CHNG I: TCH,TINH CH,THU CH PHM ENZYME 1.1.

Bn cht ha hc v cc tnh cht u vit ca Enzyme * Bn cht ha hc: Enzyme l cht xc tc sinh hc, c bn cht protein,ha tan trong nc v trong dung dch mui long. Enzyme c phn t lng ln t 20.000 1.000.000 dalton nn khng qua c mng bn thm. *Cc tnh cht u vit ca Enzyme: -Enzyme c cng lc xc tc rt ln,cc phn ng c xc tc Enzyme xy ra vi vn tc nhanh gp 108 1011 ln so vi phn ng khng c cht xc tc. - Enzyme c tnh c hiu cao: mi E ch xc tc lm chuyn ha c 1 hoc vi c cht nht nh theo mt kiu phn ng nht nh v to ra sn phm mong mun, khng to ra sn phm ph. - Enzyme tc dng trong iu kin m du: to 30-50oC, pH trung tnh v p sut thng, khng cn nng axit hay nng kim mnh,hay p sut cao. - Tt c cc Enzyme c ngun gc t nhin khng c. - Cc ch phm Enzyme c sn xut t ngun nguyn liu d kim, r tin. 1.2. Cc bc c bn thu 1 ch phm Enzyme t Vi sinh vt (ni bo), ng vt v thc vt. A. Thu ch phm Enzyme t Vi sinh vt (ni bo) Bc 1: Phn lp, tuyn chn, nh tn: Enzyme Vi sinh vt c hot tnh mnh vt xa so vi cc Enzyme khc - Phn lp, tuyn chn: mun c c chng Vi sinh vt c kh nng tng hp mt vi loi Enzyme no th cn phi tin hnh phn lp t t, nc, khng kh, t mt s thc vt, sn phm. VD: Ngun phn lp nm mc: Cc chng nm mc c phn lp t cc mu g mc thu thp rng nhit i Vit Nam. - nh tn Vi sinh vt: + Theo c im hnh thi + Theo c im sinh l, sinh ha + Phn loi theo phng php sinh hc phn t Bc 2: Ci to ging Vi sinh vt C th dng cc tc nhn t bin tc ng ln b my di truyn ca t bo Vi sinh vt to ra cc dng bin chng c kh nng sinh tng hp c bit cao mt loi Enzyme no . C 2 phng php t bin to bin chng: * t bin bng cc tc nhn vt l hoc ha hc:

- Cc tc nhn vt l: gm cc tia cc tm (tia t ngoi), tia X (tia Rnghen), tia hoc bn ph bng ht notron, electron. Trong thng s dng tia cc tm (UV) - Cc tc nhn ha hc: Cc hp cht cha nit nh: nitrozmetyllguanidin. * t bin bng phng php sinh hc phn t: - Phng php bin np: + l s truyn AND t t bo cho n t bo nhn,c th xy ra trong ng nghim. Khi cho t bo nhn tip xc vi dch chit t t bo cho m khng cn c s tip xc trc tip gia cc t bo. + T bo nhn c th nhn bt k loi ADN no. + Cc on ADN c truyn i trong bin np c phn t lng vo khong 106 - 107 v phi c cu trc xon kp. - Phng php tip hp Gen: + Vt liu di truyn ADN ch c truyn i t t bo cho n t bo nhn khi 2 t bo tip xc vi nhau, do vy cc vi sinh vt c kh nng bin np th khng c kh nng tham gia tip hp Gen. + Hin tng tip hp mi ch c nghin cu 1 s loi vi khun nh: E.coli, Salmonellia. - Phng php ti np: + Vt liu di truyn ADN c truyn t t bo cho sang t bo nhn nh vo trung gian ca thc khun th (phage) + Trong qu trnh ti np cc on ADN c chuyn t t bo cho sang t bo nhn tip hp vi ADN ca t bo nhn do lm bin i tnh cht di truyn ca t bo nhn. Bc 3: Nui Vi sinh vt C 2 phng php nui VSV thu ch phm Enzyme: - Phng php nui cy b mt (phng php rn, ni, xp): VSV pht trin trn b mt mi trng dinh dng th rn c lm m v v trng. Mi trng dinh dng gm: cm go, kh cm, cm m, tm go, ng (90% - 95%), c b sung tru nh hoc mn ca (5% - 10%) lm xp canh trng khin oxy khng kh d xm nhp to iu kin cho VSV pht trin. Cm + tru + H2O 50% - 65% Hp thanh trng 120oC, 1atm, 1h. ngui 28oC 30oC + b sung VSV. Tri ra khay, dy 3 5 cm. Nui (pH, thi gian, to thch hp). - Phng php nui chm ( Phng php lng, phng php b su). + phng php ny: VSV pht trin trong mi trng dung dch lng c sc kh lin tc. Mi trng dinh dng phi c nhng thnh phn chnh sau:
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 Ngun cacbon: glucose, maltose, r ng hoc tinh bt thy phn s b hoc c th t ngun phi gluxit nh: glyxerol, cc axit bo. Ngun Nit: Nit v c: (NH4)2SO4, NaNO3, (NH4)NO3, Ure Nit hu c: thng l nc chit malt, nc chit ng, hoc cao ng,cao nm mem, pepton, bt c, kh du. Mui khong v Vitamin: MgSO4, K2HPO4, KH2PO4, K2SO4, FeSO4Vitamin B1, B12, biotin (B8). u im ca phng php ny: C tnh lin tc nn tit kim c din tch sn xut. D c gii ha v t ng ha do nng sut cao. S dng hp l cc cht dinh dng ca mi trng. Enzyme thu c t ln tp cht. Nhc im ca phng php ny: Nng Enzyme trong canh trng thp, do phi c c nn gi thnh cao. Tn nhiu in nng do sc kh lin tc. Khi khng m bo c v trng tuyt i th d xy ra s nhim ton b khi mi trng. Bc 4: Tch tinh ch Enzyme: 1. Cc dng ch phm Enzyme:
a) Ch phm th: (s dng trong lnh vc cng nghip sn xut cn, x l mi trng).

