tc dng tng i r i vi cc loi t giu cht hu c hoc trong trng hp c bn thm nhiu cht khang.C l tc dng ch yu ca chng khng phi kha cnh c nh N m l kha cnh tng hp cc cht hot ng sinh hc c tc dng kch thch s sinh trng ca cy trng . a. Cc phng php to ch phm Azotobacter

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* Dng mi trng thch nghing Chun b mi trng thch nghing Thnh phn mi trng (g/l):Glucose-20; K2HPO4.3H2O -0,2; MgSO4 .7H2O -0,2; NaCl- 0,2; K2SO4 -0,1; CaCO3 -5,0; H20 -1 lt ;Thch 20 pH: 6,5-7. Chun b ging Azotobacter :Cc ng ging c phn lp v tuyn chn t trc. To ch phm: Cy truyn ging vi khun Azotobacter t ging gc vo nhng ng nghim thch nghing. m nhit : 28-30 0 C trong thi gian 2-6 ngy, loi b nhng ng b nhim vi sinh vt khc ta thu c chng Azotobacter thun khit. * Dng dch th Chun b mi trng dch th Thnh phn mi trng (g/l): Glucose 20 0,2 K2HPO4.3H2O MgSO4.7H2O 0,2 NaCl 0,2 K2SO4 0,1 CaCO3 5,0 1 lt H2O pH 6,5-7 Chun b ging vi khun Azotobacter : Ly 5-10 ml mi trng dch th (C thnh phn nh trn khng c thch) kh trng cho vo mi ng nghim 14 5ml Dng que cy gt nh, a dch vi khun vo bnh nn (250500ml) ng 150ml mi trng v nui chng trn my lc(220 dao ng/pht) . Khi khi dch nui t mt 109 t bo/ml th c th em s dng * Dng bt Nui cy ging vi khun Nhn ging Hp th ging vo cht mang vi khun im quan trng cua phng php ny l la chn cht mang vi khun, cht mang vi khun phi t yu cu thch hp v s tn ti ca Azotobacter trong qu trnh bo qun v sng st trn ht ging khi c tm vo, cht mang vi khun c th l than bn, t bt giu cht hu c, c ni s dng c tru, b bia lm cht mang vi khun

190

Ngy nay cht mang vi khun c s dng bi than bn thch hp trong vic s dng ch phm vi khun ni chung v ch phm Azotobacter ni ring. Ngoi c tnh ca than bn l khng c, c tnh hp th cao, gi nc tt , n cn l nguyn liu r tin sn c nhiu ni. 1.2. Clostridium pasteurianum v mt s VSV c nh N khc C.pasteurianum l trc khun k kh bt buc, c bo t, bo t nm gia lm t bo phnh ln nh hnh thoi. C. pasteurianum l mt loi vi khun butyric, chng c th s dng cc hydratcarbon thng thng v lm ln men suinh ra acid butyric, acetic, CO2 v H2. Chng c th s dng hp cht N v c v hu c lm ngun thc n N, khi nhng hp cht ny y th tc dng c nh N ca chng h thp hoc hon ton khng c. Tc dng c nh N ca C. pasteurianum thp hn Azotobacter. C mi khi tiu th 1g hydratcarbon th chng c nh c 2-3 mg N. Tuy nhin C.Pasteurianum rt nhiu v phn b rng ri hn Azotobacter nn ngun N m chng c nh cho t l rt quan trng. Chng li pht trin c rung ngp nc, yu cu pH khng gt gao nn thch hp cho cc loi rung ca ta. Ngoi hai loi trn, trong cc VSV c nh N cn c mt s vi khun thuc chi Clostridium, Bacillus v Azotomonas. Tuy nhin, v nhng loi ny phn b khng nhiu v hiu lc c nh N cng thp nn tc dng thc t khng ln lm. 2. Vi khun c nh N cng sinh. Phn ln cc cy thuc h u khng cn thc n hp cht, chng c th s dng c N khng kh. Nhng kh nng ny khng phi bn thn cy h u c m do nhng vi khun nt r cng sinh vi cy h u mang li. 2.1. Vi khun nt r Ngi u tin chng minh rng cc cy b u c th sinh trng trong t khng cha N l H. Hellrigel v H. Wilfarth (1886). Nm 1886 M.W.Beijerinck phn lp c vi khun t nt r mt s cy u. Ging (chi) Rhizobium (Franck, 1889) l loi vi khun n bo t, rt d nhm vi Pseudomonas, trc khun t do c kch thc 1,2(m x 0,5 chuyn ng nh nhung mao v tin mao cc, hiu kh. Khi hnh thnh nt sn th trc khun bin dng (c hin tng a hnh thi), n thay i hnh dng ty theo la tui v iu kin sng. Khi cn non chng l trc khun nh b, khng c bo t, di ng nh tin mao. Trong qu trnh pht trin, chng s chuyn thnh hnh cu v nhng dng phn nhnh hnh ch Y, ch V gi l gi khun th (Bacteroids).

191

Nm 1963, Manil chia cc vi khun nt sn thnh 3 nhm: -Rhizobium leguminosa (u H Lan), R. trifolii (c ba l), R. phaseoli (u c ve, u xanh). -Rhizobium lupini (mc tc, linh lng), gp nhm 1 v 2 thnh Rhizobium mc nhanh. - Rhizobium lupini (u a, lupin), R. Japonicum (u nnh) vi nhiu loi ph. Nhm ny hin nay c nh loi l Bradyrhizobium (gm tt c cc chng mc chm. Hin nay, ngi ta xp vi khun nt r vo mt chi chung l Rhizobium.Chng c th s dng nhiu hydratcarbon khc nhau lm ngun C. Khi sng ring l trong t hoc trong mi trng nui cy chng khng c kh nng c nh N. V h hp, chng l loi vi hiu kh. Rhizobium c nhiu loi v c tnh cht chuyn tnh, ngha l mi loi ch c th xm nhp v to thnh nt r trong mt s cy h u nht nh. Vi khun nt r khi gp cy h u thch hp s xm nhp vo t bo r, kch thch r to thnh nhng nt r. Nt r thng thng bng ht go, nt r to bng ht u nh, trong nt r cha y vi khun. Sau khi hnh thnh nt r gia vi khun v cy h u lm thnh mt th cng sinh c li cho c hai bn. Ngoi cc cy h u, mt s r cy khc cng c th cng sinh vi vi khun c nh N. Gn y ngi ta cn thy nt sn l mt vi loi cy nhit i. Trong nt sn cng cha vi khun c nh N gn ging r. 2.2.Phn bn nt r (nitragin) Phn bn vi sinh do Noble Hiltner sn xut u tin ti c nm 1896 v c t tn l nitragin: Sau pht trin sn xut ti mt s nc khc nh M (1896), Canada (1905), Nga (1907), Anh (1910) v Thy in (1914). Nitragin l loi phn c ch to bi vi khun Rhizobium do Beijerink phn lp nm 1888 v c Fred t tn vo nm 1889 dng bn cho cc loi cy thch hp ca h u. T cho n nay c nhiu cng trnh nghin cu nhm ng dng v m rng vic sn xut cc loi phn bn vi sinh c nh nit m thnh phn cn c phi hp thm mt s vi sinh vt c ch khc nh mt s x khun c nh nit sng t do Frankia spp, Azotobacter spp, cc vi khun c nh N sng t do Clostridium pasteurianum, Beijerinkia indica, cc x khun c kh nng gii cellulose, hoc mt s chng vi sinh vt c kh nng chuyn ha cc

192

ngun d tr phospho v kali dng kh ho tan vi s lng ln c trong t mn, than bn, trong cc qung apatit, phosphorid v.v... chuyn chng thnh dng d ho tan, cy trng c th hp th c. Trong cc loi vi sinh vt k trn, vi khun nt r c ngha ht sc to ln trong nng nghip. Cng c nh N cng sinh ln hn nhiu so vi cng c nh N khng cng sinh. Sau khi trng cc cy h u t giu thm N rt nhiu. Chnh v vy, t u th k XX ngi ta nghin cu s dng phn bn nt r vi mc ch l tip ging nhn to, lm cho t khng cha vi khun nt r thch ng s c vi khun thch ng, lm cho cy h u khng sinh nt r s c th sinh nt r v nhng cy c th sinh nt r th cng sinh nhiu hn, c nh N cng mnh m hn. Cch s dng phn bn vi khun nt r l em trn phn vi ht ging, ri kh, sau em gieo. Cng c th dng nt r tt, nghin nt trn vi ht ging. Khi s dng c th tng sn lng cy h u ln 15- 20%. Ngoi hai nhm vi khun c nh N sng cng sinh vi thc vt v sng t do trong t nh ni trn, cn mt s vi khun c kh nng c nh N sng trn b mt r v n su vo vo lp t chc b mt r ca mt s cy ha tho nh la, ng, ma,... l mt loi vi khun c dng xon c pht hin t nm 1974 thuc chi Azospirillum. Ngoi cc nhm vi khun c nh N ni trn ra, cn c mt s loi vi to n bo cng c kh nng c nh N. V d nh vi khun lam sng t do v sng cng sinh trong bo hoa du. Cc loi ny cng ng gp khng nh vo qu trnh c nh N khng kh.

Cu hi n tp chng 9
1.Hy nu tng qut chu trnh chuyn ha N trong thin nhin v vai tr ca vi sinh vt trong chu trnh ? 2.Cc nhm vi sinh vt c nh N ? ngha thc t ca vic nghin cu vi sinh vt c nh m. 3.Nu mt s thnh tu hin nay v nghin cu vi sinh vt c nh m. 4. Naza l g ? C ch hin bit ca qu trnh c nh N phn t. 5.Cc loi phn bn vi sinh vt ? c s khoa hc ca vic sn xut v s dng cc loi phn bn vi sinh vt? 6. Trnh by cc bc ch yu iu ch Azotobacterin v Nitragin. Trong iu kin hin nay Vit Nam c th sn xut 2 ch phm ny c khng ? Ti sao ?

193

Chng 10

Di truyn hc vi sinh vt
Di truyn l c tnh chung ca mi sinh vt gi li nhng v truyn cho con chu nhng c im v cu to v pht trin ca t tin, hay ni cch khc, l hin tng cc c th trong mt gia nh c nhng thuc tnh cu to v pht trin ging t tin, vi cha m hoc gia con ci vi nhau. Bin d l c tnh chung ca mi sinh vt c th mang nhng s khc bit v nhiu chi tit tnh trng so vi b m ca chng v vi cc c th khc cng loi. i tng nghin cu ca di truyn hc khng ch l hin tng di truyn m c hin tng bin d. Tnh bin d c v nh c lp vi tnh di truyn nhng thc ra s khc bit gia cc c th trong mt loi trong nhiu trng hp lin quan n s bin i hoc trong trng hp khc n s phn ng ca vt cht di truyn ca sinh vt. vi sinh vt bin d th hin mc ln hn vi sinh vt bc cao nh s c th trong mt qun th ln, n allele, sinh sn ng lot, giai on sinh dng ngn, tn s t bin v ti t hp cao v c kh nng trao i di truyn ngoi loi. D c ch xut hin khc nhau nhng phn ln cc trng hp bin d u to ra nhng dng hay tp on c s thch ng tt nht vi iu kin ngoi cnh vn lun bin ng.

I. C s vt cht di truyn vi sinh vt


1. Vt cht di truyn vi khun Acid nucleic l c s vt cht di truyn ca tt c cc dng sinh vt. tt c cc sinh vt nhn s (prokaryote, cn gi l sinh vt nhn s s, bao gm cc vi khun) hay nhn chun (eukaryote, cn gi l sinh vt nhn chun, bao gm nm, tc chn khun, nguyn sinh ng vt, to, thc vt v ng vt bc cao), tr virus v cc yu t sinh hc n gin hn nh viroid v prion, tnh trng c m ha v tn tr di dng m ha l trnh t thng ca cc nucleotide trong thnh phn acid deoxyribonucleic (DNA). Vt cht di truyn ny c th hin thnh tnh trng ca c th thng qua qu trnh tng hp t khun DNA thnh phn t RNA thng tin (qu trnh phin m) v sau RNA thng tin ny li lm khun tng hp protein (cu trc, enzyme, th th, kch thch, km hm,...) trong qu trnh gi l dch m (transcription) dn n biu hin tnh trng. Vt cht di truyn c truyn t th h t bo ny sang th h t bo khc (v t th h ny sang th h khc) nh qu trnh t sao

194 (replication) ca phn t DNA. Quy tc ny c gi l quy tc trung tm biu hin di truyn. vi khun DNA c th gp hai dng: DNA nhim sc th v DNA plasmid. Nhn (th nhn) vi khun l mt nhim sc th vi khun cu to t mt phn t DNA duy nht xon kp (gm hai mch xon), khp kn (khng c u t do), phn b trong t bo cht. Mi t bo vi khun ch c mt nhn (th nhn) duy nht, mc d trc khi phn bo s lng nhn (nhim sc th) thng thy l 2, 4 hoc nhiu hn do qu trnh phn bo din ra chm hn qu trnh phn nhn. V vy, thng thng nhim sc th c m hnh ha trong t bo vi khun di dng mt vng trn.

Hnh 10.1: M hnh cu trc mt on phn t DNA: mt dn xut purine lin kt qua cu ni hyr vi mt dn xut pyrimidine nn khong cch gia hai sn ng ribose - Acid phosphoric ca hai chui l khng i, guanine lun kt hp vi cytosine, adenine kt hp vi thymine ca chui song i nn trnh t hai chui khng bao gi ging nhau nhng trnh t mt chui quy nh trnh t ca chui kia, hn na cc sn c nhiu nhm i thy nn DNA tan tt trong nc.

195 Hai mch xon ca phn t DNA thc cht l hai chui polymer v c mi quan h cht ch vi nhau v thnh phn ha hc cng nh trnh t sp xp ca cc monomer c gi l mt nucleotide. Mi chui polymer c cu to t bn loi monomer c cu trc tng qut gm ba thnh phn: baz nit d vng (dn xut purine hoc pyrimidine), ng deoxyribose (C5) v Acid phosphoric. DNA, c bn loi nucleotide: adenine (A), thymine (T), guanine (G) v cytosine (C) (hay xitzin). Cc nucleotide khc nhau bi gc baz khc nhau. Chui polymer ca mi mch (si) DNA c hnh thnh nh lin kt phosphoester gia Acid phosphoric v ng deoxyribose to nn b "sn" ca phn t ((-P-C5)n). Cn cc gc baz nit gn vo nguyn t C 1' ca phn t ng deoxyribose. Phn t Acid phosphoric trong mt nucleotide gn vo v tr C 5' ca phn t ng deoxyribose, trong khi nguyn t C 3' gn vi Acid phosphoric ca nucleotide k tip. Do trnh t hot ng ca cc enzyme tng hp DNA t u C 5' n u C 3' ca mi mch DNA nn ngi ta quy c m t mch theo hng C 5'C 3'. V d, nu khng c ch gii khc th mch ATT CGC GCA TCA GCT cng c ngha l 5'ATT CGC GCA TCA GCT-3' hay 5'-ATT CGC GCA TCA GCT. Hai chui ca mt phn t DNA gn kt vi nhau nh cc mi lin kt hyr gia cc gc baz, mt gc purine c gn kt vi mt gc pyrimidine theo nguyn tc "tng b": adenine gn vi thymine bng hai mi lin kt hyr, cn guanine gn vi cytosine bng ba mi lin kt hyr. Hai chui nh vy khng i xng m chy ngc hng (i nghch song song, hay song i) t 5'3' hoc ngc li. Do mt gc purine ca mt chui lin kt vi mt gc pyrimidine ca chui kia nn khong cch gia hai sn (-P-C5-)n ca phn t DNA mi im khng i. V nh vy m sn ny hng ra pha ngoi v xon quanh trc tng tng xuyn qua cc lp gc baz nit vng, nn vi tnh i thy ca Acid phosphoric v ng deoxyribose, DNA l cht tan tt trong nc v kh bn vng trong dung mi ny. Do s lng mi lin kt hyr gia guanine (G) v cytosine (C) cao hn nn DNA c nhiu G+C thng c mc nhit bin tnh hay "nhit nng chy" (melting point) cao hn hay bn vng hn. Hn na, bn cnh cu trc xon phi (B-DNA) nu trn, nhng vng hm lng G+C cao DNA cn c cu trc xon tri (Z-DNA) l ni DNA c s gp khc mnh v xut hin on DNA xon chui ba. C th nhng Z-DNA ng vai tr quan trng trong ti t hp gen cng nh iu ha hot ng ca gen. Mt on DNA m ha mt RNA (nh RNA ribosome, RNA vn chuyn) hay m ha mt protein (qua RNA thng tin) c gi l mt gen. Gen m ha phn t protein c gi l gen cu trc. vi khun ton b trnh t nucleotide gen cu trc c phin m thnh RNA thng tin v sau dch ton b thnh trnh t Acid amin trong phn t protein.

196 Mc d c cu to bi hai chui nhng vi khun (cng nh sinh vt nhn chun) gen ch c mt mch "hiu lc", tc ch mt mch lm khun tng hp nn RNA thng tin. Hip hi sinh ha quc t quy c chui lm khun tng hp nn RNA thng tin (hay tng hp RNA hot ng, nh RNA ribosome,...) l chui m. Nh vy, chui dng ca mt gen l chui c trnh t nucleotide tng ng vi trnh t nucleotide ca RNA thng tin. Ngoi ra, do mt polypeptide c khi u tng hp vi Acid amin methionine (m AUG trn RNA thng tin), kt thc bi mt trong nhng "m dng" (UAA, UAG v UGA) v ch c th c thuc tnh protein nu ln (di hn 50 Acid amin), ng thi, thng tin di truyn c gii c tng "khung" b ba nucleotide (tc ba nucleotide lin k nhau m ha cho mt Acid amin trong sn phm protein) nn ch c nhng on DNA bt u t b ba ATG (theo c ba cch c ca mi chui DNA) n mt m dng (TAA, TAG, TGA) di (hn 150 nucleotide) mi c th l (nhng khng nht thit phi l) mt gen cu trc. on DNA ny c gi l mt "khung kh phin" (hay mt "khung c m" - open reading frame). Ngoi ra, khng phi tt c cc on DNA u mang gen cu trc, chc nng ca nhiu on cn cha bit, ng thi cn c cc on DNA c bit tham gia vo iu ha hot ng ca cc gen cu trc. Nh vy, cc tnh trng quy nh trong genome ch biu hin trong iu kin mi trng nht nh, ng thi qu trnh tng hp cc c cht nguyn liu cho qu trnh tng hp cc thnh phn t bo cng din ra v nh ch mt cch hiu qu v tit kim. sinh vt nhn s cc gen cu trc (cc cistron) c gn lin nhau thnh tp hp v cng chu s kim sot ca cc gen iu ha gm gen (hay vng) khi ng (promotor - P), gen (hay vng) iu hnh (operator - O, cn gi l gen ch huy) v gen iu ha (regulator - R). Gen iu ha (R) nm v tr no trong nhim sc th xa vi cc gen cu trc v iu khin vic tng hp mt protein c bit c tc ng (phong ta hoc gii ta) trc tip hoc gin tip ln cc vng P hoc O. C tp hp bao gm cc vng khi ng, vng iu hnh v cc gen cu trc to thnh mt n v hot ng gi l operon. RNA gm ba loi khc nhau: RNA ribosome l thnh phn ch yu cu to nn ribosome, c chc nng hot ng nh mt enzyme tc xc tc qu trnh sinh tng hp protein. (Nhng RNA xc tc phn ng sinh hc thng c gi l ribozyme). RNA thng tin c sao chp t chui m ca gen c vai tr sao m di truyn t nhim sc th cho RNA ribosome gii m thnh phn t protein c mch polypeptide c thnh phn v trnh t Acid amin xc nh. Tham gia vo qu trnh sinh tng hp protein cn c nhm cc RNA vn chuyn. y l nhng phn t RNA tng i ngn c tc dng hot ha Acid amin nguyn liu v vn

197 chuyn Acid amin ny n RNA thng tin cng c hot ha nh gn kt vi ribosome. Trnh t RNA thng tin quyt nh trnh t Acid amin trong thnh phn polypeptide, nhng qu trnh nhn bit trnh t Acid amin m ha trong RNA thng tin l nh RNA vn chuyn. Mi Acid amin c m ha bi mt b ba nucleotide trn RNA thng tin, v b ba ny tng b vi trnh t b ba nucleotide nhn bit ca RNA vn chuyn. Nh mi Acid amin c loi RNA vn chuyn ring nn trnh t m b ba c dch mt cch chnh xc.
Bng 10.1 : M di truyn trn nhim sc th (biu hin trn mRNA, chiu 5' n 3') Ch th hai ca m u U C A G
5'

Ch th nht ca m (u 5')

UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG

Phe Phe Leu Leu Leu Leu Leu Leu Ile Ile Ile Met Val Val Val Val

UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG

Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala

UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG

Tyr Tyr Stop Stop His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu

UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG

Cys Cys Stop Trp Arg Arg Arg Arg Ser Ser Arg Arg Gly Gly Gly Gly

U C A G U C A G U C A G U C A G

Khc vi DNA, cc RNA u cu to ch t mt chui nucleotide. Ngoi ra, trong thnh phn ca cc RNA cn c nhng im khc bit khc: uracine thay th thymine, ng deoxyribose c thay th bi ribose trong sn ca mch polymer cng nh s c mt mt s baz dn xut khc ca purine v pyrimidine. Chnh v c thm mt gc -OH v tr C 2' (ca ng ribose) m cu ni phosphoester gia nguyn t C 3' v Acid phosphoric ca nucleotide tip theo thng yu hn so vi cu ni C 3' vi Acid phosphoric trong DNA. RNA, v vy, cng km bn vng hn DNA. Tuy cu to t ch mt chui nucleotide nhng cu trc khng gian ca cc loi RNA li n nh nn chc nng ca chng cng n nh. Tnh n nh ca cu trc c c nh cc mi lin kt hyr xut hin gia hai on trnh t tng b ngc hng ca cng chui nucleotide. Hai on c trnh t tng b ngc hng to thnh mt on xon kp,

Ch th ba ca m (u 3')

198 trong khi on nucleotide nm gia chng hnh thnh mt "thng lng" mch n. V vy, chng hn, cc phn t RNA vn chuyn c dng "l ba ch" vi chc nng c hiu. Bn cnh DNA nhim sc th vi khun cn c th gp c s vt cht di truyn ngoi nhim sc th gi l plasmid. V bn cht ha hc, plasmid cng l mt phn t DNA xon kp, khp kn, nhng c phn t lng thp hn nhiu (khong 106 - 108 Dalton, hay khong 1/100 n 1/200 ln nhim sc th vi khun). Mt vi khun c th khng mang, hoc mang mt hoc mt s plasmid. Plasmid c th phn b trong t bo cht trng thi t do v c th sao chp mt cch c lp vi nhim sc th hoc nm trong nhim sc th (ti t hp), khi plasmid c gi l episome. trng thi ti t hp trong nhim sc th (hay episome) plasmid thng c sao chp ng thi vi nhim sc th v c truyn t th h ny sang th h khc ca vi khun. Tuy vy, trong nhiu trng hp (nh chng Hfr) plasmid ti t hp li c th iu khin sao chp c lp, lm cho bn thn plasmid hoc mt on plasmid km theo mt on DNA nhim sc th c tng hp ng lot vi tn sut cao. C loi plasmid c s phin bn cao (high-copy plasmid), trong khi c loi plasmid c s phin bn thp (low-copy plasmid).