- Ch phm th t canh trng lng: Mi trng nui VSV sau khi lc nng Enzyme rt thp nn bc u ngi ta phi c c. Dch lc t canh trng c nng cht kh t 4 6 g/l c c chn khng. - Ch phm th t canh trng b mt: Canh trng rn sau khi nui VSV c sy nhit 40oC n m cn khong 12% thu c ch phm th dng kh.
b) Ch phm k thut: l ch phm c tinh ch s b,trong 1 s Enzyme v Protein tp

c tch ra. S dng trong cng nghip thc phm gi mu sc, hng v v cht lng sn phm.
c) Ch phm Enzyme tinh khit: c loi b hon ton cc Protein v Enzyme tp,ch cn li

duy nht 1 Enzyme mong mun. Dng trong y hc, nghin cu khoa hc v phn tch. 2. Khu tr ca Enzyme: Da vo v tr khu tr ca Enzyme trong t bo, ngi ta chia Enzyme thnh 3 nhm chnh:
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- Enzyme ngoi bo: l Enzyme c sinh tng hp ra trong t bo, sau tit ra ngoi mi trng trong qu trnh nui cy chm. Ch yu l Enzyme hydrolase. - Enzyme ni bo: l Enzyme c tng hp v s dng bn trong t bo di dng lin hp, lin kt hoc di dng b nht trong cc bo quan ca t bo. - Enzyme Periplasmid: l Enzyme nm ngoi mng sinh cht nhng trong mng t bo. 3. Cc phng php trch ly cc Enzyme: * C s chung trch ly cc Enzyme: - trch ly cc Enzyme ra khi t bo phi ph v thnh t bo, mng t bo v nhng cu trc di t bo bng nhng phng php l hc hoc ha hc khc nhau. * Phng php ph v t bo: a) Phng php c hc: - Ph v t bo bng sng siu m: + Nguyn l: Sng siu m chuyn ng theo hnh sin vi tn sut cao (18KHz 1MHz) to nn cc vi bt. Cc vi bt s ln ln trong pha gin ca sng m thanh v b xp i trong pha nn. Vic xp i cc bng bt s chuyn nng lng m thanh thnh nng lng c hc. - Ph v t bo bng my ng ha cao p: My s dng ph v t bo ph bin l dng Manton Gaulin APV. Bm y a dch huyn ph t bo qua 1 van kim tra ti xilanh bm. T bo c y vi p sut 150 Mpa v vi vn tc dng ti 10.000 l/h qua 1 van x c ming hp. Do c ming hp l phn then cht ca my nn n khng thch hp khi s dng ph v cc c th c dng si m thng c s dng ph v cc c th n bo. - Ph v t bo bng phng php lnh ng: + Huyn ph t bo di dng bt nho c lm lnh ng -20oC ri nn di p sut cao qua cc l hp ca my nn hughes th t bo s b ph v do s thay i pha v th tch cng nh lc ct ca cc tinh th . b) Phng php khng c hc: - Sc thm thu: c th lm dung gii t bo bng sc thm thu khi cho dung dch huyn ph t bo vo trong 1 mi trng nhc trng (20% saccaroza). - X l bng kim: pH = 11,5 12,5 s lm thy phn mng t bo v do s gii phng cc Enzyme. - S dng cht ty ra: - Dung gii Enzyme:
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4. Phn on v tinh ch Enzyme: - Cc phng php kt ta. - Cc phng php phn tch lng lng. - Phng php lc qua mng siu lc. - Phng php sc k. Bc 5: Kim tra tinh khit ca Enzyme bng phng php in di. Bc 6: nh gi ch phm Enzyme (xc nh hot v hot ring Enzyme). B. Thu ch phm Enzyme t ng vt. Tch, tinh ch pepsin: Nguyn liu (Mng nhy d dy) X l (ra) Nghin T phn (HCl 5%) to = 42oC, thi gian: 40 42h (1h khuy 1 ln) Gn, lc dch t phn Kt ta (= NaCl 22%) Tch ta B sung cht n nh Sy (38 40oC) Ch phm Pepsin C. Thu ch phm Enzyme t thc vt: Tch tinh ch Tyrosinase nm n:
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Bc 1: + Kho st hot tnh t 7 loi nm n: nm hng, nm s + Xc nh hot tnh Tyrosinase bng phng php khuch tn trn a thch. Bc 2: Chit Enzyme t nm m Agaricus bisporus: Dung mi: nc ct, hu c (butanol, mui long, axeton) Nm lnh qua m 20oC trc khi chit Enzyme. Ra nm bng axeton (1,3 l/kg nm) 2 ln. Nghin nm 1 pht (axeton 30%, 1 l/kg nm + 5% axit ascorbic 0,5 M). Ly tm 6000 vng/pht, 5 pht, 4oC. Bc 3: Tinh sch Enzyme: + Kho st tc nhn kt ta Tyrosinase + Tinh sch Tyrosinase bng phng php sc k trao i anion ( DEAE sepharore HE 16/10) trn h thng FPLC. * Tinh sch Enzyme: Chn nha trao i ion. Dung dch m n nh ct VD: Dung dch m axetat pH = 4,7 (1) V = 0,5 ml/pht Dung dch m y Enzyme VD: Ging dung dch (1) nhng b sung thm NaCl 0,5M. Phn on chit: v = 0,4 ml/pht 0,5 2 ml/pht Bc 4: Kim tra tinh sch Protein bng phng php in di trn Gel Poly acrylamide SDS PAGE Bc 5: nh gi Enzyme 1.3. NGUYN TC CA CC PHNG PHP KT TA: a) ng in: - pH ca protein Enzyme ti tng in tch = 0 gi l im ng in (PI), tc l khng c lc y tnh in nn cc protein enzyme s kt hp vi nhau to ra kt ta. b) Kt ta bng (NH4)2SO4:
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- ha tan ca mt s Protein enzyme tng cng vi lc ion (M) ca mi trng. M= .Ci.

Trong : Ci: nng ca loi ion : in tch ca loi ion . - Khi lc () vt qua 1 ngng no th ha tan li gim nhanh v protein enzyme c th kt ta Y= Trong : Y: nng amon sulffat bo ha G: lng (gam) amon sulffat trong 1000 ml dung dch bo ha. - u im: khng lm bin tnh Enzyme - Nhc im: + Mt lm sch thp + Phi loi mui (bng 2 phng php): Lc qua gel Sephadex G25 Thm tch bng mng xelophan. c) Kt ta bng dung mi hu c: - Khi thm dung mi hu c trung tnh vo dung dch Enzyme lm gim hng s in mi, lm tng lc ht tnh in gia cc Protein Enzyme lm cho phn t protein lin hp li to kt ta. - Dung mi hu c c dng ph bin nht kt ta l: etanol, izopropanol, axeton. - Mi Enzyme kt ta nng dung mi hu c khc nhau. - u im: + Mc lm sch cao + D loi dung mi hu c (cho bay hi). - Nhc im: D lm bin tnh Enzyme nn phi lm nhit thp < 0oC d) Phng php siu lc: - Da vo khi lng phn t ca Protein Enzyme - S dng mng c kch thc l ln loi tp cht c M > M protein enzyme - Sau dng mng c kch thc l nh, loi tp cht c M < M protein enzyme 1.4. NGUYN TC CC PHNG PHP SC K * Sc k trao i ion:
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- Sc k trao i ion da vo s trao i ion gia protein tch in v cc ion ca cht nha trao i ion, protein tri du c gi li trn ct cn protein cng du b ra tri ra khi ct - Sau dng dung dch m thch hp v NaCl c nng tng dn y protein enzyme ra khi ct. Protein enzyme c i lc vi nha yu nht s b y ra khi nha trc nht. * Sc k lc Gel: - S lc gel l s tch cc phn t c kch thc khc nhau, c tin hnh da trn c s kh nng chui vo bn trong l cc ht gel. Gel c s dng rng ri nht l gel sephadex v gen polyacrylamide. Sephadex: Khi cho 1 hn hp nhiu cht chy qua ct cha sephadex c trong nc th: + Cht c ht nh chui vo bn trong l gen ra chm hn + Cht c ht ln chui ra ngoi ra trc Gel polyacylamide: l polyme ca acrylamit v bisacrylamit c cu trc cc lin kt ngang to ra 1 mng xp ( ging bt bin ). Cc cht phi chui vo l gel mi ra c v vy nhng cht c khi lng phn t nh ra trc v ln ra sau. * Sc k i lc: - Sc k i lc da vo kh nng lin kt c hiu v thun nghch ca 1 Enzyme vi 1 phi t c gn bng lin kt ng ha tr vo 1 cht mang khng ha tan cha trong 1 ct. - Khi cho 1 hn hp c cha Enzyme mun lm sch i qua th ch c Enzyme quan tm b gi li, cn tt c cc Enzyme khc khng tng tc c vi phi t s b tri ra khi ct. - Sau s dng cht ra gii y Enzyme quan tm ra khi ct 1.5. PHNG PHP NH GI HIU SUT V TINH SCH CA CH PHM ENZYME: * nh gi thng qua xc nh n v o hot xc tc ca Enzyme: - n v hot Enzyme l: lng Enzyme c kh nng xc tc chuyn ha 1 lng c cht nht nh trong 1 thi gian nht nh iu kin tiu chun. + n v IU: l lng Enzyme c kh nng xc tc chuyn ha c 1micromol c cht sau thi gian 1 pht iu kin tiu chun. 1IU = 1 M c cht ( 10-6M/pht) + n v Katal (Kat): l lng Enzyme c kh nng xc tc chuyn ha 1mol c cht trong thi gian 1 giy iu kin tiu chun. 1 Kat = 1 mol c cht/giy 1IU = x10-6 kat = 16,67nkat