Hnh 10.2: Bn enzyme hn ch ca plasmid pBR322, l mt plasmid c s dng ph bin trong k thut DNA ti t hp. Plasmid ci bin nhn to ny mang hai gen khng thuc (tetR khng tetracycline v ampR khng ampicillin) v mt lot v tr ct ca enzyme hn ch trong mi enzyme ny ch c mt v tr phn ct, nh vy plasmid vector ny p dng c vi nhiu DNA khc nhau.

199 Thm hoc mt mt plasmid thng dn n mt hoc bt mt hoc mt s tnh trng, mc d nhiu plasmid khng th hin tnh trng (plasmid "cm"). Tnh trng m cc plasmid iu khin a dng nhng nhn chung plasmid khng phi l yu t thit yu i vi s sng cn ca vi khun. Da vo s tng hp ca cc sn phm ca plasmid m ngi ta chia cc plasmid thnh mt s nhm: plasmid gii tnh, plasmid khng, plasmid iu khin sinh tng hp (nhm cht no ),... v thng lin quan n tnh gy bnh ca cc vi khun. V d, xc nh c rng mt s loi (hoc cn c coi l dng huyt thanh hc) vi khun Salmonella c c c tnh nh mang plasmid.
Bng10.2 : Plasmid c tnh c hiu mt s loi (dng huyt thanh) Salmonella ln (kb) Biu hin tnh gy bnh ch yu Salmonella 90 c tnh gy cht, gim thiu tnh thm thu S. typhimurium ca cc cht k thy c tnh gy cht, tnh khng huyt thanh, 75 S. dublin nng lc tng trng trong t bo h li ni b 75 Ging nh plasmid S. dublin ( hay serovar S. naestved Dublin) 54 c tnh gy cht S. enteritidis 50 c tnh gy cht, gy chng mu nhim S. choleraesuis khun chut S. gallinarum85 c tnh gy cht pullorum

Plasmid gii tnh cn gi l yu t sinh dc hay yu t F (fertility factor), l plasmid iu khin s hnh thnh cc pili gii tnh (nhung mao gii tnh), s tip hp v s vn chuyn thng tin di truyn (gen plasmid hay gen nhim sc th) mt chiu t t bo cho sang t bo nhn. Trong trng hp ny t bo mang plasmid F l t bo cho thng c gi l t bo c hay t bo F+, cn t bo nhn c gi l t bo ci hay t bo F-. Yu t F c th nm trng thi t do trong t bo cht hay trng thi lin kt vi nhim sc th hay trng thi episome. Trong trng hp sau qu trnh vn chuyn thng tin di truyn thng din ra vi tc v tn sut cao nn nhng chng vi khun ny thng c gi l chng Hfr (bt ngun t ting Anh: high frequency of recombination). Yu t khng, hay yu t khng thuc, hay plasmid R (resistance factor), l plasmid iu khin s khng ca vi khun i vi cc cht khng sinh v thuc chng vi khun khc. C ch khng ny l s iu khin vic tng hp cc enzyme (nh penicillinase,...) phn

200 hy thuc khng sinh thnh cc sn phm v hot hoc c ch s xm nhp ca thuc khng sinh vo t bo (nh chn cc l khng trn mng t bo cht i vi tetracycline hoc thuc khng sinh h macrolide,...). Yu t R lan truyn c (transmissible R factor) l yu t F khng thuc, gm hai min gen. Mt min gen iu khin s khng. S lng yu t b khng c th l 1 n 10 hoc nhiu hn na, trng hp sau thng gi l plasmid khng ng lot. Min gen th hai (min RTF) l gen iu khin s vn chuyn cc plasmid vo t bo khc. Thng thng s vn chuyn cc plasmid ny c tc cao, nhng yu t vn chuyn cng c th t c ch hoc b c ch bi yu t khc. Yu t R khng lan truyn c (non-transmissible R factor) ch mang min gen iu khin tnh khng. Cc plasmid ny khng t vn chuyn sang t bo khc m ch lm cho t bo mang chng c tnh khng thuc khng sinh. Chng c th tn ti v ph bin trong qun th vi khun nh qu trnh sinh sn ca vi khun hoc nh cc qu trnh vn chuyn vt cht di truyn qua dch th tc qu trnh bin np (transformation) hoc s vn chuyn ca virus ca vi khun (phage, hay thc khun th) tc nh qu trnh ti np (transduction). 2. Vt cht di truyn virus Khc vi cc sinh vt khc, virus ch mang mt trong hai loi Acid nucleic (hoc DNA hoc RNA), c loi virus mang genome di dng DNA (tng t genome ca vi khun, ng vt v thc vt) nhng c loi li mang genome di dng RNA. Hu ht cc virus thc vt c genome RNA, cn hu ht virus ca vi khun c genome DNA, trong khi cc virus ng vt c genome hoc loi ny hay loi khc. Cng nh bn cht ha hc, cu trc genome ca virus rt a dng, c loi virus mang DNA mt si (chui) m hoc dng, c loi DNA hai si, c loi mang RNA mt si m hoc dng hoc lng ngha (ambisense RNA: c on m c on dng, hay ng thi vi khung kh phin mt gen cu trc cn mang khun ca mt khung kh phin mt gen khc ngc hng). Genome nhiu virus l mt si acid nucleic duy nht nhng cng c loi virus genome bao gm mt s on. Thng tin di truyn trn genome ca virus cng c s khc bit khc so vi genome vi khun. l hin tng gen chng lp (overlapping genes): c th c hai RNA thng tin khc nhau c sao chp t mt on genome. Di y trnh by ngn gn v cu trc genome mt s virus. Genome cc virus thuc chi Parvovirus l virus cha DNA mt si m nhng cng thy khong 1 - 50% s virion cha DNA mt si dng. cc chi Dependovirus v Densovirus th s lng virion cha

201 DNA chui m v virion cha DNA chui dng tng b v cn bn tng ng nhau. Genome ca cc Adenovirus (h Adenoviridae) l mt phn t DNA 2 si chui thng. Genome cc virus h Papillomaviridae l mt phn t DNA hai si chui vng khp kn, phn t lng khong 5 kbp . Genome ca cc baculovirus (h Baculoviridae) rt ln, tng ng genome ca cc t bo cn trng, l mt phn t DNA duy nht hai si, trong c cha mt gen cho tiu n v ln ca enzyme ribonucleotide reductase (RR1). Genome ca cc hepadnavirus (h Hepadnaviridae) l mt phn t DNA mch vng, c vng gm 2 si, c vng ch mt si. Si DNA m l si y , c chiu di ton b l 3,0 - 3,2 kb c khc (nick) c hiu mt s ch. Cn si DNA dng th c di khng hon ton v khng n nh, thng t 1,7 n 2,8 kb. Genome ca cc orthomyxovirus (h Orthomyxoviridae) l RNA mt si gm 8 phn on (ring virus cm C c 7 phn on). Cc bunyavirus (h Bunyaviridae) c genome gm 3 phn t RNA mt si m c kch thc khc nhau (gi l cc RNA L, M v S). Ring RNA S ca chi Phlebovirus c mt on c c nng ca si dng, nn thng gi "RNA lng ngha (ambisense RNA"). Genome cc arenavirus (h Arenaviridae) c 2 phn t RNA mt si. Ngoi ra, bn trong virion cn c cc RNA 28 S, 18 S, 4 - 6 S c ngun gc t t bo k ch. Cc RNA 4 - 6 S cha RNA thng tin di genome (subgenomic mRNA) m ha protein Z c th l nhn t iu tit sao chp. RNA genome cng lng ngha (ambisense). Genome picornavirus (h Picornaviridae) cu to t 1 phn t RNA mt si dng chui thng, phn t lng 2,4 - 2,7 x 106 (khong 7,5 - 8,5 kb), c chui poly-A u 3', cn u 5' kt hp cng ha tr vi phn t protein VPg c phn t lng khong 2.400 Da. Genome ca cc birnavirus (h Birnaviridae) l phn t RNA hai si, gm hai on (on A c kch thc 3,1 kbp v on B 2,9 kbp). 3. Vt cht di truyn vi sinh vt nhn chun vi sinh vt nhn chun (eukaryote) vt cht di truyn cng c bn cht ha hc l acid nucleic DNA, RNA. Chc nng hai loi acid ny tng t t bo nhn s. DNA mang mt m thng tin di truyn trong khi RNA tham gia chuyn ha mt m ny thnh protein m th

202 hin tnh trng. Qu trnh t sao chp DNA cng l c s di truyn t th h t bo ny sang th h t bo khc hay th h ny sang th h khc. im khc bit ln gia sinh vt nhn chun v sinh vt nhn s l nhn chun c mng nhn v genome thng gm hai b (lng bi) hoc i khi nhiu hn nhim sc th ( thc vt: tam bi, t bi, a bi) cng cu to t DNA xon kp nhng khng khp kn (c u t do). Tuy nhin nhiu nm v thc vt cn tn ti chu trnh n bi, khi trong t bo nhn ch cha mt b nhim sc th. Th n bi ny nhiu nm tn ti song song vi th lng bi v dng t bo song nhn c hai nhn n bi ring r. cc nm chuyn hnh hon ton (holomorphous fungi) th lng bi thng l im khi u ca s hnh thnh c quan sinh bo t hu tnh t sn sinh cc bo t n bi. Cn cc nm chuyn hnh khng hon ton (imperfect fungi) khng c giai on lng bi. im khc bit na l, DNA nhim sc th nhn chun cn qun quanh cc phn t protein histone. T chc gen sinh vt nhn chun cn c s khc bit khc so vi sinh vt nhn s. Gen cu trc bao gm nhiu on v ch mt s on m ha cu trc protein. Trong qu trnh phin m, gen trn nhim sc th c sao chp thnh RNA thng tin s cp trong nhn t bo nhng tr thnh RNA thng tin thnh thc c hiu th RNA phi tri qua qu trnh bin i. Mt s on ca RNA thng tin s cp b ct b khi on RNA thng tin (trong qu trnh gi l splicing - "ct xn", hay "tch ghp"), ng thi u 5' c methyl ha, cn u 3' c b sung on di adenine (poly-A). Methyl ha u 5' lm cho RNA thng tin c th nhn bit i vi ribosome, cn qu trnh poly-A ha RNA thng tin gp phn y RNA ny ra khi l khng mng nhn m i vo t bo cht. Phn gen tng ng phn cn li ca RNA thng tin thnh thc l phn c biu hin thnh tnh trng nn c gi l exon, phn b ct b gi l intron. on DNA m ha RNA thng tin s cp c gi l mt n v gen. Cu trc gen khng lin tc ny ng vai tr quan trng i vi s bit ha t chc trong qu trnh sinh trng ca c th. Ngoi l c th gp l gen histone v interferon khng c intron. (Ngoi ra, genome nhim sc th ng vt bc cao cn c trnh t lp giu G+C u nhim sc th, gi l telomer, thng b ngn dn sau mi ln phn bo do on RNA mi khng c tng hp b p bng DNA, c th lin quan n qu trnh lo ha ca c th). Bn cnh genome nhim sc th ( trong nhn), cc eukaryote cn c h thng tn hiu di truyn khc l DNA trong cc ty th v lp th. B gen ny iu khin s tng hp mt s protein ca cc c quan ny v c bn cht tng t b gen sinh vt nhn s (DNA cng c cu to mch xon kp, khp kn) nhng c ct bt i nhiu. sinh vt

203 sinh sn hu tnh (hp t hnh thnh t giao t c v giao t ci) b gen ny mi c th u c ngun gc t m do trong qu trnh th tinh giao t c khng truyn c cc ty th (v lp th thc vt) vo giao t ci. Genome ca cc tiu c quan (ty th v lp th) c phin m thnh RNA thng tin tng ng vi qu trnh din ra tng t t bo nhn s. Gen nhy (transposon) l on DNA c bit gp c sinh vt nhn s v sinh vt nhn chun, khng c v tr n nh trong nhim sc th. on ny c th tch ri khi v tr vn c ca n v di chuyn n mt v tr khc ca cng nhim sc th hoc sang nhim sc th khc. Tuy khng phi l gen cu trc nhng transposon nh hng rt ln n qu trnh biu hin tnh trng. N c th khng ch hoc gii phng mt gen cu trc no dn n vic mt tnh trng hoc xut hin tnh trng quy nh bi gen . Qu trnh khng ch c th din ra khi mt gen nhy chn vo khong gia gen khi ng (promoter) v gen cu trc cng nh chn vo gia gen cu trc lm cho qu trnh phin m khng th khi ng hoc b gin on.

II. Di truyn v biu hin tnh trng


1. T sao hay ti to DNA (replication) Qu trnh tng hp DNA trong nhn t bo gn cht ch vi qu trnh bo ton v truyn t thng tin di truyn t th h ny sang th h khc. Nguyn l b sung cc baz trong chui xon kp DNA p ng qu trnh nhn i v ti bn chnh xc trnh t cc baz trong phn t gc hay DNA khun. Trong qu trnh ti to DNA, phn t ny c tch i thnh hai mch n. Mi mch n ca DNA gc c dng lm khun tng hp mt mch mi c cc nucleotide lin kt hyr tng b vi cc nucleotide ca chui gc. Phn t DNA, nh vy, c mt chui l chui gc cn chui th hai hon ton c tng hp mi. Do , c ch t sao DNA l c ch bn bo ton. y l qu trnh phc tp c s tham gia ca nhiu enzyme v protein khc nhau. c bit DNA sinh vt nhn s, do DNA xon kp, khp kn nn hai si phi xon bn v lng vo nhau khng th tch ri nn qu trnh t sao DNA li cng phc tp v c s tham gia ca nhiu yu t. V tr bt u qu trnh ti bn DNA c gi l im khi u ti bn, vng c mch gc c tch i gi l chc ti bn. S pht sinh mt chc ti bn t im khi u c gi l s khi u ti bn. Khi khi u ti bn enzyme topoizomerase (tc gyrase) c chc nng ct t mt si ca chui DNA v khi phc si ny li nhanh chng lm cho DNA xon kp c th tch i c m vn gi nguyn cu trc. Qu trnh tho dui xon ca chui i thnh chui n c s tham gia ca enzyme helicase. on mch n duy tr c

204 cng ca n l nh cc enzyme c bit gi l cc protein lin kt DNA mch n (ssDNA binding protein). Trong t bo cc nucleotide nguyn liu c tng hp sn di dng cc phn t cao nng nucleotide triphosphate (dATP, dTTP, dGTP v dCTP). Cc nucleotide ny khng th t lin kt vo chui DNA mt si d tha mn tnh tng b m phi cn cn c mt oligonucleotide (tc mt on Acid nucleic mch n) gi l mi (primer) gn kt mt cch tng b vi on DNA mt si c dui sn. on oligonucleotide mi ny thng l mt on RNA ngn c enzyme primase xc tc tng hp trc tip trn khun DNA v sau cung cp u 3'-OH cho mt nucleotide nguyn liu lp rp vo. Nh , mch ca chc ti bn lun duy tr c u 3'-OH t mt nucleotide mi khc lp rp vo. Qu trnh lp rp tip din di s xc tc ca enzyme DNA-polymerase III. Mi RNA sau c phn hy khi DNA khun v ch trng ny c lp y nh enzyme DNApolymerase I. Qu trnh tng hp DNA t chc ti bn, nh vy, tip din nh ni di u 3'-OH ca mi mch mi theo hng 3'5'ca si khun. Chnh v vy, qu trnh tng hp DNA trn mt si khun l lin tc, cn trn si kia (si i nghch song song vi si trc) qu trnh tng hp li din ra gin on, trong khi chc ti bn chuyn ng lin tc v mt hng. Mch c s lp rp lin tc gi l mch tin cn trn mch kia s tng hp li din ra theo hng ngc vi chiu chuyn ng ca chc ti bn gi l mch li hay mch gin on. Mt khi on khun mi c m ra th trn mch li ny li din ra s tng hp mt mi mi nh enzyme primase, ri t mi ny qu trnh lp rp cc nucleotide tng b vi trnh t ca mch khun li din ra. Sau nh tc dng endonuclease ca enzyme DNA-polymerase I m mi b loi b, v cng di s xc tc ca enzyme ny ch trng c lp bi s lp rp cc deoxyribonucleotide. Nhng on mch ny c gi l on Okazaki (gi theo tn ngi pht hin). Mi lin kt gia cc on Okazaki c kt ni sau nh enzyme ligase. Cc protein trong tp hp cc protein tham gia qu trnh ti bn DNA gi l replisome. Nh h thng ny m qu trnh ti bn DNA din ra vi tc cao ( E. coli khong 30000 50000 i nucleotide trong mt giy). sinh vt nhn chun qu trnh t sao DNA din ra theo qu trnh tng t nhng c ch c th phc tp hn v cn nhiu im cha r. DNA nhim sc th t bo nhn chun rt di v s khi u ti bn c th ng thi t nhiu im khi u ti bn v thng din ra theo c hai chiu. on DNA gia hai im khi u ti bn c gi l n v ti bn. Trung bnh di mt n v ti bn n 40000 bp. Khi bt u ti bn t im khi u ti bn, enzyme topoisomerase ct DNA mt si v nhanh chng khi phc kha t chui siu xon kp c tho xon v dui thng. Enzyme DNA-helicase tham gia vo tho dui xon

205 ca chui kp to mch n. Bn cnh cc DNA-polymerase, topoisomerase, helicase v primase, trong t hp replisome ca t bo nhn chun cn c cc protein khc gi l cc yu t ti bn A, B, v C,... (RFA, RFB, RFC,...) cng nh khng nguyn hot ha nhn t bo (PCNA). Cng pht hin c bn loi DNA-polymerase khc nhau, k hiu theo ln ca phn t lng l , , , v . DNA-polymerase , v pht hin thy trong nhn t bo cn DNA-polymerase th ch pht hin trong cc ty th v lp th. Qu trnh ti bn DNA nhn chun trn chc ti bn din ra tng t t bo vi khun. DNA-polymerase hot ng tng ng DNA-polymerase III vi khun: xc tc ko di qu trnh lp rp. S khi u mi on ti bn cng do enzyme primase m nhn, n tng hp mt on RNA trn khun DNA chui n v nh cung cp on chui i cho DNA-polymerase nhn bit v u 3'-OH cn thit cho qu trnh lp rp. Enzyme ny xc tc qu trnh lp rp trn c mch tin (mch lin tc) ln mch li (mch gin on). Tng t DNA-polymerase I vi khun, mch li qu trnh kh b cc RNA mi v lp ch trng ca mi bi deoxyribonucleotide do DNApolymerase m nhn. (Vai tr ca hai DNA-polymerase khc cn cha r). Cui cng vt khc gia cc on Okazaki cng c kt ni nh s xc tc ca enzyme ligase. Tc ti bn DNA ph thuc vo tc lm vic ca cc h thng ti bn trn mch li v qu trnh ti bn trn mch ny thng chm tr so vi mch tin. Bn cnh c ch ti bn ph thuc primase to mi ( sinh vt nhn s v nhn chun) nu trn cn c c ch ti bn DNA khc gi l c ch ti bn theo vng ln thng thy plasmid v c th din ra nhim sc th vi khun cng nh nhim sc th sinh vt nhn chun. plasmid hoc vi khun, chng hn, t mt im ct bi enzyme endonuclease trn mt si hnh thnh u 3'-OH v u 5'-P. u 5'-P tch dn ra khi vng v c tng hp gin on (c s tham gia ca primase), cn u 3'-OH c DNA-polymerase nhn bit v xc tc qu trnh lp rp khng cn mi RNA. sinh vt nhn chun ti bn kiu vng ln din ra trong trng hp khuych i gen. Khi mt mch n ca on gen ny b ct ti mt im, tch ra ri vng li, u 3'-OH hot ng nh mt mi cho DNA-polymerase tng hp lin tc nhiu vng on nhim sc th ny. Kt qu l sau ln ti bn DNA tip theo s phin bn ca gen tng cao trong thnh phn ca nhim sc th. cc virus RNA, qu trnh t sao genome c c ch a dng ty thuc loi virus DNA mt si hay hai si, virus RNA mt si hay hai si, hay virus c qu trnh phin ngc (hepadnavirus v retrovirus). Cn cc virus DNA hai si, nhn chung, c qu trnh sao chp tng t prokaryote v eukaryote. virus DNA c genome chui thng c cu