2 n v ny thng s dng nh gia Enzyme mnh hay yu. Xc nh hot UI/ml, g (U)
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Xc nh hm lng protein mg/ml, g (tng) Xc nh hot ring UI/mg protein * nh gi tinh khit bng phng php in di: - in di l qu trnh dch chuyn ca cc phn t tch in trong in trng. Tc dch chuyn ca cc phn t ph thuc vo in tch, kch thc v khi lng phn t. - Gel thng s dng trong in di: + Gel polyacrylamid + Gel tinh bt Thc t, in di trn gen polyacrylamid c mt SDS (SDS PAGE) hay c s dng nht.

1.6. ENZYME TI T HP L G? CC BC THU ENZYME TI T HP. a) Enzyme ti t hp (Protein ti t hp): l protein do 1 sinh vt c DNA bin i sn xut ra nh thm vo h gen ca sinh vt on DNA m ha cho protein quan tm. b) Cc bc thu Enzyme ti t hp: 1. Tch tinh sch DNAtng s, ARNtng s 2. Nhn DNAtng s = k thut PCR hoc RT PCR (nhn CDNA) 3. Tch dng DNA vo vector tch dng s dng k thut in di. 4. Biu hin vector tch dng mang gen m ha Enzyme vo vector biu hin Thng ghp vector tch dng vi vector biu hin lm mt. 5. Biu hin vector biu hin mang gen m ha Enzyme thnh t bo ch. 6. iu kin nui VSV ti t hp sinh tng hp Enzyme mong mun cao nht: iu kin v mi trng dinh dng. B sung cht cm ng: IPTG (vi khun); metanol (nm men); ng glucose (nm mc). 7. Tch, tinh ch Enzyme: ch yu dng sc k i lc. 8. Kim tra tinh sch bng phng php in di. 9. nh gi ch phm thu c: - Xc nh hot Enzyme, Protein, hot ring, hot tng. - Mc tinh sch.

CHNG II: ENZYME C NH 2.1. KHI NIM ENZYME C NH: - Enzyme c nh l enzyme c gn ln cc cht mang khng ha tan hoc c gn vi nhau bng lin kt ng ha tr to nn i phn t enzyme khng ha tan. 2.2. U IM , NHC IM CA ENZYME C NH * u im: - Mt lng enzyme c th c s dng lp i lp li nhiu ln trong mt thi gian di. - Enzyme khng tan ln vo trong sn phm nn khng gy nh hng xu n mu sc, mi v sn phm. - C th lm ngng nhanh chng phn ng khi cn thit bng cch tch Enzyme ra khi c cht. - Bn vi pH, to, dung mi hu c. * Nhc im: - S chuyn khi chm. - C th mt hot tnh sau khi c nh. - Hiu qu km vi c cht c M ln. - Mt tnh thch nghi hnh th. 2.3. CC PHNG PHP C NH ENZYME a) Gn bng lin kt ng ha tr: * Gn cc Enzyme ln cc cht mang: - iu kin ca cc cht mang: + ha tan thp v bn vng i vi cc tc ng c hc, ha hc, sinh hc. + Khng gy tc dng km hm Enzyme. + Hp ph chn lc i vi cc protein khc. + Cht mang c bn cht ho nc v cht mang k nc c th c ch Enzyme va c lin kt. + Cht mang tt nht l cht mang c cha in tch tri du vi Enzyme. - Mt s cht mang thng c s dng: + Cht mang hu c: Chitoxan, Dextran, Agarose, Sepharose, Collagen. + Cc dn xut ca Cellulose: Cacboxyl metyl cellulose (CM cellulose) Dietyl aminoetyl cellulose (DEAE cellulose)
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 DEAE Sephadex, CM Sephadex. + Cc polyme tng hp: polyacrylamit, polystirol + Cc cht v c: Silicagel, nhm hydroxyl - Phng php gn Enzyme ln cht mang bng lin kt ng ha tr: (2 phng php) + Gn 1 giai on: C th gn 1 giai on nu cht mang c cha cc nhm c kh nng gn trc tip vi nhm NH2 ca protein enzyme. + Gn 2 giai on: Giai on 1: Hot ha cht mang Giai on 2: Kt hp Enzyme vo cht mang hot ha. - u im ca phng php: C nh Enzyme ln cht mang to bn cao cho lin kt gia protein v cht mang, thch hp trong cc phn ng lin tc. - Nhc im ca phng php: + Lng Enzyme c nh c thp. + Hot tnh Enzyme c th b gim do s bin i cu trc hnh th Enzyme. * Gn cc Enzyme vi nhau nh Glutaraldehyt: - Tc nhn kt gn Glutaraldehyt vi t l tc nhn Enzyme khong 1 10% w/w to lin kt cho ng ha tr gia cc phn t Enzyme hnh thnh i phn t Enzyme khng ha tan. - u im: Lin kt to ra rt bn trc cc tc nhn pH, to. - Nhc im: Chi ph cao, km hiu qu (hot tnh Enzyme gim).

b) Phng php gi Enzyme trong khun Gel: * Gi di dng ht: - Cho dung dch Enzyme vo dung dch Na alginat, caraghenan, chiltosan. Sau cho hn hp Enzyme Gel vo thit b to ht (ng knh 0,5 4mm); Cc ht cha Enzyme v Alginatnatri cho vo dung dch CaCl2. * Enzyme b nht trong cc l nh dng si: - Cho hn hp Enzyme polyme (collagen) vo thit b p n c p sut to si, cc si c cho vo b c cha toluol lm bn si. - Hoc c th nht Enzyme trong cc l nh ca si tng hp (cellulo tralaxetat) bng cch cho cc dng lng protein enzyme chy tun hon bn trong si. * Gi enzyme trong bao vi th:
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- Cho dung dch H2O Enzyme vo monome ho nc (Glycol) to thnh nh tng, sau thm monome k nc to mng. - B sung cc tc nhn hot ng b mt lm bn nh tng v iu chnh kch thc l t 1 100 m. - Mng ny c kch thc l xp nh ngn chn s khuch tn Enzyme ngoi nhng ln cho c cht i vo v sn phm i ra. * Phng php tin polyme: - To lin kt cho gia cc tin polyme bng chiu quang gi Enzyme. - Khi chiu tia cc tm gn (360nm) ln dung dch c cha cht tim polyme, Enzyme v cht nhy quang (Benzoin etylete, Benzoin izobutylete) 3 5 pht s to ra lin kt cho gia cc gc ca tin polyme, t Gel c hnh thnh v bao ly Enzyme. Phng php tin polyme Urtan: - Trn cc cht tin polyme urtan vi dung dch nc ca Enzyme. Trong dung dch nc, cc gc chc nng izoxinat 2 u urtan phn ng vi nhau to thnh lin kt cho ur - Gel c hnh thnh sau vi pht v bao ly Enzyme.

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2.4. CC C TNH ENZYME C NH a) Enzyme c nh c hot ring thp hn hot ring ca Enzyme t do bi: - Khi gn Enzyme vo cht mang di nh hng ca in tch cht mang lm cho hnh th enzyme thay i.=> lm kh nng xc tc thay i - Cc Enzyme c gi trong khun Gel: mt s c cht c kch thc ln khng xc tc c vi Enzyme. - Do tng tc protein protein gia cc phn t Enzyme c nh lm bin dng 1 phn cu trc khng gian ca cc phn t Enzyme. b) Hng s ng hc Enzyme c nh tun theo ng hc Michaelis Menten V= Km nh i lc E v S ln v ngc li Tuy nhin c 1 s sai khc: - Nu cht mang tch in cng du vi S th: Km c > Km t do - Nu cht mang tch in tri du vi S th: Km c < Km t do - Nu cht mang khng cha in tch th: Km c Km t do c) Enzyme c nh bn vi nhit hn so vi Enzyme t do. VD: bn ca Enzyme t do v c nh ca _D_Glucosidase.