206 trc kp tc (hair pin) u qu trnh ti bn DNA c th theo c ch ti bn theo vng ln. Cc virus RNA hai si cng c qu trnh tng t DNA hai si nhng din ra trong t bo cht t bo k ch vi s xc tc ca enzyme RNA-polymerase ph thuc RNA v s sao chp RNA khng cn n on mi oligonucleotide. cc virus mt si (DNA hoc RNA), qu trnh ti bn genome u thng qua dng ti to (replicative form) l dng Acid nucleic (NA) hai si gm mt si NA virus v mt si NA tng b c tng hp trong t bo k ch (nhng khng c trong thnh phn virion). Si tng b tip tc lm khun tng hp ng lot cc si NA virus. Trng hp c bit xy ra vi cc virus thuc h Hepadnaviridae v h Retroviridae. Genome hepadnavirus gm DNA hai si mch vng, si m c cu trc kh y nhng c mt im khuyt thiu (khc) c hiu chi, cn si dng c di khng hon ton v khng n nh. Nh DNA-polymerase sn c trong virion, si dng khng hon ton c tng hp thnh si dng hon ton ( tng hp RNA thng tin). Cn si m lm khun tng hp si RNA dng. Sau , nh s xc tc ca enzyme RT (enzyme phin ngc - reverse transcriptase), hng lot si DNA m ca virus c tng hp t khun l si RNA dng. Nhng si DNA m ny li lm khun tng hp DNA dng nhng qu trnh ny cha kp hon thin th DNA hai si ny b nhi vo v capsid cng ng thi hnh thnh. cc retrovirus, genome l phn t RNA gm hai on c u 5' ging nhau. Di tc dng ca enzyme RT v mi l RNA vn chuyn chui DNA m tng b c tng hp. Sau khi chui RNA b phn gii si DNA m c dng lm khun tng hp si DNA dng tng b. Cu trc DNA hai si ny c cc on di trnh t lp u cc (LTR - long terminal repeat). Nh LTR ny m sau khi di nhp vo nhn DNA retrovirus ti t hp vo DNA nhim sc th t bo k ch (tr thnh tin virus - provirus). Provirus nh tn hiu sao chp trong LTR di tc dng ca RNA-polymerase ca k ch m tng hp RNA genome virus (ng thi vi vic tng hp cc RNA thng tin c kch thc khc nhau). 2. Phin m (Transcription) Phin m l qu trnh sao chp trnh t nucleotide trn DNA gen cu trc thnh trnh t nucleotide trn RNA thng tin (mRNA). Thng tin di truyn trn DNA nh vy c truyn t sang h thng tng hp cc yu t hnh thnh tnh trng. Qu trnh ny thng din ra trn chc ti bn DNA nn khi hon thnh qu trnh t sao DNA th t bo cng chun b sn lng ln RNA thng tin cn thit. ng thi, trn mt khun DNA c nhiu RNA thng tin c tng hp. V nguyn l tng

207 hp RNA thng tin cng theo nguyn tc tng b nucleotide. Mt adenyl ribonucleoside triphosphate s tm n lp rp tng b vi thymine trn khun DNA. Cc ribonucleoside triphosphate khc cng hot ng tng t: A b T, C b G, G b C nhng uracyl ribonucleoside triphosphate thay th thymyl ribonucleoside triphosphate gn kt tng b vi adenine trn khun DNA. Qu trnh phin m c xc tc bi enzyme RNA-polymerase v khng cn c s tham gia ca mi. Qu trnh phin m din ra trong bn giai on: s nhn bit on khi u, m u s hnh thnh chui, ko di chui v kt thc. RNApolymerase vi khun gm bn tiu phn: hai tiu phn ging nhau v hai tiu phn khc v 1. Enzyme nhn bit c im khi u khi c yu t . on khi u c trt t nucleotide giu A v T nn thng gi l trnh t ATAT (hay min ATAT) l mt vng ca min khi ng (promoter) trong cu trc operon (gm cc on gen cu trc vi cc gen iu ha (R) v gen iu hnh (O) phn b trc chng). Gn vo im khi u phin m, RNA-polymerase nh hng ti ng ch bt u tng hp RNA. im bt u qu trnh lp rp mt ribonucleoside triphosphate tng b c lp vo. Sau RNA-polymerase tip tc trt dc theo khun (chui n) DNA, nh cc ribonucleoside triphosphate tng b tip theo cng c lp vo chui RNA. Hng ko di ca mch lun l 5'3'. Khi chy n vng kt thc, RNApolymerase c tc ng vi yu t , v xy ra qu trnh kt thc phin m: c RNA-polymerase ln chui RNA c tch khi chui DNA khun. Trong nhiu trng hp vng kt thc phin m c cu trc c bit v thng l vng c cu trc lp ngc hng (palindrome) to nn cu trc thng lng hay cu trc un palindrome. vi khun c mt loi RNA-polymerase. Enzyme ny xc tc tng hp c ba loi RNA: RNA thng tin, RNA ribosome v RNA vn chuyn. Thng tin di truyn thng kt chui thnh mt n v phin m (operon): mt s gen cu trc (cistron) c phn b lin tip sau mt on gen khi u phin m (t hp R v O) v phin m cng nhau thnh mt RNA thng tin a cistron gm mt s RNA thng tin phn cch nhau bi mt on trt t nm khng m ha Acid amin gm khong vi chc nucleotide. sinh vt nhn chun c ba loi RNApolymerase I, II v III, theo trnh t tng hp RNA ribosome (I), RNA thng tin (II), cc RNA vn chuyn v cc RNA ribosome c phn t lng nh (III). Cc RNA thng tin sinh vt nhn s c s dng trc tip ngay trong qu trnh phin m tng hp protein trn b mt cu trc mng (mng t bo cht, rt c th l vng mesosome), nhng sinh vt nhn chun th RNA thng tin c tng hp trong nhn phi tri qua qu trnh thnh thc ha (processing) gm methyl ha u 5' v poly-A

208 ha u 3'-OH v ct xn hay tch ghp (splicing: vng intron ca gen cu trc b ct khi RNA thng tin ch cn li phn exon) tr thnh RNA thng tin thnh thc hay RNA thng tin dch m. Sinh vt c c ch iu ha qu trnh phin m cng nh dch m, bo m cho qu trnh s dng thc n cng nh tng hp cc hp cht nguyn liu ng ha mt cch tit kim. vi khun gen iu ha I, chng hn, tng hp protein iu ha c mt trong t bo vi liu lng thp c tc ng bao vy vng iu hnh (O) gy c ch sao m cc gen cu trc (iu ha m tnh). Khi c mt trong mi trng, yu t cm ng (nh lactose i vi Lac operon) lm cho protein iu ha thay i cu trc khng gian, mt hot tnh bao vy vng O. Vng O c gii ta cho php RNA-polymerase nhn bit v xc tc qu trnh tng hp cc RNA thng tin. Cc gen cu trc trong tp hp operon c phin m cng nhau thnh mt RNA thng tin a cistron, sau qu trnh dch m thnh cc protein trn ribosome tin hnh theo tng on ng vi cc gen cu trc. Bn cnh c ch iu ha m tnh nu trn, trong t bo cn c c ch iu ha dng tnh. Khi cht gi vai tr trng yu l adenosyl monophosphate vng (cyclic AMP - cAMP) tc dng gin tip qua protein c bit c tn l CAP (catabolic gene activator protein) kt hp vi v tr khi ng lm tng i lc ca RNA-polymerase dn n tng cng qu trnh phin m. Bn cnh hai c ch cm ng nu cn c c ch iu ha khc: c ch km hm. C cht nguyn liu c tng hp ra mt cch d tha (v d cc Acid amin) tham gia vo t hp km hm vng operator lm dng qu trnh phin m. 3. Dch m (translation) Dch m l qu trnh tng hp mch polypeptide ribosome trn c s khun RNA thng tin. Trnh t c b ba nucleotide (hay khung c b ba - triplet reading frame) quyt nh trnh t cc Acid amin trong mch polypeptide. Tham gia vo qu trnh dch m l mt h thng phc tp: RNA thng tin (mRNA), ribosome v cc Acid amin c hot ha bi RNA vn chuyn (tRNA) thnh dng aminoacyl-tRNA di s xc tc ca enzyme aminoacyl-tRNA-synthetase, mt lot cc protein khc v c bit l cn c s tham gia ca cu trc mng. vi khun cu trc mng tham gia vo qu trnh sinh tng hp protein l mng t bo cht m c th l vng mesosome. Trong khi cu trc mng c chc nng tng ng sinh vt nhn chun l mng li ni cht (endoplasmic reticulum). Mng li ni cht khng c ribosome bm ln gi l mng li ni cht nhn cn mng li ni cht c ribosome bm ln gi l mng li ni cht c ht. Ribosome ch hot ng khi bm ln cu trc mng. Nh vy (v cn nh cc cht to khun gi l chaperon) cc

209 protein c tng hp ra c c cu trc khng gian xc nh, phn b trong cu trc mng theo hng xc nh (u NH3+ hng ra ngoi mng t bo cht v hng vo pha trong ca mng mng li ni cht) v, nh , c chc nng xc nh. Ribosome cu to t hai tiu th c hng s lng khc nhau: 30S v 50S vi khun v trong ty th hay lp th, cn trong nhim sc th t bo nhn chun ribosome ln hn v cu to t tiu th 40S v 60S. Cc ribosome u c hai v tr gn tRNA: v tr A gn aminoacyl-tRNA v v tr P gn peptidyl-tRNA. RNA vn chuyn (tRNA) ch gm 70 - 90 nucleotide, cha nhiu baz nit ci bin (dihydroutidine, pseudoutidine, thioutidine, inosine, ribothymidine,...), c dng l ba ch vi cung v ba thy. u 5' ca cung l v tr gn Acid amin, cn thy gia l i m (anticodon). Qu trnh dch m bao gt cng bt u t mt methionine. vi khun tRNA vn chuyn methionine u chui cng khc vi tRNA vn chuyn methionine vo gia chui. Sau khi gn vi tRNA methionine u chui thng phi formyl ha (nh nhn gc formyl t Acid N10tetrahydrofolic) thnh formyl-methionine-tRNA. V vy, RNA vn chuyn methionine vo u chui v gia chui c k hiu ln lt l RNAfmet v RNAmmet. Trc ht nh s kch thch ca protein IF-3, mRNA lin kt vi ribosome 30S. Tip n t hp fMet-RNAfmet dng phc hp cao nng (fMet-RNAfmet-IF-2-GTP) c chuyn vo v tr P trn ht 30S ng vi codon m u UAG ca methionine. Tn hiu UAG ca methionine trn mRNA l ca methionine m u ch khi n nm gn trnh t Shine-Dalgarno (3 - 9 nucleotide cha trnh t trung tm GAG) trc . Nh tn hiu ny codon AUG m u c chuyn chnh xc vo v tr m u trn ribosome thay th v tr ca IF-3 trn ht 30S ca ribosome. Sau ht ny lin kt tip vi ht 50S thnh th ribosome 70S. ng thi, IF-2 (trong t hp hot ha vi IF-1) b y ra ngoi nh nng lng thy phn GTP. Kt qu l phc hp m u (70S-mRNAfMet-RNAfmet) c hnh thnh. Qu trnh ko di cn cc yu t ko di: EF-T v EF-G. Yu t EFT gm hai protein: Tu v Ts. Tu v GTP hot ha tt c cc Acid amin tham gia ko di chui polypeptide. T hp Acid amin hot ha c dng chung l aan-tRNA-Tu-GTP. Trc ht t hp ny gn vo v tr A vi codon tng ng, ri GTP b thy phn bi Tu v cng b y ra ngoi. T hp Tu-GTP nng lng cao c phc hi nh yu t Ts. Lin kt peptide th nht c to thnh nh hot tnh peptidyl-transferase ca ribozyme 23S trong thnh phn ht ribosome 50S. Nhm -COOH ca fMet v -NH2 ca Acid amin tip theo (aa2) tham gia phn ng trng ngng gii phng mt phn t nc, nhn in t (nng lng) t t hp

210 tRNA-Tu-GTP. dch m c tip tc fMet-aa2-tRNA c dch chuyn t v tr A sang v tr P ca ribosome, cn tRNAfMet b tch khi c hai lin kt: lin kt vi codon mRNA v lin kt vi methionine m tr nn t do. S chuyn ch ny cn yu t EF-G v GTP: EF-G thy phn GTP cung cp nng lng cho s chuyn ch ng thi y EF-G ra ngoi. sinh vt nhn chun Acid amin m u s dch m cng l methionine nhng khng cn formyl ha. Cc chui polypeptide c tng hp nh ribosome phi din ra trn cu trc mng. vi khun c cu ny ch yu l mng t bo cht to thnh np cun ca mesosome, cn sinh vt nhn chun chnh l mng li ni cht. V thc cht hai c cu mng ny u l sn phm ca qu trnh tng hp mng vn din ra lin tc trong t bo v cung cp phn din tch mng mi bao bc th tch ca t bo ang tng trng. Mng c hnh thnh ch yu nh qu trnh chuyn ha lipid trung tnh d tr thnh lipid phn cc (ch yu l phospholipid) to nn c cu mng n v (hay mng in hnh) hai lp c cc ui Acid bo hng vo nhau nh lc k thy. Mng n v c b sung cc phn t protein ang c tng hp trn ribosome bm dnh trn mng ny m tr thnh cu trc mng sinh hc c trng: cc phn t protein khc nhau hoc thm nhp sut dy ca mng, thm nhp ch pha trong hoc pha ngoi ca mng, hoc nm gia hai lp mng n v ph thuc vo tnh phn cc v lc k thy ca ton b hay mt phn cu trc phn t ca chng. C cu mng sinh hc t trng thi mng li ni cht chuyn dn ra ngoi ri dung hp vi mng t bo cht lm din tch mng t bo cht cng rng. Mt khc, cc protein tit xut cng c tng hp trn c cu mng v nh qu trnh dung hp mng tiu bo (th Golgi phnh rng) vi mng t bo cht m c tit xut ra ngoi t bo. Chnh nh nhng c cu ny m c th c nhng tnh trng c trng. vi khun, qu trnh hnh thnh mng bt u t mesosome v qu trnh tng hp protein cng din ra ng thi. Cc phn t protein c tng hp tri dt dn trong cu trc linh ng ca mng. cc t bo ng vt hay thc vt b cm nhim virus, cc qu trnh tng hp protein virus v hnh thnh mng ca t bo cng din ra ng thi theo phng thc tng t. V vy, nu pht trin virus lun l xut cc khng nguyn ca mnh trn b mt t bo k ch trc khi gii phng ra khi t bo.

III. Sa cha DNA


T bo c c cu sao chp thng tin di truyn hon thin nh c ch sao chp bn bo ton v lp rp nucleotide tng b vo khun mch n. Tuy vy, c ch ny cn c b sung thm c cu sa cha DNA.

211 C th gp ba c ch sa cha DNA l sa cha trc tip, sa cha ct baz v sa cha ghp i sai. Sa cha trc tip thng gp khi trn DNA hnh thnh dimer thymine, l kt qu ca s chiu x t ngoi vo la cy t bo. Trong c th c mt loi enzyme gi l photolyase m trch vic sa sai ny. Trong thnh phn c FADH2 v 5,10-dimethyl tetrahydrofolate, photolyase c hot ha bi nh sng nhn thy. Khi n c th nhn bit cu trc dimer thymine, phn gii dimer ri tch khi DNA. V d khc v sa cha trc tip l sa cha phc hi guanine b methyl ha trn nguyn t O6. O6-methyl guanine (O6MG) hnh thnh khi t bo pht trin trong mi trng c yu t ankyl ha. Khi sao m, O6MG c khuynh hng ghp i vi T hn l vi C, v vy gy t bin GC thnh AT. Enzyme DNA-methyl transferase xc tc loi b nhm methyl khi O6MG lm phc hi guanine bnh thng trong chui DNA. (Do nhm methyl lin kt cht vi Acid amin cystine ca trung tm hot ng ca enzyme nn enzyme b v hot sau phn ng sa cha DNA). Sa cha trc tip cn nh cc exonuclease l nhng enzyme ct DNA t hai u. Sa cha ct baz c thc hin nh enzyme DNA-glycosylase. Enzyme ny nhn bit nhng ch h hng ph bin trn DNA nh adenine v cytosine khuyt thiu amin hoc guanine v adenine methyl ha. Cc enzyme ny ct b nucleotide sai bng cch ct b mi lin kt glycosyl gia baz nit v nguyn t C 1' ca deoxyribose. Kt qu l to nn nhng im khuyt thiu purine hoc pyrimidine, k hiu l AP (apurinic/apyrimidineic site). Sau khi AP xut hin cc enzyme APendonuclease ct b deoxyriboso-5'-phosphate st li, sau DNApolymerase I tng hp DNA b sung v ligase hn ch trng trong mch. Bn cnh sa cha im nu trn cn c qu trnh sa cha ghp i sai. H thng sa cha ny E. coli, chng hn, c t nht 9 protein. S phn bit si da vo tc dng ca enzyme dam-methylase methyl ha DNA cc v tr N6 ca tt c cc adenine (*A) gp trong trnh t 5'GATC hay enzyme dcm-methylase methyl ha cytosine (*C) nm k adenine hoc k thymine trong trnh t CCATGG hay CCTAGG, hay ng vt bc cao mt s cytosine trong trnh t CG. Sau thi im sao chp ch si khun c methyl ha cn si mi cn cha methyl ha. Nh t bo phn bit c si c v si mi. Trong sa cha DNA, cc protein MutS, MutH v MutL ng vai tr quan trng. MutS lin kt cc cp baz ghp i sai, MutH lin kt vi trnh t GATC, cn MutL kt hp vi c hai protein kt hp DNA ni trn thnh mt phc hp lm cho phn t DNA un cong trong khong di 1000 bp pha u 5'

212 ca trnh t GATC. Khi , nu mt trong hai mch khng c methyl ha MutH s tc dng nh mt endonuclease phn gii si khng c methyl ha pha 5' ca guanine thuc GATC. Si khng methyl ha s c tip tc b ci xon v b phn gii theo hng 3'5' t v tr nh du n v tr ghp sai ri c thay th bng si DNA mi vi s xc tc ca h thng replisome gm helicase II, protein SSB, exonuclease I, polymerase III v ligase.

IV. C ch chng s xm nhp ca gen l (hn ch v ci bin)


T bo c c ch t sao chp v sa sai bo m cho vt cht di truyn n nh. Tuy nhin trong nhiu trng hp t bo chu s xm nhp ca gen l, nh cc phage xm nhp vo t bo vi khun. Khi t bo cn c c ch khc chng li s xm nhp ca DNA t ngoi vo. S hn ch (restriction) ny l nh cc t bo k ch c h thng enzyme nhn bit v ct cc DNA l (gi l cc enzyme hn ch - restriction enzyme) trong khi cc DNA t bo ch khng b ct l nh s ci bin (modification). Hn ch v ci bin ph bin vi sinh vt v c nghin cu k vi khun. Mt mt chng c tc dng nh du DNA t bo, mt khc phn hy DNA l. H thng gm hai hot tnh enzyme trn cng mt phn t protein hay trn hai protein ring r: hot tnh endonuclease v methyltransferase. Cc endonuclease ny nhn bit mt trnh t c hiu di ngn khc nhau ty loi enzyme v phn ct DNA ny ti v tr hoc cch xa im nhn bit . Do DNA ca mnh c methyl ha ti cc im adenine (ti N-6) v cytosine (ti N-5 v N-4) bi hot tnh methyltransferase tc dng phn ct ca hot tnh endonuclease b tr ngi. Cc endonuclease u ct DNA si i v c th chia thnh hai loi. Loi I nhn bit trnh t DNA c bit v ct DNA ny xa v tr nhn bit. Endonuclease ca phage P1 thuc loi ny. Loi II nhn bit v phn ct DNA trong trnh t nhn bit l mt vng c trnh t i xng quay kp (trnh t i xng o v hay trnh t lp ngc hng palindrome). Do phn ct ti nhng im c hiu to ra nhng mnh DNA xc nh nn enzyme hn ch loi II c s dng trong k thut ti t hp v to dng gen. Bng di y gii thiu mt s enzyme hn ch. Ch mt s enzyme hn ch (Eco RI, Hha I) ct DNA hai si to ra u gm mt s nucleotide mt si (u dnh - cohesive), trong khi mt s enzyme hn ch khc (Ba II, Hae III) ct DNA hai si to ra u bng (u ty).

213
Bng 10.3: Mt s enzyme hn ch, dng vt ct v ngun gc ca chng Enzyme Eco RI Hha I Ba II Hae III Bam HI Hind III Kpn I Mbo I Trnh t im nhn bit 5'-GAA*TTC 5'-GC*GC 5'-TGGC*CA 5'-GG*CC 5'-GGATCC 5'-AAGCTT 5'-GGTACC 5'-GATC Ngun gc Escherichia coli RY 13 Haemophilus haemolyticus Brevibacterium albidum Haemophilus aegypticus Bacilus amyloliquefaciens H Hemophilus influenzae Rd Klebsiella pneumoniae Moraxella bovis

Hnh 10.3 : Hai dng u ct ca enzyme hn ch loi II: a) ct hnh thnh u ty v b) ct hnh thnh u dnh.

V. Bin d
1. Bin d kiu hnh Bin d kiu hnh cn gi l thng bin, l hin tng c s khc bit v cu trc cng nh c im pht trin gia th h con ci so vi th h b m cng nh gia chng vi nhau di tc ng ca mi trng v khng lin quan n s thay i vt cht di truyn. y l nhng bin d tm thi, thun nghch v khng n nh ca ton b qun th sinh vt. Nhng bin d ny xut hin chm v mt i khi cc yu t lm xut hin chng mt i. Bn cht ca bin d kiu hnh l hin tng tri - ln ca gen sn c trong t bo di tc ng cm ng ca mi trng. Khi mi trng mt i yu t cm ng th tnh trng tng hnh thnh cng tr nn khng biu hin na (gen tr nn ln). Bin d kiu hnh, nh vy, c nhng tnh cht sau: tnh tm thi (khng n nh), tnh thun nghch v tnh ton b. vi khun, bin d kiu hnh c th biu hin di mt s hnh thc. Bin d hnh thi trong qu trnh sinh sn thng xy ra bin d v kch thc ca t bo. T bo vi khun E. coli nui cy c 5 - 6 gi trong mi trng v iu kin thch hp s di ra trc khi phn bo. Tri

214 li chng s tr nn nh hn khi vo trong giai on pht trin ti a, v c kch thc ti thiu khi mt kh nng sinh sn. Hoc bin d hnh thi di nh hng ca ngoi cnh nh cu trc ha hc ca mi trng lm cho vi khun c nhng hnh thi khc nhau, vi khun tr nn mp hn nu c nui cy trong mi trng giu cht dinh dng, tri li hnh thnh nhng dng t bo mnh hoc dng cu bt thng khi nui cy trong mi trng ngho cht dinh dng. Sc cng b mt ca mi trng cng nh Acid (pH) mi trng tng lm hnh thnh nhng vi khun mp hn, ngn hn. nhit 37 C Brucella, chng hn, c dng rt ngn, hnh thoi hoc dng cu khun nh trong khi nui cy nhit thp hn t bo thng di, mnh. Nhng cht c ch nng thp c kh nng bin i hnh thi ca vi khun. Tng t, nhiu nm gy bnh thng biu hin hai dng hnh thi khc nhau, thng gi l nm nh hnh. Chng c dng hnh cu (dng nm men) khi trong ni quan ng vt mc bnh nhng c dng si khi nui cy trn mi trng nui cy nhn to hay mi trng ngoi, v d, Aspergillus fumigatus, Coccidoides immitis, Histoplasma farsimosa,... Bin i hnh thi do thng bin cn biu hin dng khun lc. Nhng vi khun cng loi khi pht trin trn mi trng c c th hnh thnh khun lc lng (dng S) hoc nhm (dng R) hoc nhng dng trung gian. Bin d kiu hnh cn th hin hin tng dung np thuc v kh nng hnh thnh enzyme s dng cc yu t mi trng. Tip xc lu ngy vi nng thp cht khng sinh c th dn n vic hnh thnh nhng chng vi khun khng thuc khng sinh do gen ln c cm ng tr nn tri (mc d trong a s trng hp s xut hin tnh khng vi thuc khng sinh l do t bin hoc vn chuyn v ti t hp thng tin di truyn). Trong trng hp thng bin khng thuc cc gen tng hp yu t khng (thng l enzyme phn gii thuc khng sinh hoc cc hp cht lm thay i cc l khng trn mng t bo cht) t trng thi b c ch c cm ng m chuyn thnh dng hot ng tc tr thnh khun tng hp cc protein tng ng. 2. Bin d kiu gen Khi nim bin d kiu gen sinh vt a bo thng c coi l ng nht vi khi nim t bin (mutation). Tuy vy, vi sinh vt, thng l n bo, th khi nim bin d kiu gen phi c hiu rng hn v nh l s hnh thnh cc bin chng c th l kt qu ca t bin (bin i cu trc genome sn c) hoc ca qu trnh tip nhn vt cht di truyn t bn ngoi (thng tip theo sau l qu trnh ti t hp thng tin di truyn vo genome nhim sc th).

215 2.1. t bin t bin l s hnh thnh cc c th c nhng chi tit (cu trc, pht trin) khc bit so vi th h cha m lin quan n s bin i vt cht di truyn. Bin d ny l nhng bin i t ngt, xut hin mt cch ngu nhin, khng d tnh trc c v khng lin tc mt s c th him hoi (tn sut xut hin vi khun: 10-8 - 10-6) ca mt qun th vi sinh vt. Chng tng i c lp vi mi trng chung quanh, c tnh c nh, tnh bn vng v tnh di truyn. Tnh gin on ca t bin l s thay i xy ra mt cch hon chnh trong mt thi hn, thng l mt ln phn bo. Tnh him ca t bin l hin tng tt c cc t bin u xy ra vi tn sut ht sc thp v xy ra khng u. Trong nhng iu kin nht nh xc sut xut hin t bin no c biu din nh l t s t bo hnh thnh t bin trong tp on vi khun gia hai ln phn chia t bo. Tn sut ny vi khun khong 10-7 (10-8- 10-5). Tnh vng bn ca t bin l s xut hin mt bin chng nu tn ti c trong iu kin ngoi cnh xc nh th tnh trng mi c duy tr v truyn sang th h tip theo. Mc d vy, trong nhiu trng hp c th xut hin nhng t bin bin o (conversion). Bin o khng nht thit c cng tn sut xut hin ca t bin ban u nhng chng cng him, gin on v di truyn. Tnh ngu nhin ca t bin c ngha l s hnh thnh bin chng t bin trc khi c nhng yu t chn lc tc ng n, mc d cng c nhng t bin do cm ng vi cc yu t chn lc. Trong nghin cu vi khun, kim tra s hnh thnh mt bin chng no ngi ta phi s dng yu t chn lc tuyn chn nhng dng vi khun c thuc tnh khc bit nh km hm nhng vi khun khng c thuc tnh khng cht chn lc. Th nghim ca Lederberg (1952) vi mt chng E. coli pha long phn nh ra ng nghim v pha long m khng phn nh (vn trong bnh ln) nui cy chun b trc khi cy ln a thch mi trng c cht cm ng chn lc (streptomycin) chng minh rng t bin xy ra ngay c khi khng tip xc vi yu t chn lc (cht khng sinh). Tnh c hiu ca t bin cn hiu l tnh c lp ca t bin. Mt tnh trng mi xut hin thng khng nh hng n s hnh thnh tnh trng t bin khc. Tn sut xut hin mt t bin kp (v d, t bin khng c penicillin v streptomycin) l tch s cc tn sut tng ng. V d, tn sut xut hin bin chng khng mt cht khng sinh l 10-7 v tn sut xut hin bin chng khng cht khng sinh khc l 10-

216 th tn sut xut hin mt bin chng khng c hai thuc trn l 10-15. Nh vy, phi hp hai thuc trn c th lm gim kh nng chn lc nhng bin chng khng thuc hn l s dng cc thuc khng sinh ring r. Nguyn nhn t bin c th l do cc yu t ha hc v vt l ion ha hoc gn kt ha tr vi cu ni gia hai chui monomer ca DNA xon kp (tia t ngoi to dimer thymine, actinomycin D, ethidium bromide, bromouracine, 2-aminopurine, ethyl- hoc methylmetasulfonate, dimethyl- hoc diethylmetasulfonate, N-methyl-N-nitroN-nitroso-guanidine, ethyleneimine, acid nitr,...). Tuy nhin, trong nhiu trng hp kh c th pht hin v kim sot c cc nguyn nhn gy nn nhiu t bin. Ngi ta gi chng l t bin do ngu nhin. Bn cht ca t bin: B nucleotide ca DNA thuc mt gen l mt mt m thng tin, n tng ng vi cu trc mt protein c hiu. Mi thay i ca b ny dn n s thay i cu trc v chc nng ca protein , hnh thnh mt tnh trng t bin. t bin l nhng thay i tm phn t. Mi t bin d l ngu nhin hay do cm ng th s thay i ca b nucleotide c th xy ra hai kiu: thm, bt hoc thay th mt i baz bng mt i baz khc (t bin im) hoc thm, bt hoc thay th mt on DNA bng mt on DNA khc (t bin on). Mc d t bin on thng kh gp, nh hin tng khuych i gen, nhng do tnh ph bin ca t bin im nn ngi ta thng ng nht khi nim t bin im vi t bin. t bin ny thng khng bin o. t bin im tuy nh nhng thng gp phn quan trng trong qu trnh tin ha. Khi mt baz b mt hoc c chn thm vo trnh t ca gen cu trc s xut hin s thay i mnh m trong cu trc protein do thay i ton b trnh t Acid amin trong thnh phn chui polypeptide k t v tr thm im hay mt im (c theo hng 5'3' ca RNA thng tin). Bin i ny c gi l s chuyn dch khung (frame shift). Trong a s trng hp, c bit t bin im v tr u 5' ca si dng ca gen cu trc, cng l u 5' ca RNA thng tin, nhng bin chng ny thng mt kh nng sng nu bin d xy ra trn gen thit yu (house-keeping genes) ca sinh vt do cu trc (ha hc cng nh khng gian) protein thay i dn n mt chc nng vn c (cu trc, enzyme, kch thch, th th,...) ca n. t bin th im (GCAT v ngc li) ngc li l t bin thng gp nhng t biu hin ra bn ngoi v thng kh pht hin. Khi DNA gen c phin thnh RNA thng tin, im t bin th im khng lm thay i khung c ca phn ln chiu di ca chui RNA thng tin. V vy thnh phn ca chui polypeptide ch thay i ti mt v tr tng ng vi im t bin trn gen. Khi , nu Acid amin mi khc nhiu
8

217 so vi Acid amin nguyn thy v thuc tnh phn cc, tnh i thy v tnh phn ng th tnh cht nguyn thy ca protein b thay i dn n chc nng c ca protein b mt. Hin tng ny biu hin trm trng nu acid amin thay th nm trung tm hot ng ca mt enzyme thit yu, kt qu c th l bin chng khng th tn ti. Trong trng hp Acid amin thay th khng c s khc bit nhiu so vi acid amin nguyn thy hoc t bin to mt m thoi ha (nhiu m b ba nucleotide cho mt acid amin) th t bin khng biu hin tnh trng v tch ly ln trong qun th. y thng l nguyn nhn ca s hnh thnh loi mi trong qu trnh tin ha. Chng loi t bin thng gp kh phong ph. C th gp t bin hnh thi nh t bin dng khun lc, thay i kh nng sinh sc t hoc kh nng hnh thnh nha bo. c bit c th gp cc t bin khuyt dng hay t dng (auxotrophic mutants): cc chng t bin ny mt kh nng pht dc nu trong mi trng thiu mt yu t no . Ngc li, mt s t bin dn n vic tng kh nng tng hp cc yu t sinh trng hay cc hp cht khc nh Acid amin,... (ng ngha vi vic mt kh nng iu tit sn xut tha). Cc t bin khc cng thng gp vi khun l t bin thay i kh nng ln men, kh nng s dng cc ng, t bin gy vng bn i vi cc nhn t c ch dn n thay i c lc, thay i tnh khng nguyn hoc s mn cm i vi khng sinh hoc tia t ngoi, v.v... 2.2. S vn chuyn v ti t hp thng tin di truyn C ba c ch vn chuyn di truyn khc nhau vi khun: Bin np (cn gi l chuyn th - transformation), ti np (transduction, cn gi l chuyn np) v tip hp (conjugation, cn gi l tip np hay giao phi). Nhng c ch u c c im l vn chuyn di truyn theo mt chiu t t bo ny sang t bo khc m khng c chiu ngc li, v t bo cho (donor) ch vn chuyn mt phn di truyn, tr mt vi ngoi l t gp trong nhng trng hp vn chuyn nhim sc th do s tip hp. Mi c ch vn chuyn ny u c c tnh ring quyt nh s truyn nhng yu t di truyn t t bo cho sang t bo nhn. Trong qu trnh bin np cht liu di truyn l nhng mnh DNA t do. Hin tng chuyn np l do cc phage a mnh nhim sc th t mt t bo phage k sinh v dung gii trc y cho mt t bo khc. Khc vi s vn chuyn di truyn trong bin np v ti np, hin tng giao phi l s phi hp gia hai vi khun c gii tnh khc nhau, hay l s vn chuyn t vi khun c sang vi khun ci. 2.2.1. Bin np (Transformation)

218 Bin np (hay chuyn th) l s vn chuyn thng tin di truyn nh s xm nhp ca cc phn t DNA t do (c ngun gc t t bo cho phn gii) vo t bo nhn lm cho genome ca t bo nhn thay i. Kt qu ca bin np ph thuc vo mc tng ng trong cu trc phn t DNA ca t bo cho v t bo nhn. V vy s bin np thng c kt qu hn trong gii hn mt loi c cc c th c nhng tnh trng khc nhau. iu kin bin np: Qu trnh bin np ch xy ra trong khi cc vi khun nhn mt tnh trng sinh l c bit ( khong 1 - 15% t bo qun th, thng thng giai on pht trin gim). Tnh trng ny lm thay i cc tnh cht b mt t bo nhn c cc yu t cm ng enzyme c bit trn cc on phn t DNA c th xm nhp c. Trn b mt t bo nhn c cc yu t cm ng cho on DNA c th c nh. C chng 30 - 70 vng cm ng nh th. on DNA nhp vo t bo c tch i, v mt mch n c ghp vo nhim sc th ca t bo nhn. Qu trnh ny c gi l s ti t hp (recombination). Tn sut ti t hp ph thuc vo c tnh cc on nhim sc th khc nhau, vo s tng ng cc phn t DNA. Bin np t nhin thng din ra h Enterobacteriaceae. Trong iu kin th nghim tn sut bin np tng cao nh t bo c x l lm thay i mng t bo cht (nh x l polyethylene glycol) v gy sc nhit (chuyn t bo t nhit 0C sang nhit 40 - 50C trong vng 50 - 60 giy). Griffith l ngi u tin pht hin s bin np ph cu khun (Streptococcus pneumoniae, tn c l Diplococcus pneumoniae hay pneumococcus) vo nm 1928. Vi khun ny c hai dng khc nhau. Dng gy bnh cho ng vt hnh thnh khun lc lng k hiu l S. Dng khng gy bnh hnh thnh khun lc nhm k hiu R. Nu tim vi khun sng chng S th lm chut nht cht, tim S cht do qua x l nhit (h nng) th chut vn sng, cn tim vi khun chng R sng cho chut th chut vn sng, nhng tim R sng ng thi vi tim S cht li lm chut cht v sau trong mu chut cht phn lp c chng S c trng. Cc th nghim sau (Avery, MacLeod v McCarty, 1944) cho thy nu x l dch S cht c tinh ch vn c nng lc bin np in vitro v nu x l bng DNase th lm mt hot tnh bin np ca dch ny trong khi x l bng protease vn gi nguyn tnh bin np. iu chng minh rng DNA, ch khng phi protein, l vt cht mang thng tin di truyn. Cho n nay, nhng tnh trng c bin np c bit l kh nng tng hp yu t sinh trng, kh nng s dng ng sinh trng, kh nng khng thuc, kh nng sinh c tnh,... Hin tng ny c vn dng rng ri trong k thut di truyn.

219 2.2.2. S ti np (chuyn np - transduction) Ti np l s vn chuyn vt cht di truyn nh phage (bacteriophage hay thc khun th) l cc virus k sinh vi khun. C hai loi phage. Cc phage c lm tan vi khun k ch c hiu trong khi cc phage n ha khng lm tan vi khun. Tuy vy, cc phage n ha c hai giai on thay th nhau trong chu trnh pht trin, chng c th tr thnh phage c khi mi trng c tc nhn (ha, l) gy t bin. Khi phage hp bm ln mng t bo vi khun c hiu bng th th lng ui, ATP-ase ng ui ca phage c hot ha phn gii ATP gii phng nng lng lm co rt v ui, kt qu l ng ui chc thng mng t bo cht vi khun v lm DNA phage theo ng ui ny xm nhp vo t bo cht vi khun. Sau khi xm nhp DNA, phage c tn ti c lp trong t bo cht, iu khin qu trnh tng hp protein phage, DNA phage v lm phn hy DNA ca vi khun. Trong qu trnh lp rp v protein phage thng bao bc DNA phage nhng trong nhiu trng hp v protein phage li cha mnh DNA vi khun (v to thnh phage khuyt tn hay phage thui: abortive phage). Khi gii phng ra ngoi cng cc phage khc do dung gii t bo, phage khuyt tn ny li tip tc hp bm ln t bo mi v a on DNA ca t bo c vo t bo mi. mt s phage c gi l phage n ha, sau khi xm nhp vo t bo cht vi khun, DNA n nhp vo cu trc phn t DNA nhim sc th ca vi khun dng tin phage (prophage). trng thi episome ny b gen ca phage n ha c truyn cho cc th h sau ca vi khun tng t cc gen khc ca nhim sc th. Cc vi khun mang prophage c gi l vi khun dung nguyn (lysogenic bacteria) do nhng vi khun ny mang mm mng ca qu trnh dung khun sau ny, khi chuyn sang trng thi c. Vi khun dung nguyn tip tc sinh trng bnh thng, tuy nhin khi gp iu kin mi trng c cc yu t gy t bin th phage chuyn sang trng thi c lm vi khun dung gii. Khi prophage t sao chp thnh nhng phin bn trng thi c lp trong t bo cht v bt u chu trnh dung gii vi khun. Trong qu trnh sao chp cc phage mi hnh thnh c th mang theo DNA ca phage v on DNA (nm k bn) ca nhim sc th vi khun. Khi xm nhp vo t bo mi, cc phage mang gen nhim sc th thc hin chc nng vt mang cht di truyn t t bo cho (k ch trc) sang t bo nhn (k ch sau). S ti np ph bin rng ri v c nghin cu k vi khun h Enterobacteriaceae. E. coli l cc phage T v P-1, Shigella P-1, Salmonella typhimurum P-22. Cc phage ti np Staphylococcus, Pseudomonas v Proteus cng bit n. Qu trnh ti np c th vn chuyn mt hoc mt s gen bt k nh nu trn c gi l ti np

220 chung hay ti np khng c hiu (nh ti np do phage P-1 v P-22 E. coli, Shigella v Salmonella. Nhiu phage ch c th ti t hp vo mt v tr (locus) nht nh trong genome k ch. Chnh v vy chng ch c th vn chuyn nhng gen phn b gn locus ny m thi. Hin tng ny gi l ti np c hiu. Phage lambda () E. coli, v d, ch vn chuyn nhng gen nm k locus galactosidase (Gal) l v tr phage ny ti t hp vo genome k ch. Tng t, phage -80 cng E. coli c th ti t hp vo gn locus iu khin tng hp tryptophan v sau ti np yu t ny mt cch c hiu. Khi nghin cu s ti np Salmonella ngi ta cn pht hin c s ti np khuyt (abortive transduction). Chng hn, trng hp mt on nhim sc th iu khin tng hp cc purine c phage ti np sang t bo nhn khng ti t hp vi nhim sc th m tn ti t do trong t bo cht dng gen ngoi (extragene) v khng t sao chp. Khi vi khun tr nn c kh nng pht trin trn mi trng khng c hp cht ny v th hin kiu hnh l cc khun lc nh mc trn mi trng tng hp khng cha purine. L do ca qu trnh ny l gen tng hp purine khng sao chp c trong mi ln vi khun phn bo nn ch mt t bo vi khun duy nht mang tnh trng tng hp purine trong qun th ( pht trin v c th h tr t bo bn cnh). Mt s phage n ha khi lm dung nguyn ha vi khun c kh nng lm thay i cc khng nguyn vi khun nhn. Khi ti np, mt on nhim sc th iu khin tng hp khng nguyn thn c truyn theo iu khin tng hp khng nguyn thn t bo nhn, sau khi c n nhp vo t bo nhn (vn khng c khng nguyn ny). Hin tng ti np on gen iu khin c t c pht hin Corynebacterium diphtheriae. loi vi khun ny ch c nhng t bo dung nguyn c kh nng tng hp ngoi c t biu hin c tnh: gy st v cc hin tng bnh l khc ngi (bnh bch hu). Ni chung, vi khun chuyn np ng vai tr trong lan truyn c tnh, lan truyn khng nguyn thn (gy phn ng cho min dch) v nng cao kh nng tng hp cc cht yu t sinh trng. 2.2.3. Tip hp (hay ti np, giao phi - conjugation) Tip hp l qu trnh vn chuyn thng tin di truyn bng cch tip xc trc tip cc t bo cho v nhn nguyn vn v mt sinh l. Qu trnh truyn vt cht di truyn ch din ra mt chiu t t bo c (F+) sang t bo ci (F-). iu ny c Lederberg v Tatum chng minh ln u tin vo nm 1964 trn cc bin chng E. coli K-12. Kh nng cho ca t bo c quyt nh bi s c mt ca yu t di truyn ngoi nhim sc th l