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d) pH ti u ca Enzyme c nh thng b chuyn sang min kim hoc axit so vi pH ti u ca Enzyme t do.. - Nu Enzyme lin kt vi cht mang c in tch m th pH ti u s chuyn sang pha pH kim 1 2 n v so vi Enzyme t do. - Nu Enzyme lin kt vi cht mang c in tch dng th pH ti u s chuyn sang pha axit. e) Enzyme c nh c thi gian bo qun lu hn v bn vi cc cht km hm cng nh cc tc nhn bin tnh. VD: Tripsin lin kt ng ha tr vi Sephadex bng brom cyanua gi trong dung dch ur, sau 24h vn gi c 75% hot so vi ban u. 2.5. NG DNG ENZYME C NH a) Trong Y hc: - L-Asparaginase c nh trong microcapsule iu tr bnh bch cu, c ch s pht trin ca u c tnh do s c mt ca L-Asparagin. - Chymotrysin, Superoxit dismutase chng vim. - Streptokinase, Urokinase, Lumbrokinase lm tan cc cc mu ng gy tc nghn ng mch, vim tnh mch. - Urokinase gn trong microcapsul c s dng c hiu qu loi ur mu trong chy thn nhn to. b) Trong cng ngh ha hc: - Sn xut L-axit amin s dng aminoacylase c nh trn DEAE Sephadex. - Sn xut Acrylamit. - Sn xut khng sinh: Penicillin. c) Trong cng ngh mi trng: - Enzyme c nh c s dng mt cch c hiu qu trong cng ngh mi trng ( x l nc thi ) bng phng php sinh hc. d) Trong cng ngh thc phm: - Sn xut Glucose: S dng -Amylase v Glucoamylase c nh sn xut dch Glucose nng cao (92 96%) t tinh bt. - Sn xut sa khng c lactoza (LACT ATD): + S dng -galactosidase (lactase) thy phn lactose thnh glucose v galactose l nhng ng d tiu ha, ngt cao hn. + Lactose t nm men c c nh bng cch gi trong si Cellulose triacetate, c th s dng thit b phn ng gin on (STR).

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+ Lactose t nm mc c c nh trn xp Silicdioxit, b sung glutaraldehyt s dng thit b phn ng dng ct (PBR). - Sn xut siro ng vi nng Fructose cao (HFCS): + S dng gluco isomerase c nh chuyn ha glucose thnh fructose c ngt gp 2,5 ln so vi glucose. + Nng glucose 95%, pH = 7,5 8,5; to = 55 60oC thu c 42% fructose + 50% glucose + 8% oligosaccarit. - Sn xut ng nghch o: S dng Invertase chuyn ha saccarose thnh glucose v fructose. e) Trong cng nghip ty ra: + S dng protease c nh, lipase c nh, cellulase sn xut cc loi x phng. - Sn xut Bio diesel: lipase c nh chuyn ha du v m to bio diesel. - Sn xut vin nang du n: Lipase c nh chuyn ha du n to vin nang (cho ngi b nhim m mu). f) ng dng to KIT enzyme: - KIT Ach Enzyme (Acetyl cholin esterase) - KIT Tyrosinase: S : Cht mang C nh TYROSINASE 40 50 u/ml, 10pht Sy 35oC, 30pht KIT TYROSINASE - Enzyme c nh trong sn xut pin sinh hc Glucoza H2O2 A.glucomic (H+) + H2O2 O2 + H2O

CHNG III: IN CC SINH HC ( BIOSENSORS) 3.1. KHI NIM IN CC SINH HC: 1. B in cc sinh hc thc cht l mt thit b phn tch chuyn i cc p ng sinh hc thnh cc tn hiu in, quangT cc tn hiu , ngi ta c th o c nng cc cht cn phn tch.
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S: c cht cn o (glucose, ure) a: mng sinh hc: l mng c c nh cc cht c hot tnh sinh hc, nh enzyme, ADN, VSV hoc phn t min dch (khng nguyn, khng th). b: thit b chuyn i: l mt thit b chuyn i tn hiu nhm chuyn i p ng sinh hc thnh tn hiu (in p, dng in, in th, nhit, quang) c: b khuch i tn hiu: thng s dng vi cc trng hp tn hiu in hoc n gin ch l thu cc tn hiu gc a vo b x l tn hiu. d: b x l tn hiu: x l tn hiu a ra kt qu, thng qua vic so snh vi cc mc chun hoc cng thc tnh ton. e: biu hin kt qu 3.2 CC LOI IN CC SINH HC 3.2.1: Khi nim in cc in ha: + Nguyn tc in cc in ha: - in cc Enzyme c ch to di dng 1 mng m 1 mt tip xc vi mi trng c cht cn mt kia tip xc vi vng o. - Phn ng gia c cht enzyme xy ra trn lp mng. - Kt qu s thay i nng lng t do c in cc thu nhn ri truyn qua h thng o di dng o dng in hoc o in th. * in cc o pH: VD: Xc nh glucose D-glucose D-gluconat + H+