221 F (fertility factor, sex-factor, yu t gii tnh). Cc thut ng "t bo c" v "t bo ci" c dng theo h thng nh danh hin i t nm 1976. Trn b mt ca t bo F+ c cc nhung mao c bit - pili gii tnh, c th hp th cc phage c bit. S lng pili gii tnh rt t (khong 2 3). ng knh trong khong 25c th cho php si DNA ca t bo i qua khi n tip xc vi t bo nhn. Yu t ngoi nhim sc th iu khin qu trnh tip hp gi l cc plasmid tip hp (conjugative plasmid). Nhng plasmid ny c th iu khin s vn chuyn DNA ca chnh n hoc nh hng vn chuyn nhim sc th khi n nm trng thi lin kt vi nhim sc th . Ngoi yu t gii tnh F cn c cc plasmid tip hp khc nh plasmid iu khin s khng ca vi khun i vi mt s thuc (plasmid R). Plasmid iu khin tng hp colicin (col j, col v2, colv 3), plasmid iu khin tng hp enterotoxin E. coli, plasmid Hly iu khin s tng hp hemolysin, hemotoxin, c plasmid c t vir, cng nh plasmid iu khin tng hp khng nguyn K-88 (tc F4), K-99 (tc F5) v mt vi plasmid khc. Qu trnh tip hp ca plasmid F ti t hp trong DNA mch vng ca nhim sc th bt u t khi hai t bo c v ci tip xc nhau. Khi , mt mch ca on nhim sc th k bn yu t F b ct v dui ra m tr nn c cu trc thng. u nhim sc th t do ny tch ra v t sao thnh hai si, hng v pha pili gii tnh v c chuyn theo pili ny sang t bo F-. Bi v plasmid F nm cui chui thng nhim sc th nn n khng c truyn qua t bo F- v trong qu trnh tip hp ngn ngi DNA nhim sc th thng b t on, v nh ch mt phn nhim sc th ca t bo F+ c chuyn sang t bo F-. Nhng chng c tn sut bin np gen nhim sc th cao c gi l chng Hfr (chng c tn sut bin np cao). Trong trng hp pili gii tnh t do trong t bo cht, hai hin tng c th gp l bin np mt phn genome plasmid F hoc bin np ton b genome plasmid. Trong trng hp u ch mt s gen ca plasmid F c chuyn sang v c th ti t hp vo nhim sc th t bo nhn. Trong trng sau, t bo nhn tr thnh t bo F+. Hin tng ny c gi l s hng ha ("c ha") t bo ci. 2.3. Tng tc cc virus Tng tc cc virus l hin tng thng gp v gp phn thay i tnh trng cng nh kh nng pht trin ca virus khi trong mt t bo ng thi cm nhim mt s virus. Hin tng ny c th lin quan n kiu gen nhng trong nhiu trng hp cc virus ch xy ra lin quan n kiu hnh. Tng tc kiu gen c th l: 1) ti t hp, hot ha ng

222 lot, d hp ha, chuyn v v ti hot ha cho. Tng tc kiu hnh c th l pha trn kiu hnh, ti hot ha phi kiu gen, tng b, kch thch v cn nhim (hay can thip cm nhim). Ti t hp l s trao i vt cht di truyn gia cc th virus khc bit nhau v mt s tnh trng cng pht trin trong mt t bo. Kt qu l hnh thnh nhng th lai mang mt phn tnh trng ca chng gc ny v mt phn tnh trng ca chng gc khc. virus ng vt xng sng hin tng ti t hp ch din ra khi cc chng virus gn gi cng cm nhim mt t bo v tn sut xut hin ph thuc vo chng loi h thng t bo k ch. Ti t hp l c tnh chung ca mi virus nhng vic pht hin c th lai l kh khn v ph thuc vo nhiu iu kin. Tn sut lai cao din ra mt s virus RNA (orthomyxovirus, reovirus, oncornavirus) v tt c cc virus DNA hai si. Ti t hp c th truyn hng lot tnh trng: hot tnh ngng kt hng cu, tnh khng nhit v cht c ch, c tnh i vi ng vt, hot tnh sinh sn trong phi g, hot tnh enzyme v min dch. C th xy ra, chng hn qu trnh ti t hp gia cc virus ng vt v ngi trong th nghim in vitro cng nh in vivo, iu ny gii thch s xut hin dch cm ngi cng nh kh nng ngi mc bnh cm ng vt do cm ng vt c th tip nhn mt s tnh trng (nh mang th th i vi t bo ngi) bo m tip tc xm nhp v pht trin trong t bo ngi. Bn cht ca qu trnh ti t hp gen virus c th l s trao i tng on v trao i trong on kt qu l th lai mang thng tin di truyn ca c hai chng gc. Hot ha ng lot l qu trnh thng din ra trong mt t bo cm nhim ng thi nhiu th virus b v hot bi bc x t ngoi. Do t ngoi ch gy t bin mt s phn ca genome virus nn, trong mt t bo, cc phn gen cn nguyn vn ca cc genome c th trao i (ti t hp) dn n hnh thnh th c y nhng gen nguyn vn. D hp ha l hin tng khi mt s th virus c tnh trng khc nhau ti sn ng thi trong mt t bo c th hnh thnh nhng virion mang ton b genome ca mt virus v mt phn hoc ton b genome ca virus th hai. Mc d s kt hp genome trn khng di truyn nhng n cho php cc virus sn sinh th h mi, trong mt s virion mang tnh trng ca mt virus khi nguyn loi ny cn mt s khc mang tnh trng ca virus khi nguyn loi khc. Nhng virus trn c gi l d hp th v khc vi cc virus ng th ch cc virion th h con khng ng nht. Hin tng d hp ha c th quan st trong cc th nghim vi virus cm v virus bnh Newcastle. Th nghim cng ch ra rng c th un nng lm bin tnh DNA virus lm mch i chuyn thnh mch n v khi ngui th mch i li t phc hi. Nu trong dung

223 dch tn ti hai loi virus th c th hnh thnh nhng th d hp mang genome hn hp ca hai virus khi u. Chuyn v (transcapsidation) l hin tng genome ca mt virus c n nhp vo v ca virus khc c kh nng bo tn trng thi n nh trong t bo mn cm vi virus khi ngun. Hin tng ny c th gp khi nui cy vo t bo ng thi adenovirus v virus SV-40 ca kh. Hot ha cho l hin tng genome tng t ti t hp nhng mt trong hai virus tham gia dng nguyn vn cn virus kia b v hot do h tn mt phn genome (un nng hoc chiu t ngoi). Trong trng hp ny phn genome ca virus b v hot c th tham gia ti t hp vi genome virus bnh thng. Kt qu l hnh thnh nhng virion c tnh trng hn hp ca hai virus khi nguyn. Dng ti t hp thng d hnh thnh hn trong trng hp hot ha cho so vi vic kt hp hai virus nguyn vn. Hot ha cho thng din ra trong t bo nu tim virus v hot (bng nhit hoc tia t ngoi) vo h thng mn cm khong 24 - 48 gi trc khi tim virus c hot tnh. C n 70% genome virus cm gia cm v hot A1 c hot ha nh virus cm A2. Virus u th cng c chng minh c hot ha cho. Pha trn kiu hnh l hin tng hai virus khc nhau khi ti sn ng thi trong mt t bo lm xut hin nhng virion mang genome ca ch mt virus khi nguyn nhng li mang khng nguyn ca c hai virus khi nguyn . Trong trng hp ny khng din ra qu trnh tng tc kiu gen m cc virion ch tha hng chung cc protein cu trc c tng hp ng thi trong mt t bo. Hin tng pha trn kiu hnh thng gp phage T2 v T4 v cng nh mt s virus ngi v ng vt (nh virus cm, virus bnh Newcastle, bi lit, v cc "arbovirus" typ A). Ti hot ha phi kiu gen l hin tng mt virus b v hot protein cu trc nhng c th pht trin nh hot tnh ca protein-enzyme c ngun gc t virus khc, v d nh enzyme "ci v" ca virus u. Hin tng ti hot ha phi kiu gen c th din ra nh mt virus u nguyn vn nhng cng c th nh mt virus u genome khuyt tn. Tng b l hin tng khi nhim ng thi hai virus c genome tn thng do bin d hai min gen khc nhau v u mt kh nng ti sn, th chng cng s dng chung sn phm (cc protein) ca hai genome m cng c kh nng thc hin chu trnh ti sn. Chng hn, gen khuyt tn do bin d mt virus c th l protein dch sm (RNApolymerase) cn virus khc khuyt tn gen protein cu trc. Khi tch bit hai virus ny u khng th ti sn nhng nu cm nhim hn hp th

224 qu trnh ti sn ca c hai u xy ra. Do khng c s ti t hp di truyn nn cc virion th h con chu cng c genome khuyt tn. Kch thch l hin tng mt virus nguyn vn khi cm nhim ng thi vi virus khuyt tn th cung cp mt hoc mt s nguyn liu cn thit cho virus khuyt tn thc hin chu trnh ti sn. V d virus u tht Raus khng th cm ng tng hp protein cu trc, m s dng protein ca virus bch cu g trong thnh phn o ngoi. Khi trong mi trng c mt ca virus bch cu g, t cc t bo g bin np (c gen ti t hp) virus u tht Raus xut hin cc virion virus u tht Raus. Virus bch cu g trong trng hp ny gi l virus tr gip. Virus cm typ 3 c th l virus tr gip i vi virus Newcastle trong la cy t bo thn kh, virus Sendai i vi virus bi lit trong la cy t bo s phi chut vng, adenovirus i vi cc virus ty thuc adeno (hay AAV, hay adenosatelite). Cn nhim (hay can thip cm nhim, can nhim - interfering) l hin tng mt t bo b cm nhim mt loi (typ) virus c kh nng khng li s xm nhp sau ca virus khc. Hin tng ny lin quan n s hnh thnh protein c bit gi l interferon do genome t bo ng vt chi phi, gip t bo b nhim cng nh cc t bo bn cnh tr nn khng ti cm nhim virus. Ph thuc vo chng loi virus b cn nhim m chia thnh ba loi cn nhim: t cn nhim, cn nhim ng loi, cn nhim d loi. im c bit ca interferon l ch bo v c cc t bo cng loi vi t bo tng hp nn n (tnh c hiu k ch) v chng li s xm nhp ca cc loi virus khc nhau (tnh khng c hiu vt k sinh). Do kh nng hnh thnh nhanh chng (khong 24 48 gi) v to s khng i vi nhiu loi virus mt cch nhanh chng nn interferon c coi ch ng dng. Tc dng ca cc vaccine virus nhc c (attenuated vaccine) trong giai on sm l do hin tng cn nhim. Ngy nay, hp cht ny c sn xut bng cng ngh di truyn v p dng trong iu tr bnh nhim virus ngi.

VI. K thut di truyn


K thut di truyn bt u nh mt khoa hc t 7/1972 khi nhm nghin cu ca Paul Berg (Vin hn lm khoa hc quc gia M) hon thnh vic ch to b gen lai gia virus SV40 ca kh vi phage lambda. Ln u tin kt ni hai virus khc nhau hon ton v mt di truyn thnh mt chnh th v c nhn ln di dng plasmid trong t bo vi khun. Phn t DNA lai cha cc thng tin di truyn ca virus SV40, thng tin iu khin qu trnh t sao ca DNA phage lambda cng nh ton b chc nng ca operon galactosidase ca vi khun E. coli. Ban u khi nim k thut di truyn ch cc k thut ti t hp DNA nhng

225 dn dn ni dung ca thut ng ny m rng ti cc k thut lin quan nh PCR vi cc bin tng PCR, k thut lai phn t (Northern hybridization, Southern hybridization, microarray,... Nguyn tc chung ca cng ngh DNA ti t hp gm mt s bc. Trc ht DNA genome mc tiu v DNA chuyn ti (vector) c tinh ch v ct thnh on nh mt loi enzyme hn ch. Trong nhiu trng hp (c bit i vi sinh vt bc cao) DNA genome mc tiu c thay th bng DNA tng b (cDNA) ca RNA thng tin thnh thc. DNA ny c tng hp t khun RNA thng tin nh s xc tc ca enzyme sao chp ngc (reverse transcriptase, RNA-dependent DNA-polymerase). DNA vector c th l ca mt plasmid hoc phage (nu vt mang, dng ha gen l vi khun), plasmid Ti ca vi khun Agrobacterium tumefaciens (nu vt mang dng ha gen l t bo thc vt), l virus (retrovirus hoc adenovirus khng gy bnh, nu vt mang dng ha gen mc tiu trong t bo ng vt). Bc th hai l chuyn vn DNA ti t hp vo c th vt mang, dng ha v bc tip sau l sau nui cy vt mang, sn xut (chit xut v tinh ch) sn phm mc tiu. Nh c ct cng mt loi enzyme hn ch (thng l enzyme u dnh) cc on DNA mc tiu v cc DNA vector (ch ct ti mt im ca mch vng xon kp) c th ngu nhin hnh thnh mi lin kt hyr tng b cc u dnh, ri c enzyme ligase hn gn cc u khc. Kt qu l trong hn hp hnh thnh th ti t hp vector vi mt hoc mt s mnh DNA ca genome mc tiu (bn cnh cc sn phm khng mong mun nh vector t khp vng, cc vector t ti t hp vi nhau v cc mnh DNA khng ti t hp hoc t ti t hp). Tt c cc sn phm trong hn hp u c trn vi cc vt mang to dng, sau cc vt mang c nui cy trong mi trng dinh dng c cha yu t chn lc (cht khng sinh i vi vi khun, ha cht gy cht i vi t bo ng vt v thc vt). Trong iu kin , ch cc t bo vt mang mang vector sng st. Hn na, do plasmid b enzyme hn ch ct tho vng ti mt v tr xc nh trong mt gen iu khin mt thuc tnh nhn bit khc, nhng plasmid ti t hp mt kh nng to tnh trng vt mang (chng hn, mt kh nng tng hp beta-galactosidase), nn ch nhng vt mang sng st v khng biu hin tnh trng c chn nui cy to dng. Bc tip theo l chn nhng dng biu hin tnh trng ca gen mc tiu hoc nhng dng mang gen mc tiu (nh lai phn t: cc phng php th nghim min dch nh ELISA, Western blot analysis hoc vi cc d DNA hoc RNA - Southern blot analysis, Northern blot analysis), hoc PCR. Nui cy to dng nhng c th vt

226 mang tha mn cc bc th nghim trn gip to ra dng thun cc sinh vt mang c th sn xut cc sn phm mc tiu. Cho n nay, k thut di truyn t c nhng bc tin vt bc. Nhiu sn phm c ch c sn xut t cc sinh vt ti t hp hay sinh vt bin i gen (genetically modified organisms - GMO) nh ging ng cc, u tng, ht c du, bng,... mang gen chng su bnh hoc khng thuc chng c di hoc c cht lng sn phm nng cao nh khoai ty cha nhiu protein, la giu vitamin A v st,... to ra c nhng vaccine ti t hp c th d sn xut (d nui cy trn la cy t bo hay phi g) v d s dng (cho n, cho ung), hoc cc ng vt ti t hp sn sinh nhng sn phm hu ch nh thuc cha bnh, thm ch to ra nhng n ln mang khng nguyn ngi hy vng c th l vt cho tim hoc ni quan khc iu tr nhng trng hp ngi bnh cn phi thay th ni quan,... Tuy nhin, k thut di truyn cng t loi ngi vo tnh trng c th phi ng u vi nhng e da mi lin quan n sc khe ca ngi v sinh thi - mi trng. Cha th chng minh c tnh v hi ca GMO thc phm i vi con ngi, cng nh cha th ph nhn kh nng lan truyn gen khng thuc (ca plasmid vector) trong tp on cc vi khun t nhin ca c th (v d, trong ng rut) ngi v ng vt. Chm sc nhng loi cy trng khng thuc dit c v thuc dit su bnh c th dn n lm dng nng dc v nu thoi ha tr thnh c di th thc vt khng thuc v khng su bnh s l mt nguy c mi i vi nng nghip. Tng t, nguy c nhng sinh vt GMO ln t sinh vt t nhin l rt cao v c th dn n s tuyt dit ca nhiu loi sinh vt hin c, nh hng xu n mi trng sinh thi,... PCR (phn ng chui trng hp: polymerase chain reaction) l mt k thut ring bit c sng to khuych i DNA in vitro da trn c s phn ng t sao ca DNA nh DNA-polymerase t cc deoxyribonucleoside triphosphate lun cn mi (primer) l on oligonucleotide (trong t bo l on RNA do primase xc tc tng hp) gn kt sn mt cch tng b trn khun si n ca phn t DNA. Hin thc ha nguyn l trn p dng trong ng nghim c xc tin nh nhng tin b trong k thut nh: tng hp c cc mch oligonucleotide c trnh t c hiu bit trc t cc deoxyribonucleoside triphosphate, thnh cng trong vic ch to my chu k nhit hay my lun nhit (thermocycler) v tinh ch c enzyme DNA-polymerase chu nhit (t vi khun u nng Thermus aquaticus - Taq polymerase, sau t E. coli ti t hp gen ny ca Thermus aquaticus - rTaq polymerase). Trong my lun nhit, hn hp gm DNA, dNTP, DNA-polymerase, cp oligonucleotide mi v dung dch m c gia nhit (94 - 96 C), khi cc DNA mch i bin tnh thnh chui n, sau khi my h nhit

227 xung khong 40 - 60 C th cc oligonucleotide mi s gn vo cc v tr c hiu (c trnh t nucleotide tng b) v cung cp u 3'-OH cho DNA polimerase gn vo, khi gia nhit ln khong 60 - 75 C polymerase hot ng gn kt cc dNTP tng b ko di si mi theo hng 5'3' lm cho si n tr thnh si i bt u t im gn mi. Tip tc cc chu k gia nhit gy bin tnh (denaturation), h nhit gy gn mi (annealling) ri nng nhit gy ko di (elongation) ta c th tng hp c lng ln si n mi. Do hai mi trong hn hp c thit k c th gn c hiu vi hai si n khc nhau sao cho mch mi tng hp ca mi ny cung cp ch gn cho mi kia nn sau mt s kh ln (25 40) chu k tng hp on DNA nm gia hai mi c tng hp vi s lng ln p o. Nu cho sn phm phn ng di ng trong gel nh dng in mt chiu bn cnh cc on DNA c phn t lng (DNA molecular weight marker) bit trc v nhum ethidium bromide ri soi di tia t ngoi, ta c th nhn ra cc bng DNA v xc nh c phn t lng ca sn phm (cng nh s thun khit ca sn phm). Do hai mi c thit k mt cch c hiu vi trnh t DNA bit trc hay do mi sinh vt c trnh t DNA c hiu nn sn phm c PCR tng hp c phn t lng c hiu (do tnh c hiu ca trnh t nucleotide). Nh c th dng phn ng ny nhn bit s hin din ca mt on gen cho trc hoc chn on bnh (ng nh: nhn bit s hin din ca mm bnh) hoc nhn din (ng nh) c th. tng hiu qu ca PCR, m rng phm vi s dng ca n hoc p dng nhn DNA t khun RNA, hng lot bin tng ca k thut ny c thit lp (nested PCR, LA PCR, hot start PCR, RAGE, panhandle PCR, inverse PCR,...). Ngoi k thut PCR thng thng (conventional PCR) nu trn cn c PCR thc thi (real-time PCR) c pht trin trn c s phn ng PCR thng thng vi cp mi c hiu v mt d ch hp th v pht x (bc x bc sng c hiu) khi gn vi DNA on gia hai mi t hp vi thit b c bit trc nh cng pht x ca d ngay trong qu trnh phn ng. PCR cn c s dng trong vic xc nh trnh t nucleotide (sequencing) ca DNA. Khi , c th s dng trc tip sn phm khuch i nh PCR hoc cc on DNA c dng ha. Nguyn tc chung l khuych i mt on DNA nh mt mi vi hn hp gm DNA khun, mt mi duy nht bt cp vi u 5' ca on DNA cn c trnh t, Taq DNA-polymerase, cc dNTP cng vi mt trong bn loi ddNTP (dideoxyribonucleotide) c nh du hunh quang (hoc phng x,...) trong mi l trong bn l phn ng (hoc bn loi nh du khc nhau trong mt l phn ng chung). Phn ng xy ni di bt u t mi v kt thc khi gn kt vi mt ddNTP nh du (do ddNTP thiu gc 3'-

228 OH cn thit cho phn ng tip tc). Do vic gn kt vi cc phn t ddNTP l tng b c hiu v ngu nhin gia mt ddNTP vi dNTP cng loi nn sau khi bin tnh kt qu phn ng (bng nhit, c thm formamide) ta c hn hp cha cc on DNA si n di ngn khc nhau. Khi in di (trong polyacrylamide gel bo ha urea) trong bn ln (lane) cnh nhau (vi trng hp nh du cng loi) hoc trong mt ln (vi trng hp nh du khc loi) cc on di ngn khc nhau s di ng vi tc khc nhau. Kt qu l ta c cc bng (band) phn b lin tip (ln lt, c t bng di ng nhanh nht) ph thuc vo di ca mch n nucleotide tc ph thuc vo v tr gn kt ca mt loi ddNTP. Trnh t c c chnh l trnh t nucleotide ca on DNA bt u t mi. Lai phn t (hybridization) c th gip nhn bit s hin din ca mt phn t c hiu no , gm lai Southern (phn tch DNA) hoc Northern (phn tch RNA) v cc k thut min dch hc nh Western blot analysis v ELISA. Mt trnh t mch n (DNA hoc RNA) bit trc gi l d (probe) c nh du (bng cht phng x) c chun b sn v cho tip xc vi sn phm RNA hoc DNA si n c nh trn gi th (mng nylon hoc mng nitrocellulose), nu c trnh t tng b d s gn kt mt cch c hiu. Sau khi ra k mng gi th, nu pht hin c d (nh k thut phng x t k) trn mng chng t trong sn phm c cha trnh t nucleotide tng b. Western blot analysis l phng php lai khng th gn kt vi mt enzyme c kh nng chuyn mu c cht thch hp (nh du bng peroxidase hoc phosphotase kim) vi sn phm c nh trn mng, trong khi ELISA l phng php lai khng th nh du vi sn phm c nh trn b mt l khay. Nu trong sn phm c khng nguyn c hiu th khng th nh du s kt hp c hiu v khng b ra tri. Kt qu l nu pht hin c yu t nh du (nh k thut chuyn mu c cht enzyme) th chng t trong sn phm c khng nguyn hay protein c hiu.