o lng H+ to thnh (o pH sau khi gii phng H+) s xc nh c hm lng D-glucose. * in cc nhn bit kh: VD: Xc nh ure: NH2CONH2 + H2O NH2CONH2 + H2O + 2H+ 2NH3 + 2 + H+ + CO2

o lng gii phng ra qua o pH s xc nh c hm lng ure ban u. Khi xc nh ure qua CO2 th in cc enzyme s c kt hp vi in cc CO2. * in cc ENFET: VD: o nng Penicillin

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Penicillin + H2O

axit penicilloic

Penicillinase ng vai tr l cht xc tc cho phn ng ny. Nu c nh penicillinase ln mng v ngm n vo trong dung dch c penicillin th ion H+ s c sinh ra, bi penicillin l 1 axit mnh. S thay i pH c o bng thit b FET nhy ion H+.

+ Nguyn tc in cc o dng in: - Khi t 1 hiu in th gia 2 in cc (650 mV) cng dng in chy gia 2 in cc t l vi mt ca cc ht in tch. - Cc in cc o dng in: in cc Oxy, in cc hydroperoxyt (CO2 v H2O2) u l nhng sn phm c sinh ra trong phn ng c cc cht trung gian Oxy ha kh n nh dng in.

* in cc H2O2: o hm lng glucose: Glucose H2O2 a.glucomic + H2O2 O2 + 2H+ + 2e-

- Di tc ng ca in p phn cc H2O2 s b oxy ha mt phn cch gia anod v mng. - Cng dng in t l vi mt cc ht tch in v xc nh c lng H2O2 b oxy ha t suy ra c lng glucose c trong mi trng nghin cu. VD: o axit lactic s dng lactatoxydase (LOD) Lactat + O2 * in cc O2 (Clark) - o hm lng ng glucose: Glucose + O2 A-gluconic + H2O2 pyruvat + H2O2

- Di tc dng ca in p phn cc 650 mV t vo gia 2 in cc, oxy khuch tn qua mng s b kh ti catod theo phn ng: O2 + 4e- + 4H+ 2H2O - Cng dng in t l vi lng oxy b kh, do t l vi hm lng oxy v glucose.

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3.2.2. Nguyn tc in cc o nhit (in cc nhit in tr). - Cc phn ng gia c cht v cht xc tc sinh hc thng gii phng ra nhit v ngi ta c th o lng nhit gii phng ra bng 1 thit b o nhit (in cc o nhit) s tnh c lng c cht ban u. - Khi lng nhit ta ra cng ln th nhy pht hin cng ln, cng chnh xc. - Mun nhit ta ra nhiu th ngi ta phi gn trn in cc t 2 n vi Enzyme. VD: S dng ng thi glucooxydase oxy ha glucose v catalase. Glucose 2 H2O2 Gluconic acid + H2O2 2H2O + O2

Khi phn ng xy ra cng lc th lng nhit ta ra nhiu hn. - Bt buc h thng thit b phi cch nhit tt trnh tc ng ca nhit khng phi do phn ng sinh ra. 3.2.3. Nguyn tc in cc o quang: - Dung dch phn tch tip xc vi mng sinh hc v khi phn ng Enzyme xy ra th s c hin tng nh sng b hp th hoc pht ra nh sng mu. - Do hp th nh sng (phn x quang) hay s pht quang nh sng ca cc cht to thnh bc sng nht nh s bit c nng cc cht cn phn tch. * o hp ph, phn hp ph (phn quang) - Nguyn l:+ C cht phn ng vi Enzyme to sn phm. P tc dng vi cht mu to mu. o nh phn x bit nh sng c hp ph bi cng mu P. Bit c nng cht cn o. + Nng sut phn x t mt phn x tun theo phng trnh: %R = . R0

Trong : R: Nng sut phn x. R0: Hng s phn x ca mi trng chun. I: Cng ca tia ti. I0: Cng ca tia phn x. Trong R c quan h vi nng cht hp th mu theo phng trnh:

=
Vi: C: nng cht hp ph.
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k: hng s hp ph. : hng s tn sc. VD: o glucose trong mu nh thuc hin mu MBTH v DMAB. Glucose mu + O2 axit gluconic + H2O2 MBTH DMAB (mu xanh)

H2O2 + MBTH + DMAB

Nu H2O2 c nng cao th phi dng Enzyme catalase. Mu phn tch sau c em i o nng sut phn x bc sng thch hp lng glucose. tnh c hm