Cu hi n tp chng 10
1. 2. 3. 4. 5. 6. 7. 8. 9. Vt cht di truyn sinh vt nhn chun, sinh vt nhn s v virus? S t sao DNA? Qu trnh phin m? Qu trnh dch m? S iu tit phin m v iu tit dch m? Ci bin v hn ch? Bin d kiu hnh? t bin? Bin np (chuyn th) vi khun?

229
10. 11. 12. 13. 14. Ti np vi khun? Tip hp vi khun? Tng tc di truyn virus" K thut di truyn v cng ngh DNA ti t hp? PCR cn nhng yu t ha hc g, nhng iu kin phn ng nh th no? 15. Cc k thut lai phn t da trn tnh cht c bn g ca cc yu t tham gia phn ng?

230

Chng 11

MIN DCH
Min dch (MD-immunity) l trng thi bo v c bit ca c th chng li cc tc nhn gy bnh (vi sinh vt v cc c t ca chng, cc phn t l...) khi chng xm nhp vo c th. S bo v c th do rt nhiu cc phn t v t bo nm ri rc khp c th tham gia theo c ch bo v khng c hiu v bo v c hiu. MD khng c hiu c hnh thnh t khi mi lt lng v trc khi c s xut hin ca khng nguyn. MD c hiu bao gm MD th dch do hnh thnh khng th trong th dch ca c th v MD t bo (to Protein c tiu dit cc t bo mang khng nguyn l)

I.C ch bo v khng c hiu


L min dch t nhin mang tnh cht bm sinh, bao gm cc tuyn phng th t ngoi vo trong ngn chn khng cho vi sinh vt (VSV) xm nhp vo c th. - Hng ro vt l: Da v nim mc ngn cch ni mi ca c th vi ngoi mi xung quanh. Trn mt biu b cha keratin khng tan trong nc, khng b VSV phn gii, do khng cho VSV thm qua. Pha ngoi, biu b ch yu gm t bo cht nn virus khng nhn ln c. Nim mc bao ph b mt trong c th nh ng h hp, tiu ho v sinh dc. Cht nhy ca nim mc l ci by bt gi VSV, h thng nhu mao chuyn ng ht VSV ra bn ngoi qua ng mi hoc ming. Lng mi, phn x ho, ht hi, nn cng gip ngn chn VSV xm nhp vo c th. * Dch c th nh nc mt, nc bt, nc tiu...cng gip ra tri VSV khi chng bm trn b mt cc m, y ht nh ta tm ra lm tri bi bm. - Hng ro VSV bao gm cc vi khun bao ph b mt hoc khu tr bn trong c th, to nn khu h VSV bnh thng. y l mt khu h VSV phc tp, c s lng gp 9 ln tng s t bo ca c th. Chng chim trc cc v tr m VSV gy bnh n, cnh tranh thc n v tit ra cht ho hc tiu dit VSV gy bnh. Ngoi ra nhiu vi khun sng trong ng tiu ho cn tit ra biotin, riboflavin v cc vitamin khc cung cp cho c th. Khi ph v s cn bng trong khu h, mt s s c th tr thnh VSV gy bnh c hi.

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- Hng ro ho hc: trong dch tit ca cc m trong mu, trong dch lympho u cha cc nhn t c ch hoc tiu dit VSV gy bnh. * pH acid c ch s sinh trng ca VSV. VSV phn gii lipid thnh acid bo lm tng acid ca da. Vi khun lactic ln men glycogen to acid lactic trong m o. Dch v do tuyn trong d dy tit ra cha acid clohydric c pH 1-2. Sn phm ln men ca cc vi khun thng tr trong ng tiu ho l acid lactic v axetic. Ngoi ra VSV cn tit ra bacterioxin l cht dit khun mnh. * Lysozym l enzyme c trong nc mt, nc mi, nc bt, dy tai, m hi, sa, dch m o...v.v.., c kh nng dit vi khun Gram dng nh c ch phn gii thnh t bo, do c coi l mt loi cht khng sinh. Protein lin kt vi st nh lactoferrin c trong tinh dch, nc mt, sa m, mt, nc mi, dch nhy rut...v.v.., transferrin c trong gian bao cc m, trong huyt thanh lm gim trng lng st t do gy tnh trng thiu st cn thit cho VSV sinh trng, do ngn cn c nhim trng. Inteferon (IFN) l glycoprotein do cc t bo m nhim virus hoc vi khun (IFN- v IFN-) hoc do cc t bo lympho T sinh ra (IFN-). IFN c ch s nhn ln ca virus trong t bo bng cch hnh thnh protein cm ng ngn cn qu trnh dch m v phn hu RNA thng tin ca virus. Ngoi vai tr bo v, IFN cn lm nhim v iu ho mng li bo v chng nhim trng v s nhn ln ca cc t bo ung th. B th (Complement) l glycoprotein c kh nng lm tan t bo v tng cng hin tng thc bo. - Hin tng thc bo: Cc bch cu tham gia vo c ch bo v khng c hiu bao gm: Bch cu trung tnh cha cc ht khng bt mu thuc nhum, c tnh ho ng ng bt gi v tiu dit cc vt l, k c VSV v cc mnh t bo phn hu, do vy ng vai tr quan trng trong chng nhim trng. Bch cu a kim cha cc ht bt mu xanh methylen, cha cc hot cht nhu histamin, heparin, serotonin. Bch cu a acid cha cc ht bt mu eosin, cha enzyme phn gii histamin v heparin, do vy iu ho hot ng ca bch cu kim. Bch cu ny c kh nng gy c cc u trng giun. i thc bo ng vai tr quan trng trong chng nhim trng, nht l virus. i thc bo thu tm VSV nh gi tc, to khng bo

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(phagosom). Cc khng bo dung hp vi lysosom to phagolysosom. Cc VSV trong phagolysosom b tiu dit do nhiu nguyn nhn. - Cc enzyme ph thuc oxy chuyn ho oxy phn t thnh peoxit gy c cho VSV. - S thay i kiu h hp, to acid lactic lm gim pH, pH thp lm tng hot tnh nhiu enzyme trong lysosom. - C ch thc bo khng ph thuc oxy bao gm hot ng ca cc enzyme phn gii ca lysosom nh lyzozim, photpholipase, ribonuclease, deoxyribonuclease..v.v.. St l c ch bo v t nhin, lm tng phn ng enzyme phn hu VSV, lm tng hot ng ca IFN v lm gim nng st t do trong mu, do gp phn tiu dit VSV. Vim khng c hiu c tc dng khu tr VSV vo mt ni khng cho chng lan rng thm, 4 tnh cht ca vim l: , au, sng, nng l do gin mch, tng dng mu, lm thot bradykinin v prostaglandin, bch cu trung tnh, i thc bo vo vim.

II.Cht sinh min dch v khng nguyn


1. Cht sinh min dch (immunogen) l nhng cht khi a vo c th iu kin thch hp c kh nng gy mt p ng min dch (UMD), cn khng nguyn (KN-antigen) l nhng cht c kh nng lin kt vi khng th hoc th th c hiu ca t bo limpho T (TCR). Tt c cc cht sinh min dch u l khng nguyn, nhng khng phi tt c khng nguyn u l cht sinh min dch. V d: Hapten l khng nguyn khng trn vn, c th gn vi khng th nhng khng kch thch to khng th. Ba iu kin cn ca mt cht sinh min dch l: - Tnh l: C th khng p ng bo v vi khng nguyn bn thn, do vy cht cng l vi c th, kh nng gy p ng min dch cng cao. - Trng lng phn t ln: Khng nguyn thng c trng lng phn t trn 10.000 dalton. Nu thp hn, kh nng sinh min dch yu hoc khng c v kh b i thc bo bt gi v x l. - Cu trc phn t phc tp: nhiu cht c trng lng phn t ln nh polylisin, polisaccharid khng gy hoc gy p ng min dch yu v cu trc n gin do lp i lp li, trong khi hapten c trng lng phn t thp nhng gn vi protein th li tr thnh cht gy p ng min dch.

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2. Tnh c hiu ca khng nguyn (KN) Khng phi ton b khng nguyn tham gia vo kch thch h thng min dch m ch c mt phn nht nh ca khng nguyn gi l quyt nh khng nguyn hay epitop, mi lin kt vi khng th hoc t bo lympho. Phn tng ng vi quyt nh khng nguyn nm trn mi khng th gi l v tr kt hp khng nguyn hay paratop cn phn tng ng nm trn mt t bo lympho l th th, v d th t bo T (TCR Tcellreceptor). S lin kt gia epitop vi paratop mang tnh c hiu cao, tng ng nh gia enzyme v c cht hay gia kho vi cha.

III. Cc c quan v t bo tham gia vo h thng min dch


Nhiu c quan v t bo nm ri rc khp c th hp tc vi nhau nhn din v phn ng vi khng nguyn theo nhiu cch dn n p ng min dch cui cng. 1. cc c quan lympho: l ni hun luyn v tng tr cc t bo lympho, bao gm c quan lympho trung tm v c quan lympho ngoi vi. 1.1. C quan lympho trung tm gm tuyn c v ti fabricius Cc t bo gc hay cn gi l t bo ngun i t tu xng vo tuyn c. Ti phn v ca tuyn c, tin t bo T c phn chia nhiu ln v bit ho thnh t bo T chn vi cc th th khc nhau trn b mt. Cc gen kim tra s hnh thnh cc th th ny xut hin do ti t hp DNAca t bo T non khi cn tuyn c. Phn ln cc t bo T c th th nhn din c khng nguyn bn thn s b loi tr v cht. Cc t bo cn li di c vo phn tu ca tuyn c tip tc bit ho to cc dng t bo T phn lp, sau cc t bo ny theo mu ri tuyn c vo c quan lympho ngoi vi. Ti Fabricus l c quan lympho trung tm ch c gia cm, nm gn hu mn v l ni bit ho t bo B. ng vt c v khng c ti Fabricus nn s bit ho t bo B xy ra ngay trong tu xng v cc c quan tng ng, nh c quan lympho ca h tiu ho. Cc t bo B chn ri ti n c quan lympho ngoi vi. Khi tip xc vi khng nguyn s c kch thch, bit ho tr thnh t bo plasma (tng bo) sn xut khng th. V th t bo plasma c coi l nh my sn xut khng th. 1.2. Cc c quan lympho ngoi vi Lch l c quan lympho ngoi vi ln nht, l ni by khng nguyn vo bng ng tnh mch v l c quan chnh to khng th. Trong lch c vng cha t bo T v vng cha t bo B. Trung tm mm

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nm trong nang lympho vng t bo B. Trong trung tm mm cha y t bo B v t bo plasma, mt s i thc bo v t bo tua. Hch lympho nm ri rc khp c th v hot ng nh mt h thng lc. Khng nguyn di chuyn chm theo cc mch hp v gp khc tng s tip xc vi i thc bo v t bo lympho. Phn v gm vng v nng c nhiu nang cha t bo B. Khi c khng nguyn kch thch, nang s ni rng ra to trung tm mm, cha ch yu t bo B chun b phn chia. Vng v su cha ch yu l t bo T v mt s i thc bo. Phn ty c nhiu xoang cha dch lympho. Cc t bo plasma i t vng v sang vng tu. Khi c khng nguyn xm nhp, i thc bo bt gi, x l, hp tc vi cc t bo B v T sn xut khng th. 2. Cc t bo tham gia v p ng min dch: t ngun gc chung l t bo tu xng s c bit ho to thnh cc dng t bo khc nhau: (Hnh 11.1)

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- Dng to mu bit ho thnh cc t bo dng tu bao gm t bo mono (tin thn ca i thc bo) v cc t bo a nhn (bch cu trung tnh, bch cu kim v bch cu acid). Dng hng cu to t bo hng cu v dng t bo khng l to tiu cu. - Dng lympho i vo cc c quan trung tm. Nu vo ti Fabricius, s bit ho thnh t bo B (t ch Bursa Fabricius) khi c khng nguyn kch thch t bo B s bit ho thnh t bo plasma sn xut khng th v t bo B nh p ng vi khng nguyn vo ln hai. Nu t bo ngun i vo tuyn c s c bit ho thnh cc t bo lympho T (t ch Thymus-tuyn c). Cc t bo T c phn bit bi cc th th b mt thnh cc dng t bo T4 v T8. Dng t bo T4 bao gm: - Lympho T h tr (TH) c nhim v kch thch t bo B bit ho thnh t bo plasma sn xut khng th. - Lympho T cm ng (Ti) tit ra intlkin-2 kch thch s bit ho v hot ng ca cc t bo T khc. - Lympho T gy qu mm mun (TDTH) tit lymphokin hot ho i thc bo v cc bch cu khc gy qu mm mun. - Lympho T iu ho ngc (TFR) c tc dng hot ho lympho T c ch. Dng t bo T8 bao gm: - Lympho T c (TC) tn cng trc tip cc t bo c khng nguyn l (t bo ung th, t bo nhim virus). - Lympho T c ch (TS) iu ho p ng min dch bng cch c ch s hot ng ca cc loi lympho bo khc. i thc bo ng vai tr nh l t bo khi mo cho mt p ng min dch. Trc ht chng lm nhim v ch bin khng nguyn bng cch bt gi, nut v tit enzyme phn gii bc l quyt nh khng nguyn, sau y khng nguyn ra b mt tip cn vi t bo B v t bo T. Nh ng tc ny m i thc bo c gi l t bo trnh din khng nguyn (APC-antigen presenting cell). i thc bo cn cha cc th th dnh cho phn Fc ca IgG v b th C3, do lm tng kh nng thc bo.

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IV. Khng th
Khng th (KT-antibody) l cc gamma globulin Ig) c trong huyt thanh ca ng vt c kh nng lin kt c hiu vi khng nguyn kch thch sinh ra n. y ta ch xt cc khng th min dch (KTMD) 1. Cu trc ca khng th min dch: Tt c cc khng th u c cu trc ging nhau gm 4 chui polypeptit. Hai chui nh k l L v 2 chui nng k hiu l H, gn vi nhau bi cu disulfua (S-S).

Hnh 11.2: Cu to ca khng th

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Cc amino acid nm xa nhau trn cng mt chui c th gn vi nhau nh cu ni S-S to vng gp (domain) Chui nh: Trt t amino acid ca hai chui nh ging ht nhau v c chia lm hai vng. Vng c trt t amino acid thay i gi l vng bin i (k hiu VL), nm pha u amin (-NH2) ca phn t. Vng c trt t amino acid khng thay i gi l vng c nh (k hiu CL), nm pha u cacboxyl (-COOH). Trt t amino acid vng c nh ca chui nh lun ging nhau tt c cc lp khng th, hoc theo trt t (lamda) hoc theo trt (kappa). Ngc li trt t vng bin i lun khc nhau, k c cc khng th do cng mt t bo sinh ra. Chui nng: Mi chui nng c 4 vng amino acid: mt vng bin i v 3 vng c nh. Vng bin i (k hiu l VH) nm pha u amin i xng vi vng bin i ca chui nh, to thnh v tr kt hp khng nguyn (paratop). Vng c nh nm pha u cacboxyl, chai lm 3 vng nh (k hiu l CH1,CH2,CH3). Gia vng CH1 v CH2 l vng khp ni, c tc dng nh bn l lm cho phn t c hnh ch Y v c th iu chnh cho hai paratop gn vi hai epitop ca khng nguyn. Di tc dng ca papain (protease nha qu u ), phn t khng th b phn gii ti vng bn l thnh 3 mnh: hai mnh nh cha ton b chui nh v mt na chui nng c u amin. y l ni gn vi khng nguyn v c gi l mnh Fab (Fragment of antigen binding). Mnh cn li ca chui nng pha u cacboxyl c th kt tinh c gi l mnh Fc (Fragment crystalizable). Mnh ny c th lin kt vi th th c hiu nm trn i thc bo, t bo B v b th. Di tc dng ca pepsin thu c hai mnh: mnh ln gn nh mt khng th trn vn vi hai paratop v mnh nh Fc. 2. Cc lp khng th C 5 lp khng th mang tn chui nng l IgG, IgM, IgA, IgD v IgE. - IgG chim 80% tng Ig trong huyt thanh ngi bnh thng, cu to gm hai chui nh (kappa) hoc (lamda) v hai chui nng (gama). ngi Vit Nam nng IgG trong mu l 1400 mg/100ml. IgG l khng th duy nht c truyn t m sang thai qua nhau thai. IgG gi vai tr chnh bo v c th chng tc nhn gy bnh, m nhim cc chc nng opsonin ho (gip i thc bo bt gi khng snguyn), hot ho b th, gy c qua trung gian t bo ph thuc khng th (gip t bo K dit t bo ch) trung ho ngoi c t (c t un vn, c t do

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Clostridium botulinum gy ng c thc n, nc rn, nc cn trng... gy ngng kt vi khun v trung ho virus. IgM Chim t 5-10% trong huyt thanh ca ngi bnh thng. C cu to gm hai chui nh (kappa) hoc (lamda) v hai chui nng (muy). Nm globulin chm li vi nhau thnh phn t ln hnh sao nh cu ni disulfua v chui protein J do vy c ti 10 paratop. IgM xut hin sm nht thc hin cc chc nng nh hot ho b th, ngng kt hng cu cng loi trong trng hp nhm mu AB, ngng kt vi khun. - IgA c cu to gm hai chui nh (kappa) hoc (lamda) v hai chui nng (anfa). C hai loi: IgA trong huyt thanh ch yu dng monome v IgA tit (SIgA), lun c dng dime, c trong dch tit ca c th nh sa, nc bt, nc mt, trong dch nhy ng tiu ho, sinh dc, h hp. IgA monome lm nhim v hot ho b th theo con ng nhnh. IgA tit chng vi khun trn b mt nim mc gy nhim trng ng h hp, tiu ho, ng thi chng khng nguyn nhm mu AB. - IgD c nng trong mu rt thp v cu to gm hai chui nh (kappa) hoc (lamda) v hai chui nng (delta). Hot tnh sinh hc IgD cn cha r nhng n c mt trn t bo B lm th th cho khng nguyn. - IgE c nng trong huyt thanh rt thp v c cu to gm hai chui nh (kappa) hoc (lamda) v hai chui nng (epsilon). IgE tham gia vo qu mn tc th (hay d ng) bng cch gn phn Fc vi t bo Mast (dng bo) v phn Fab vi khng nguyn. T bo Mast c hot ho s gii phng cc cht hot mch nh histamin leukotrien, serotonin gy gin mch v tng tnh thm thnh mch.

V. Th th t bo T
T bo T c kh nng nhn din khng nguyn thng qua th th b mt, vit tt l TCR (T-cell receptor). S nhn din ny mang tnh c hiu cao. Chng hn t bo Tc c th phn bit c mi loi virus khi chng xm nhp vo c th. TCR c cu to gn ging khng th, gm hai chui peptit v gn vi nhau bi cu ni disulfua. TCR cng c hai vng: vng bin i nm pha u amin ca mi chui to nn v tr kt hp khng nguyn. Vng c nh nm pha u cacboxyl v cm su vo mng sinh cht ca t bo T.

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VI. Khng nguyn ph hp t chc (MHC)


Th th t bo T ch c th nhn din khng nguyn quen, l khng nguyn "ca mnh" hoc cng loi vi mnh, gi l khng nguyn hay phc hp ph hp t chc chnh, vit tt l MHC (major histocompatibility complex). Khng nguyn y nm trn b mt t bo bnh thng v c m ho bi vng gen ring, vng gen MHC. MHC hot ng nh l phn t trnh din khng nguyn v n tng tc c hiu vi c khng nguyn l ln TCR v ng vai tr quan trng trong ton b p ng min dch. C hai loi MHC: MHC-1v MHC-2. MHC-1 c trn b mt tt c cc t bo c nhn ng vt c xng sng, cn MHC-2 ch c mt trn t bo lympho B v i thc bo. ngi khng nguyn ph hp t chc (MHC) c gi l h thng HLA tc khng nguyn bch cu ngi (human leucocyte antigen). C nhiu gen m cho MHC-1, cn ch c cc gen ph m cho MHC-2 (Hnh 11.3).

Hnh 11.3: Cu trc ca protein ph hp t chc chnh


a) Lp I; b) Lp II. (Cn nh rng cc phn t lp I nm trn b mt tt c cc t bo c nhn, cn cc phn t lp II ch c trn b mt cc t bo trnh din khng nguyn bit ho, chng hn i thc bo v t bo B).

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Protein MHC-1 c cu to gm hai chui polypeptit. Mt chui c m ho bi gen nm trong vng gen MHC, gi l chui v c cm su vo mng sinh cht cn chui kia nh hn c m bi gen nm ngoi vng gen MHC, gi l chui microglobulin -2 (vit tt l 2m). Protein MHC-2 cng c cu to t hai chui polypeptit v c hai u cm su vo mng sinh cht v nh ra ngoi mt t bo. Cu trc MHC cc c th khc nhau trong cng loi cng khc nhau, do vy khi ghp c quan gia hai c th c MHC khng tng ng th s d b thi loi. Chc nng ca MHC: MHC lm nhim v trnh din hay ni ng hn l trung chuyn khng nguyn cho t bo T. Vi mi loi MHC c mt c ch trnh din ring. - Vi MHC-1: khng nguyn sau khi c t bo k ch ch bin nh enzyme tiu ho, s c gn vi MHC-1 trong li ni cht. V d t bo nhim virus phn gii protein virus thnh cc peptit ri gn vi MHC1. Phc h MHC-1 khng nguyn s xuyn qua mng sinh cht v nm trn mt t bo, y t bo Tc thng qua TCR c hiu khng nguyn s lin kt vi phc h trn, ng thi th th CD8 trn mt t bo Tc cng gn vi MHC-1, lm cho phc h mnh hn. Sau khi c khng nguyn kch thch, t bo T tng sinh v sn ra lymphokin lm tan t bo nhim. R rng t bo T phi r sot nhn din khng nguyn ph hp vi TCR ca mnh v TCR y phi tng tc c vi epitop ca khng nguyn ln MHC.(Hnh 11.4)

Hnh 11.4: Nhn din khng nguyn nh protein MHC lp 1


Protein lp I c to thnh v tch lu trong li ni cht, cng vi cc protein khc (nh khng nguyn virus v ung th). Mt s protein ny b tiu ho v c a

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vo trong li ni cht to thnh peptit. Cc peptit ny gn vi protein lp I ri chuyn ra mt t bo. y chng tng tc vi TCR trn mt t bo Tc ng th th CD8 trn mt t bo Tc cng ng thi gn vi MHC lp I to nn phc h mnh hn, sau t bo Tc sn ra lymphokin l cc protein dit t bo ch. Bt k t bo c nhn no cng c th hot ng nh mt t bo trnh din khng nguyn (APC) cho phn t lp I.

MHC-2 c hnh thnh trong li ni cht cng vi cc protein khc. Mt chui protein khng i gi l chui I gn trc vi MHC-2 cn ngn khng cho MHC-2 gn vi cc peptit khc. MHC-2 cng chui I c chuyn vo endosom.

Hnh 11.5: S nhn din khng nguyn nh phn t MHC-2


MHC-II c to thnh v tch lu trong li ni cht cng vi protein bao vy (blocking protein) Ii . Chui ny ngn khng cho lp II gn vi cc peptit khc cng c to thnh trong li ni cht. Sau lp II i qua th Golgi vo endosom. Ti y protein Ii v protein l t bn ngopi vo s b phn gii nh enzymee protein lp II li c gn vi peptit l phn gii to phc hp a ra mt t bo. Phc hp ny tng tc vi TCR v ng th th CD4 ca t bo TH. T bo ny sn ra lymphokin kch thch t bo B to khng th. T bo APC l i thc bo hoc t bo B.

Khng nguyn l b t bo APC (i thc bo, t bo B), thu tm ri chuyn vo endosom. y nh proteinase, chng c phn gii cng vi protein I. Cc peptit l c gii phng ra s th ch cho protein I gn vo MHC-2 to phc h chui qua mng sinh cht ng trnh din cho t bo TH thng qua TCR. Th th CD4 trn mt t bo TH cng gn vo MHC-2. Khi c khng nguyn l kch thch, t bo TH hot ho v tit intlkin bit ho t bo B thnh t bo plasma sn xut khng th.

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Hnh 11.6: Cu to chi tit vng bin i ca chui nng v chui nh.

y l mt na phn t Ig, CH v CL l vng c nh ca chui nng v chui nh.

VII. C ch hnh thnh khng th min dch


Vic hnh thnh khng th l mt qu trnh rt phc tp i hi c s tham gia ca nhiu loi t bo: T bo T, t bo B v t bo trnh din khng nguyn (APC) v phi c mi tng tc gia cc phn t b mt (TCR, MHC, khng th b mt t bo B). Khi khng nguyn vo c th s theo dng mu v bch huyt vo hch lympho, lch v gan. Khng nguyn s c to thnh lch v hch lympho. Trc ht khng nguyn phi c APC trnh din. APC l i thc bo hoc cng c th l t bo B. - APC l t bo B thng qua th th l khng th b mt s nhn din khng nguyn. Khng nguyn vo t bo ri tiu ho trong endosom, cc peptit c to thnh s kt hp vi phn t MHC-2 i ra b mt t bo tng tc vi TCR. - APC l i thc bo cng hot ng nh vy nhng khng c th th dnh cho khng nguyn. i thc bo thu tm khng nguyn (theo kiu amip bt mi), tiu ho v gii phng peptit. Peptit cng c gn vi MHC-2 a ra b mt trnh din t bo TH nh trng hp vi APC l t bo B.

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Hnh 11.7: M hnh thuyt chn lc dng

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Phc h MHC-2 KN s c trnh din cho t bo TH thng qua TCR. Phn t MHC cng tng tc vi th th CD4 trn b mt t bo TH. c kch thch bi khng nguyn, t bo TH hot ho v tng sinh sn ra interlkin kch thch t bo B tng sinh v bit ho thnh t bo plasma sn xut khng th v dng t bo B nh. T bo plasma ch sng c khong mt tun nhng li sn xut mt lng ln khng th. T bo B nh c i sng di v tn ti c khi khng nguyn mt. Khi c kch thch bi khng nguyn vo ln 2, chng s bin thnh t bo plasma sn xut khng th nhiu v nhanh chng hn. chnh l p ng min dch ln 2 v l c s ca vic tim chng vaccin. Mt s khng nguyn c th kch thch to thnh khng th mc thp m khng cn c s tham gia ca t bo T, c gi l khng nguyn khng ph thuc tuyn c. Cc khng nguyn ny thng c cu to n gin, lp i lp li, nh polysaccharit. Khng th c to thnh thng thuc lp IgM, c i lc thp. t bo B p ng khng nguyn ny khng c tr nh min dch 1.Thuyt chn lc dng ca Burnette Theo Burnette th thng tin hnh thnh khng th c sn trong t bo B t trc khi c khng nguyn xm nhp. Bng con ng t bin, hng lot t bo B c to thnh. Mi loi c kh nng p ng vi mt loi khng nguyn c sn. Trong thi k bo thai, loi t bo no chng li khng nguyn bn thn s b c ch. Khi ra i, c th ch cn li cc t bo phn ng li khng nguyn l. Khi c khng nguyn xm nhp, chng s chn lc t bo no ph hp (c th th ph hp vi quyt nh khng nguyn) kch thch, phn chia theo con ng gin phn to thnh dng. Cc t bo khng ph hp vi khng nguyn th khng c kch thch to dng.(Hnh 11.7) Thuyt chn lc dng c cng nhn v n gii thch c nhiu hin tng min dch nh dung np min dch (khng to khng th chng khng nguyn bn thn) v tr nh min dch. 2.Khng th n dng Trong t nhin, khng nguyn c nhiu nhm quyt nh (khng nguyn a gi), do s kch thch c th to thnh khng phi mt m l mt phc hp khng th. Mun nhn mt loi khng th trong phc hp y th phi tin hnh tch tinh khit. Nm 1975 Milstein v Kohler tin hnh lai bng con ng dung hp t bo ung th tu v t bo lympho B. T bo ung th c kh nng phn chia nhanh nhng khng to thnh khng th, ngc li t bo B c

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kh nng hnh thnh khng th nhng khng c kh nng phn chia v chng l t bo tn cng ca qu trnh bit ho. T bo lai c c u im ca c hai t bo trn: va phn chia rt nhanh va c kh nng tng hp khng th. Tin hnh pha long lin tc trong ging ca phin nha vi lng cho n khi mi ging ch c mt t bo. T mt t bo n ch sn xut mt dng khng th thun khit. Do vy khng th n dng l khng th do mt dng t bo B sinh ra chng li mt quyt nh khng nguyn. K thut khng th n dng c p dng rng ri thay th dn mt s phng php huyt thanh thng thng nh xc nh hocmon trong nh gi chc nng ni tit, xc nh protein chn on ung th, xc nh nng thuc trong mu vi nhy cao gp nhiu ln so vi cc bin php thng thng. C th a thuc n tn mc tiu cn tiu dit nh l tn la o n. Thuc s c phng thch lin tc dit cc t bo ung th. K thut n dng cng c dng xc ng doping trong th thao.

VIII. C s di truyn ca s to thnh khng th


Mt vn t ra l lm sao cho c th c kh nng sn ra mt lng ln v a dng cc loi khng th n nh vy p ng vi cc loi khng nguyn mun hnh mun v. Nu mi protein cn mt gen m ho th c th phi cn n hng t gen sn xut khng th. iu vt qu tim nng gen c sn ca c th. Mun hiu r c ch di truyn ca s tng hp khng th, chng ta cn nghin cu chi tit hn cu trc khng th v s sp xp li gen. Cu trc khng th: Cc khng th khc nhau c trt t sp xp cc amino acid khc nhau vng bin i. Trong vng bin i li c nhng vng nh c trt t amino acid thay i rt mnh, gi l vng siu bin. chnh l v tr kt hp vi khng nguyn. Vng bin i ca chui nh cng nh chui nng c 3 vng siu bin, c m ho bi cc gen vng bin i (gen V) nm trn DNA ca t bo B trong qu trnh chn tu xng. Ti vng siu bin th 3 ca chui nng cn c mt vng c m ho bi ring gi l gen D (t ch diversity-a dng) v mt vng ni gia vng a dng (D) v vng c nh (CH) gi l vng ni (J) c m ho bi gen J, chui nh khng c vng D. Vng J ni gia vng V vi vng CL, cui cng l vng c nh c m ho bi cc gen C.

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Hnh 11.8: S sp xp li gen Ig trong t bo lympho cha chn v

c ch hnh thnh gen hot tnh (a) chui nng (b). Chui nh kapa. Chui nh lamda c m ho trong phc h gen tch bit hon ton (c). S hnh thnh mt na phn t Ig. S sp xp li gen: cc gen kim tra tnh a dng ca cc phn t ca h thng min dch nh khng th, TCR v MHC u c sn trong cc t bo lympho. kim tra s tao thnh chui nh ca khng th, trong t bo B cha chn c 150 gen vng bin i (VL), 5 on gen lin kt J, 2 gen vng c nh (CL). i vi chui nng c ~200VH, 4 gen J, 50 gen D v 5 gen CH gen xen gia cc gen V, J, D l cc gen intron, khng lm nhim v m ho. T bo B trong qu trnh bit ho xy ra ti t hp di truyn. S ti cu trc ny dn n to thnh cc gen hot tnh chui nng v cc gen hot tnh chui nh. on DNA cha cc gen ny c sao li theo c ch b sung to ra RNA s cp ri t RNA s cp cc gen hot tnh chp ni li vi nhau, loi b intron to ra mRNA dng dch m to ra protein chui nng hay chui nh, tu thuc tng vng gen, tch bit dnh cho mi loi chui. Nu s chp ni sai bao gm cc gen khng ph hp xen vo gia th cc gen sai s c enzyme phn gii. S ghp

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ni cc gen hot tnh theo nguyn tc t hp, do vy s lng cc loi khng th c to thnh s l rt ln. V d chui nh c to thnh bi cc t hp 150 gen VL, 5 gen JL, 2 gen CL s cho ra 150 x 5 = 1.500 chui khc nhau. Cng nh vy ta s c 200 x 4 x 50 x 5 = 20000 chui nng khc nhau. Hai chui nh li lin kt vi 2 chui nng nn ta c 20000 x 1500 x 2 = 6. 107 phn t khng th khc nhau. S a dng ca cc loi khng th ny tho mn vi cc loi khng nguyn. C th c trong t nhin. Tnh a dng ca th th t bo T. TCR nhn din khng nguyn do t bo APC trnh din thng qua MHC. Nh vy TCR va phi gn c vi cc vng ca MHC va phi gn c hiu vi epitop ca khng nguyn. V th s to thnh TCR ca khng nguyn cng phi rt a dng. Tnh a dng ny c c l do s sp xp li gen ca DNA trong t bo T cha chin khi ang bit ho trong tuyn c. Trong TCR cng cha cc vng VDJ, s ti t hp gia cc gen V, D, J cng s to ra cc TCR khc nhau ging nh khng th vy. C ch to thnh khng th. Theo thuyt chn lc dng (clon) ca Burnett th trong c th c sn nhiu loi t bo lympho c kh nng sn xut khng th, mi loi th nhn din v phn ng vi mt loi khng nguyn. Khi cn giai on bo thai nu t bo lympho no phn ng vi khng nguyn bn thn s b c ch. Khi c khng nguyn l xm nhp, chng s la chn t bo lympho tng ng, kch thch tng sinh to thnh dng t bo sn xut khng th. Nhng t bo khng ph hp vi khng nguyn l s khng c to thnh dng. S to thnh khng th l mt tp hp cc phn ng do khng nguyn kch thch. Khng nguyn c t bo APC x l v trnh din cho t bo TH. V phn mnh, t bo TH tit ra interleukin hot ho t bo B. T bo ny bit ho thnh t bo plasma sn xut khng th (gi l p ng nguyn pht hay ln 1) v t bo B nh. T bo B nh c i sng di, chng cn hot ng nhiu nm khi khng nguyn ht. Khi khng nguyn vo ln sau, c th s p ng nhanh v mnh hn l do t bo B nh bit ho thnh t bo plasma sn xut khng th (gi l p ng th pht hay ln 2).

IX. Min dch qua trung gian t bo


Min dch qua trung gian t bo gi tt l CMI ( cell mediated immulity) c tm quan trng chng li virus, Ricketsia, vi khun (c bit l vi khun lao Mycobacterium tuberculosis) sng bn trong t bo v trong min dch ung th cng nh trong thi b m ghp.

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m nhim vai tr trong CMI l cc t bo T, trc ht l t bo TC. T bo ny nhn mt khng nguyn khi chng gn vi MHC. Bt k t bo no mang khng nguyn l. chng hn t bo m ghp khng tng ng, t bo nhim virus, u b t bo TC tit ra perforin-cht c t bng nm trong tng bo chui qua mng sinh cht ca t bo ch tiu dit t bo ch. Lympho TDTH gy qu mn mun, chng ch yu ng vai tr trong d ng v vim mn tnh. S tng t ngt lymphokin do t bo ny tit ra gy triu chng vim da do tip xc. T bo git t nhin gi tt l t bo NK (Natural killer cells) l cc t bo nhiu ht ln trong t bo cht. Chng khng phi l t bo T hay B, nhng c kh nng git t bo ung th v t bo nhim virus nh t bo TC, ch khc ch khng cn c s kch thch ca khng nguyn c hiu. Tuy nhin cng ging nh t bo TC, trc tin t bo NK phi tip xc vi t bo cha khng nguyn l sau mi tit c t git t bo. Cho n nay ngi ta vn cha thy th th ca t bo NK cn cho vic lm tan t bo ch. Cc t bo T cng tit ra cc cht iu bin (modulator) nh cc interleukin, interferon tham gia vo s iu ho min dch kch thch t bo B sn xut khng th. Cc lymphokin khc nh yu t ho ng ng hp dn i thc bo ni c khng nguyn, yu t c ch di tn ngn cn i thc bo ra khi vng cha khng nguyn. Yu t hot ho i thc bo, kch thch t bo ny lm nhim v hiu qu hn v.v....

X. B th
B th (complement) l mt nhm protein huyt thanh c c im ho hc v min dch hc khc nhau, c kh nng phn ng vi nhau, vi khng th v vi mng sinh cht ca t bo. H thng b th bao gm 9 protein c k hiu l C (t C1 n C9), cc yu t B, D v propecdin. Cc b th mun hot ng phi c hot ho. S hot ho ny theo kiu phn ng dy chuyn. Mt b th sau khi c hot ho s kch thch hot ho b th tip theo. C hai cch hot ho b th. Cch th nht pht hin sm hn gi l hot ho theo con ng c in. Cch th hai pht hin sau gi l hot ho theo con ng nhnh hay con ng propecdin. Hai con ng ny ch khc nhau giai on u, phn cht li ging nhau. Hot ho theo con ng c in cn c s tham gia ca cc b th C1, C4, C2 v khng th. C th chia ra lm su bc:

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1. To phc h khng nguyn- khng th hot ho C1.C1 gm 3 protein hp thnh: C1q, C1r v C1s. Khi C1 gn vo phn ca FC ca 2IgG (thuc loi IgG1, IgG2, IgG3 thuc IgM) nm k nhau, th C1q c hot ho. Tiu phn ny sau khi c hot ho s kch thch C1r, C1r c hot ho s kch thch C1S, C1S hot ho s tham gia hot ho C4 v C2. 2. C1S hot ho s xc tc tch i C4 thnh C4a v C4b. Mnh C4b s gn vo mng nguyn sinh cht ca t bo v vo C2 to thnh C4bC2, C1S hot ho, tip tc tch i C2 thnh C2a v C2b. C4b kt hp vi C2a thnh C4b2a, chnh l convertase C3 (enzyme b C3). 3. Di tc dng ca C4b2a, C3 b tch thnh 2 mnh l C3a v C3b. C3b gn vi C4b2a thnh C4b2a3b chnh l convertase C5 (enzyme b C5). 4. Di tc dng ca C4b2a3b, C5 b tch thnh 2 mnh l C5a v C5b. 5. C5b gn vo t bo ch v vo C6 to phc hp C5b6 sau gn vi C7 to thnh C5b67. Phc hp ny bm vo lp lipid kp ca mng sinh cht. 6. C5b67 bm vo C8 sau bm tip vo C9 to thnh phc hp tn cng mng C5b6789 khoan l thng, lm thot ni cht v git cht t bo. Hot ho theo con ng nhnh khng cn c s tham gia ca khng th, nhng cn n yu t B, D v propecdin thay cho cc b th C1, C2, C4 dn n to thnh enzyme hot ho C3. Yu t B gn vi C3b to phc hp C3bB. Khi gn vi C3b, yu t B l v tr chu s tc ng ca yu t D v b tch ra thnh Ba v Bb. Mnh Bb c gi li to thnh phc hp C3bBb, chnh l convertase C3 dng b C3. C3bBb gn thm C3b to thnh C3bBb3b chnh l convertase C5. T y s hot ho b th s din ra ging nh con ng c in. S hot ho b th c iu ho bi cc cht c ch. Cht bt hot C1 l C1-INH, cn cht bt hot C3b l yu t I, yu t H cnh tranh vi yu t B gn vi C3b ngn cn s to thnh C3bBb. S hot ho b th dn n nhiu hu qu sinh hc, trc ht l lm tan t bo nh phc h tn cng mng (C5b6789). C3b va gn vi th th ca vi khun, virus va gn vi i thc bo, do lm tng kh nng thc bo. C3b bao ph quanh t bo ch s lm tng kh nng tiu dit t

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bo ch ca t bo K trong hiu ng ADCC (gy c t bo ph thuc khng th).

Hnh 11.9a: Hot ho v iu ho theo con ng c in


C hai con ng u to thnh convertase C3: C4b2a( con ng c in) v C3bBb (con ng nhnh). Cc enzyme ny gn vi C3b to thnh covertase C5: C4b2a3b (con ng c in) C3bBb3b (con ng nhnh).

Hnh 11.9b: S hnh thnh phc h tn cng mng (Phn Fab ca khng th c hiu bm vo t bo ch, cn phn FC bm vo t bo K, gip cho t bo K dit t bo ch). C3a v C5a l c t gy phn v, chng tham gia vo s gii phng histamin khi t bo Mast. C3a v C5a cng l cht ho ng ng, chng li ko bch cu trung tnh vo ni tp trung khng nguyn l lm nhim v thc bo.