- Vi in cc sinh hc da trn nn tng ca s phn x ton phn th b mt dy dn c ph cc khng th s tip xc vi mu phn tch. Khi sng nh sng i n lp mng nhy th ch c 1 phn nh sng nh sng b hp th vo trong mi trng, cn tt c chng b phn x li nhiu ln v dn dn ngun sng b suy gim. Da vo lng nh sng phn x, ta c th tnh c hm lng mu phn tch. * in cc o pht quang: - C cht tc dng vi Enzyme c dnh to thnh sn phm c kh nng pht ra nh sng mu. - o nh sng mu 1 bc sng nht nh s suy ra c nng c cht. VD: Xc nh cc vi khun trong thc phm theo phn ng: Luciferin + ATP Oxyluciferin + AMP + pyrophotphat + CO2 + nh sng vng

nh sng vng o bc sng 562 nm. T xc nh c lng vi khun c mt trong thc phm. 3.2.4. Nguyn tc in cc min dch: - Da vo kh nng bt cp gia khng th v khng nguyn. + Phn ng khng th khng nguyn c xc nh bng nhiu cch, trong c k thut ELISA + in cc min dch ELISA kt hp vi in cc in ha hoc o quang nh hng s bin i nng c cht trong cc phn ng xc tc bi Enzyme nh du. + Phn ng gia khng th v khng nguyn c s cn bng ng, c trng cho cn bng l hng s cn bng k: k= : hng s vn tc phn ng thun. : hng s vn tc phn ng nghch. k cng ln th kh nng kt gn gia khng th v khng nguyn cng ln.
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3.2.5. in cc VSV - c dng phn tch nhanh COD, BOD da vo s h hp v trao i cht ca VSV - C 2 loi: + in cc o s thay i hot lc h hp ca VSV c nh vi mt thit b o in th. + in cc o cc cht sinh ra t VSV. 3.2.6. in cc ADN * Nguyn tc o in cc ADN: - S dng bioreceptor l cc nucleic acid nhm pht hin cc ADN mc tiu. - p ng sinh hc c hiu ca in cc ADN l phn ng bt cp theo nguyn tc b sung gia cc trnh t ADN si n (mc tiu) v ADN d ( probe ). - Cc base lin kt bng lin kt hydro. - Hai si ADN lai vi nhau. - Cc base lin kt b sung hon ton to si i ADN bn. Khi bin tnh to 2 si n ADN tr li, mt hay vi base lin kt sai to ADN khng bn. - in cc ADN gm nhiu loi khc, c pht trin da trn cc k thut thu nhn v chuyn i tn hiu khc: in ha, quang * in cc ADN in ha trc tip: - Cc ADN mc tiu lai vi ADN probe trn lp nhn bit. - iu kin in th ph hp base (G) ca ADN mc tiu b Oxy ha gy ra hin tng tch in trn b mt in cc. - S bin i in tch c ghi bng in cc o dng in. - nhy kh cao. - S dng in cc carbon, vng hay indiunlinoxide (ITO) - Nhc im: Nhiu nn ln nn phi kh ADN bng t trng. * in cc ADN in ha gin tip: - Oxy ha ADN mc tiu gin tip thng qua cc phn t in ha trung gian: poly piridin ca Ru. Cc phn t trung gian s ly in tch t cc base (G) trn ADN mc tiu v chuyn ti in cc gy hin tng tch in in cc. o mt in tch = o in cc dng in. Ph hy mu.
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* in cc Nano (khng thi) 3.3 NG DNG IN CC SINH HC - Trong cng nghip thc phm: l mt thit b theo di v o lng nhanh, nhy cao, s dng thun li hn so vi cc phng php truyn thng. + o nng Glutamin v nng glutamat: Glutamin Glutamat glutamat + NH3 -xetoglutarat + NH3 + H2O2

+ o nng cholin bng cch s dng cholinoxydase: Cholin + O2 H2O2 + betain aldehyt Betain aldehyt + O2 H2O2 + betain + o hm lng cn bng cch s dng alcohol oxydase Alcol + O2 H2O2 + aldehyt + Pht hin s c mt ca vi khun trong thc phm: ATP + D-luciferin (Pyrophosphat) + in cc min dch o hm lng albumin, insulin. + Kim tra hm lng cloramphenicol trong sn phm thy sn + Xc nh hm lng mycotoxin trong ng cc v thc phm. - Trong mi trng: + nh lng ion kim loi nng. + nh lng phenol. + nh lng phosphat, nitrit, nitrat, sulfat. + nh lng cc cht c. - Trong y t: + o nng glucose. + o nng ur. Oxyluciferin + AMP + CO2 + nh sng vng

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