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XI. Min dch bnh l


1. Qu mn l nhng tn thng bnh l xy ra do mt p ng min dch qu mc v khng hp l, gy hu hoi m. Qu mn khng biu hin ngay sau khi tip xc vi khng nguyn ln u, m ch nhng ln tip xc sau. Gell v Combs chia qu mn ra l 4 loi. * Qu mn tip I hay qu mn tc th c c trng bi cc phn ng d ng, xy ra nhanh (<30pht) ngay sau khi tip xc vi khng nguyn vo ln hai, nguyn do l IgE c hnh thnh gn vo th th FC ca t bo mast, phn Fab ca hai IgE nm k nhau gn cho vi khng nguyn, dn n lm thay i cu trc khng gian ca th th, gy bin i ti ch mng, lm thot cc bng ri t bng gii phng cc cht trung gian hot mch nh histamin (gy co tht c trn), serotonin (tng tnh thm thnh mch), heparin (km hm ng mch, ECF-A-yu t ho hng ng bch cu trung tnh). S hot ho t bo Mast cng dn n vic tng hp cc cht mi nh phn ng chm (SRS-A lm tng tnh thm thnh mch) v yu t hot ho tiu cu (PAF, gy ngng kt tiu cu v gii phng histamin). Qu mn tp I c th biu hin ton thn nh sc phn v, sc do d ng vi thuc, sc do dng huyt thanh iu tr hoc bnh cht trong ni (khng th chng protein c trong sa b). Qu mn loi ny cng c th biu hin cc b nh hen ph qun v vim mi d ng). * Qu mn tp II hay qu mn gy c t bo xy ra khi khng th gn trc tip vi khng nguyn trn b mt t bo cng vi s tham gia ca b th v cc t bo hiu ng (t bo K, tiu cu, bch cu trung tnh, bch cu n nhn hay i thc bo) dn ti lm tan t bo ch, chng hn t bo ch gn vi khng th c hiu, hot ho b th to phc hp C5b-9 tn cng mng t bo ch, ng thi mnh C3b c th bm quanh t bo ch v bm vo i thc bo thc hin opsonin ho, gip i thc bo dit t bo ch. Qu mn tp II th hin rt r trong cc trng hp sau: Khi truyn mu khng ph hp, chng hn truyn mu nhm A cho ngi c nhm mu B th s lm tan mu ca khng th (IgM) ca nhm A s khng li khng nguyn B ngi nhn. Phn ng khng nguyn-khng th s hot ho b th gy hin tng tan hng cu ca ngi nhn mu, gy tn thng thn do tc nghn bi mt lng ln mng hng cu v gy c do gii phng phc hp hem (C34H23O4N4Fe).

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Bng 11.1: S nhm mu Nhm Khng nguyn AB A B O A&B A B Khng c Khng th Khng c khng th Khng th khng B Khng th khng A Nhm c th thn AB, O, A, B (nhn ton nng) A, O B, O

Khng th khng A, O (cho ton nng) khng B

Nhm mu Rh: khi b mang mu Rh+ v m mang mu Rh- th 50% a tr cho i mang Rh+. Nu thai nhi mang Rh+ th hng cu c th truyn sang m, kch thch to khng th khng Rh. Khng th ny c th truyn cho con qua nhau thai gy tan hng cu ca thai nhi. trnh tai ho cho ln c thai sau, ngi ta phi tim cho m khng th khng Rh (RhoGAM) loi b khng nguyn Rh c th m. Bnh ban xut huyt xy ra khi phn t thuc (hapten) gn xung quanh tiu cu, lc chng tr thnh khng nguyn mnh. Khng th chng phc hp ny gy hu tiu cu khi c mt b th. V tiu cu cn cho s ng mu nn nu thiu s gy xut huyt trn da. * Qu mn tp III hay phc hp min dch khc vi qun mu tp II ch khng th chng li vi khng nguyn ho tan phn b rng ri trong mu. Khi tim khng nguyn vo c th s kt hp vi khng th kt ta c sn to thnh phc hp khng nguyn- khng th ri lng ng vo gia cc t bo ni m v mng y mao mch. Hin tng ny ko theo s hot ho dng b th to C3a v C5a (cc cht gy phn v) lm tng tnh thm thnh mch (gay ph n). Cc thnh phn khc l cc cht ho

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ng ng, thu ht bch cu trung tnh. Cng vi tiu cu, cc t bo ny tp trung gy ghn mao mch. Bch cu trung tnh hot ho s tit enzyme protease, colagenase v cc cht hot mch gy vim lot. Kt qu l mao mch t, xut huyt dn n hoi t cc b. Hin tng ny gi l phn ng Arthus. Bnh vim cu thn l mt v d ca qu mn tp III. Phc hp khng nguyn-khng th ng li trong gian bo ni m v mng y vch mao mch gy vim. Khng nguyn c th l vi khun (sau nhim lin cu), k sinh (vin thn sau st rt c tnh ) protein l (vim thn trong bnh huyt thanh). Bnh huyt thanh: vic s dng khng huyt thanh trong iu tr mang li kt qu ln (v nh virrut di), song mt s trng hp c th gy tai bin. Nguyn nhn l do phc hp min dch lng ng trong thnh mch mu. Bnh phc hp min dch lin quan n nhim trng (Rheumatic fever): khi b vim hng do Streptococcus gy ra, khng nguyn thnh t bo cha protein M kch thch to khng th. Ti mng tim, thn, khp cng c protein ny, do vy khng th khng vi khun gy phn ng cho vi cc thnh phn ca c th gy vim (mng tim, thn, khp). V d: vim cu thn * Qu mn tp IV hay qu mn mun l tn thng do s tng tc gia khng nguyn vi cc lympho T mn cm vi cc khng nguyn gy ra. Phn ng xy ra mun, sau 24 gi hoc hn. Cc thng tn c th do t bo TC ph hu t bo ch c cc khng nguyn c hiu bm trn b mt hoc do tc ng ca lymphokin do lympho T tit ra, c tc dng lm cho i thc bo tp hp qu nhiu, nhp li thnh t bo khng l (u ht). Qu mn do tip xc l mt v d. Mt s cht nh niken, cromat, cc ho cht trong cao su, ho, m phm...c trong lng phn t (<1kDa) nn l hapten. Khi thm qua biu b, gp protein s tr thnh cht cng hp hapten protein thi (khng nguyn) kch thch lympho T, c bit l TC. Mi tng tc gia lympho T ny vi hapten bm trn da gy phn ng vim da. 2. Bnh t min Bnh thng c th khng sinh p ng min dch chng li khng nguyn bn thn. Khi tnh t dung np b mt v c th khng cn kh nng phn bit khng nguyn bn thn vi khng nguyn l th s gy p ng min dch chng li khng nguyn bn thn v dn n bnh t min.

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Bnh t min c th thuc qu mn tp II, III hoc IV. Sau y l mt s v d: + Bnh Grave (Tp II) l do khng th gn vo th th ca t bo tuyn gip dnh cho hocmon kch thch tuyn gip. Hocmon ny do tuyn yn sinh ra. Kt qu l tuyn gip b kch thch sn ra mt lng ngy cng ln hocmon tuyn gip dn n bu c do tuyn gip phng to. + Bnh nhc c (Tp II) lm cho c th suy yu. y l do khng th gn vo th th dnh cho axetylcholin ti im ni xung thn kinh c. Do khng tip nhn xung thn kinh do phn t axetylcholin, c s b yu, h hp dng, dn n t vong. + Luput ban h thng (SLE) (tp III) l bnh to trn vng ban hnh bm hay mt si. Nguyn nhn do to thnh khng th chng DNA dng xon kp hoc si n (gi chung l khng th khng nhn). Phc hp min dch lng ng cu thn, hot ho b th, thu ht bch cu ht gy vim cu thn. + Bnh tiu ng ph thuc insulin (Tp IV) do khng th c hiu tiu dit t bo tuyn tu sn sinh insulin. Vic thiu insulin lm cho lng ng trong mu tng dn n m lo v hoi th. Suy gim min dch: Suy gim min dch l s p ng min dch khng y . Nguyn nhn c th do s khim khuyt h thng min dch mang tnh bm sinh, do sai st hoc thiu cc gen ca h thng min dch mang tnh bm sinh, do sai st hoc thiu cc gen ca h thng min dch nhn t cha m hoc c th mc sau khi dng thuc, b ung th hoc sau khi nhim trng. V d: bnh Hodgkin (ung th) lm gim p ng min dch qua trung gian t bo hoc nhiu loi virus git cc t bo ca h thng min dch (HIV). Hi chng suy gim min dch mc phi (AIDS) Virus HIV l virus Retro mang genom RNA mt si v enzyme phin m ngc. Bao bc capsit l v ngoi liporotein. Trn mt v ngoi cha cc gai glycoprotein vi trng lng phn t 120 kDa nn gi tt l gp 120. Cc gai ny gn vo th th CD4 nm trn mt t bo TCD4 v i thc bo l t bo ch chnh ca HIV. Sau khi xm nhp vo t bo, virus ci b ngoi nh enzyme tiu ho ca t bo sau si RNA virus c sao thnh DNA n nh enzyme sao m ngc. Si DNA mi tng hp c dng lm khun tng hp si DNAmi theo c ch b sung. Hai si xon vo nhau to si DNAxon kp.

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DNA kp ny gn xen vo nhim sc th ca t bo ch nh enzyme integrase v nm di dng porvirus. Mt bn sao DNA c th nm im trong t bo vi thng n vi nm. Gen HIV c biu hin khi c s kch thch ca yu t phin m ca t bo ch gn vo 2 u lp li di ca provirus. S kch thch ny dn n to thnh cc RNA khc nhau, mt s l RNA genom ca virus, mt s l RNA thng tin dng tng hp protein v capsid. V capsid kt hp vi RNA genom to thnh ht virus (virion) chui ra ngoi lp li vng i mi t bo CD4 khc. Ch mt thi gian ngn sau khi nhim c 1-100 t bo cha HIV. Lc u c ch bo v ca c th lm gim tc nhn ln ca virus nhng sau chung vt qua v nhim ngy cng nhiu vo cc t bo T. Da vo s lng t bo T trong mu xc nh mc nhim. Nu lng t bo T4 trong mu xung di 600/, bnh nhn bt u mt p ng min dch qua trung gian t bo v dn n nhim bnh c hi. Khi mi bt u nhim nhiu dng t bo B c hnh thnh v trong huyt thanh cha mt lng ln IgG, IgM v IgA nhng giai on sau ca AIDS, lng khng th gim t ngt v cc khng th khng gp 120 cng gim theo. Liu php thnh cng nht chng HIV l dng Dideoxynucleozit, ch yu l azidotimidin hoc zidovudin. Cc cht ny c ch enzyme phin m ngc ca HIV v do cn tr s tng hp DNA ca virus. Cho n nay mi loi thuc cng ch lm gim s tin trin thnh AIDS. Cch tt nht vn l phng chng, ngn s ly lan theo cc con ng quan h tnh dc, tim chch ma tu v truyn mu nhim.

XII Cc phn ng huyt thanh


Khng th tn ti trong huyt thanh min dch, do cc phn ng min dch dng khng huyt thanh gi l phn ng huyt thanh hc. Phn ng huyt thanh dng chn on khng nguyn hoc khng th khi bit mt trong hai thnh phn, gi l phng php chn on huyt thanh hc. S kt hp gia khng nguyn-khng th hay ng hn l gia epitop v paratop da trn ba lc lin kt: lc Vander-wals, lc ht tnh in gia cc nhm chc v lc lin kt gia cc lin kt hydro. Phn ng xy ra theo hai pha, pha u gi l pha c hiu hay pha khng nhn thy xy ra nhanh, c trng bi s hp ph khng nguyn. Pha sau xy ra chm, biu hin ra ngoi c th nhn thy c nh kt ta hoc ngng kt. Phn ng khng nguyn-khng th ph thuc vo iu kin pH, nhit , cht in gii , cc ion Na+, Cl-, Mg++.

256

Nhiu phn ng xy ra kh nhn bit do cn cc cht ch th nh hng cu, thuc nhum hunh quang, cht phng x. Sau y l cc phn ng chnh: + Phn ng kt ta: Khi cho khng th phn ng vi khng nguyn ho tan nng chun s xut hin kt ta. Phn ng b c ch khi qu tha mt trong hai thnh phn trn. Phn ng dng xc nh khng nguyn khi c sn khng th c hiu hoc xc nh khng th khi c sn khng nguyn ho tan c hiu. C th thc hin phn ng kt ta trong thch, khng nguyn v khng th khuch tn trong thch, ni gp nhau to thnh vng cung kt ta. + Phn ng ngng kt tng t nh phn ng kt ta, nhng i hi khng nguyn hu hnh c kch thc ln nh hng cu, t bo vi sinh vt. Khng nguyn lin kt cho vi khng th kt dnh vi nhau to thnh tng cm c th nhn thy bng mt thng. Cho khng nguyn vo dy ng nghim cha khng nguyn huyt thanh vi pha long tng dn. Hiu gi khng th (titer) l pha long cao nht m vn xy ra ngng kt. V d vi pha long 1/20, 1/40, 1/80.... 1/640 u ngng kt th 1/640 l hiu gi khng th. + Phn ng trung ho xy ra khi khng th bao vy trung ho ngoi c t vi khun hoc bao vy virus. V d khng th bao quanh virus gy ngng kt hng cu do hng cu khng b virus lm ngng kt. + K thut min dch hunh quang da trn nguyn tc l thuc nhum khi b kch thch bi bc x c bc sng c bit s pht sng chng hn fluorescein pht hunh quang mu vng lc cn rodamin pht hunh quang mu da cam. Mt trong hai thnh phn (khng nguyn hoc khng th) c gn vi thuc nhum. Sau khi phn ng khng nguynkhng th xy ra, ra phn khng tham gia phn ng, ri quan st di knh hin vi hunh quang. + K thut min dch phng x da trn s cnh tranh gia khng nguyn nh du ng vi phng x (v d 125I) ( bit) vi khng nguyn khng nh du (cn bit) gn vo khng th c hiu chun. Bc 1: Trn khng nguyn nh du phng x vi khng th chun, sau , ra khng nguyn tha (khng tham gia phn ng). o phng x (A). Bc 2: Trn khng nguyn nh du v khng nguyn khng nh du vi khng th. Sau v ra khng nguyn tha khng gn vi

257

khng th. o phng x (B). So snh phng x A v B c th suy ra lng khng nguyn cn xc nh. K thut ELISA (k thut cht hp ph min dch gn enzymeenzyme-Linked Inmunosorbent Assay) nguyn tc ca k thut ny ging nh min dch hunh quang nhng khc ch thay v gn khng th vi thuc nhum hunh quang th ngi ta gn khng th vi mt enzyme. Sau phn ng khng nguyn-khng th, ngi ta cho thm c cht c mu. S xut hin mu chng t phn ng c hiu khng nguyn vi khng th. o cng mu l bit c nng khng nguyn cn pht hin.

Cu hi n tp chng 11
1.Hy phn bit min dch c hiu v khng c hiu (kh nng khng). Min dch c hiu gm nhng loi min dch no ? 2.Hy phn bit cht sinh min dch, khng nguyn v hapten. Hy nu tnh cht c bn ca khng nguyn. Quyt nh khng nguyn (epitop) l g ? 3.V s cu to khng th. Vng no ca khng th gi vai tr gn khng nguyn. Cc lp khng th c chc nng g ? 4.K tn cc t bo tham gia vo p ng min dch. Nu s khc nhau c bn gia t bo T v B. 5.Nu s khc nhau c bn gia t bo TH v TC. Chng m nhim chc nng g trong p ng min dch. 6.TRC l g ? N tham gia vo hot ho t bo T nh th no ? 7.Th no l th th t bo B, chng tham gia vo hot ho t bo B nh th no ? 8.Th no l t bo trnh din khng nguyn (APC). Hy m t qu trnh ch bin khng nguyn. 9.Phn t MHC l g ? chng c chc nng g trong trnh din khng nguyn ? 10. Hy trnh by qu trnh trnh din khng nguyn vi s tham gia ca MHC-I v MHC-II. 11.Hy trnh by qu trnh hnh thnh khng th. Ti sao c th c tim nng to ln trong vic hnh thnh khng th chng li mi khng nguyn c th c trong t nhin. 12.B th c hot ho nh th no v ng vai tr g trong p ng min dch.

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TI LIU THAM KHO


I. T I LI U TI NG VI T 1.V Triu An, Homberg,J.C. (1998). Min dch hc. Nh xut bn Y hc H Ni. 2.Nguyn Th Chnh, Ng Tin Hin, 2001. Virus hc, Nxb 3.Nguyn Ln Dng, Nguyn nh Quyn, Phm Vn Ty. 1977. Vi sinh vt hc. NXB Gio dc. H Ni 4.Nguyn Thnh t. 1999. C s sinh hc vi sinh vt. NXB Gio dc. H Ni 5.Phm Thnh H. 2000. Di truyn hc. NXB Gio dc, H Ni. 6.Phm B Kim, 2004. Vi sinh vt t. Trng i hc Cn Th. 7.V Th Thng Lan. 2002. Sinh hc phn t. NXB i hc Quc gia H Ni, H Ni. 8.Nguyn B Lc. 1997. Ho sinh. NXB Gio dc. H Ni. 9.Phm Hng Sn, Phan Vn Chinh, Nguyn Th Thanh & Phm Quang Trung. 2002. Vi sinh vt hc th y. NXB Nng nghip, H Ni. 10.Trn Th Thanh. 2001. Cng ngh vi sinh. NXB Gio dc, H Ni. 11.Nguyn Nh Thanh, Nguyn ng, Nguyn Khc Tun & Nguyn B Hin. 2004. Vi sinh vt hc i cng. NXB Nng nghip, H Ni. 12.L c Trnh. 2001. Sinh hc phn t ca t bo. NXB Khoa hc v K thut, H Ni. 13.L Xun Phng. 2001. Vi sinh vt cng nghip. NXB Xy dng. H Ni. II. TI LIU TING ANH 14.Cann A. J. 2001. Principle of molecular virology. Academic Press, San Diego. 15.Collier L. H. & Timbury M. C. (ed.) 1990. Topley & Wilson's Principles of bacteriology, virology and immunology (8th ed.), vol. 4, Edward Arnold, London.

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16.Fenner F. J., Bachmann P. A., Gibbs E. P. J. et al. 1993. Veterinary virology (2nd ed.) Academic Press, Orland. 17.Johnson L. G. 1983. Biology. Wm. B. Co. Publisher, Dubuque, Iowa. 18.Karp G. 2002. Cell and molecular biology, Concepts and experriments, 3rd edition. John Wilson & Sons, Inc., New York. 19.Lehninger A.L., Nelson D.L., Cox M.M. 1993. Principles of Biochemistry. Second Edition. Worth Publishers. New York. 20.Linton A. H. & Dick H. M. (ed.) 1990. Topley & Wilson's Principles of Bacteriology, Virology and Immunology (8th ed.), vol. 1, Edward Arnold, London. 21.Mikami T. (ed.) 1995. Juui biseibutsu gaku [Vi sinh vt hc th y] (Ting Nht), Buneidou shuppan, Tokyo. 22.Prescot Harley Klein, 2002. Microbiology. W. C. Brown publisher, USA. 23.Pham H.-S., Kiuchi A. & Tabuchi K. 1999. Methods for rapid cloning and detection for sequencing of cloned inverse PCR-generated DNA fragments adjacent to known sequences in bacterial chromosome. Microbiol. Immunol. 43: 928-836. 24.Ronald M. 1995. Principles of Microbiology University of Louisville. Kentucky. Mosby.

NHNG CH VIT TT
ADP AMP APG
A-1,3-DPG

ATP A-6PA CoA CKS DNA R-1,5-DP R-5-P RNA VSV F-6-P FAD G-6-P GAP KDPG N NAD NADH NADP NADPH PP X-5-P

Adenosine diphosphate Adenosine monophosphate Acid 3-phosphoglyceric Acid 1,3 diphosphoglyceric Adenosine triphosphate Acid 6-penicillanic Coenzyme A Cht khng sinh Deoxiribonucleic acid Ribulose-1,5-diphosphate Ribulose-5-diphosphate Ribonucleic acid Vi sinh vt Fructose-6-phosphate Flavin adenine dinucleotide Glucose-6-phosphate Glyceraldehyde phosphate 2-Keto-3-deoxi-6-phosphogluconate Nitrogen Nicotinamid adenine dinucleotide dng oxi ha Nicotinamid adenine dinucleotide dng kh
Nicotinamid adenine dinucleotide phosphat dng oxi ha

Nicotinamid adenine dinucleotide phosphat dng kh Pentose phosphate Xylulose-5-phosphate

Ngi bin son


1. PGS.TS. Kiu Hu nh 2. TS. Bin Vn Minh (Ch bin) 3. TS. Phm Ngc Lan 4. ThS. Nguyn Th Thu Thy 5. TS. Phm Hng Sn 6. PGS.TS. Phm Vn Ty

Bin son cc chng


8 1, 6, 7, v 9 4, 5 2 10 3 v 11

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