Enzyme protease M u

Ngy nay cng vi s pht trin mnh m ca Cng ngh sinh hc, cc ch phm enzyme ac sn xut cng nhiu v c s dng trong hu ht cc lnh vc nh: ch bin thc phm,nng nghip,chn nui,y tHng nm lung enzyme c sn xut trn th gii t khong trn 300.000 tn vi trn 500 triu USD,c phn phi trong cc lnh vc khc nhau. Phn ln enzyme c sn xut quy m cng nghip u thuc loi enzyme n cu t,xc tc cho phn ng phn hy.Khong 75% ch phm l enzyme thy phn c s dng cho vic thy phn c cht t nhin. Protease la enzyme c s dng nhiu nht hin nay trong mt s ngnh sn xut nh: ch bin thc phm ( ng t sa lm cho phomt,lsmf mm tht,b sung tng cht lng sn phm trong sn xut bia.x l ph ph phm trong ch bin thc phm(sn xut cht ty ra,thuc da,y t,nng nghip) Qua nhiu nm ,vic gia tng s dng vi sinh vt nh l mt ngun cung cp protease ci thin ng k hiu qu sn xut v sn phm c to ra nhiu hn.Tuy nhin gi thnh ch phm protease cn kh cao,do cng hn ch vic s dng rng ri enzyme trong sn xut.Cc ch phm thu c sau qu trnh nui cy sn xut enzyme cha phi l ch phm c tinh khit cao v protein ch chim 20-30%. V vy, vic nghin cu ci tin phng php tch v tinh ch enzyme nhm thu ch phm c tinh khit cao rt cn thit. tch v tinh ch enzyme ni chung v protease ni ring th c mt s phng php ha l khc nhau. C th chia lm ba nhm chnh sau: - Phng php kt ta - Phng php sc k - Phng php phn tch h hai pha nc

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Enzyme protease
Protease phn b thc vt, ng vt, vi sinh vt. Tuy nhin, ngun enzyme phong ph nht l ngun t vi sinh vt, c hu ht cc vi sinh vt nh vi khun, nm mc v x khun, C th ni vi sinh vt l ngun nguyn liu thch hp nht sn xut enzyme quy m ln dng trong cng nghip v i sng.

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Phn I Tng quan v enzyme protease
1.Gii thiu chung Nhm enzyme protease xc tc qu trnh thy phn lin kt peptid ( -CO-NH)n trong phn t protein, polypeptid n sn phm cui cng l cc acid amin. Ngoi ra, nhiu protease cng c kh nng thy phn lin kt este v vn chuyn acid amin. H2NCHCONHCHCO -.- NHCH-COOH R1 R RX
+H2O

H2N-CH-COOH + H2N-CH-CONH-CH-COOH R1 R2 RX

M hnh enzyme protease

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Protease cn thit cho cc sinh vt sng, rt a dng v chc nng t mc t bo, c quan n c th nn c phn b rt rng ri trn nhiu i tng t vi sinh vt ( vi khun, nm, virus) n thc vt ( u , da,..) v ng vt (gan, d dy b,..). So vi protease ng vt v thc vt, protease vi sinh vt c nhng c im khc bit. Trc ht h protease vi sinh vt l mt h thng rt phc tp bao gm nhiu enzyme rt ging nhau v cu trc, khi lng v hnh dng phn t nn rt kh tch ra di dng tinh th ng nht. Cng do l phc h gm nhiu enzyme khc nhau nn protease vi sinh vt thng c tnh c hiu rng ri cho sn phm thy phn trit v a dng.

Cu trc khng gian enzyme protease

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1.1 Phn loi Protease Protease (peptidase) thuc phn lp 4 ca lp th 3 (E.C.3.4).

Peptidase (E.C.3.4)

(protease)

Exopeptdase (E.C.3.4.11-17)

Endopeptidase (E.C.3.4.21-99)

Aminopeptidase

Serine proteinase
Cystein proteinase Aspartic proteinase

Carboxypeptidase
Metallo proteinase

S phn loi protease Protease c phn chia thnh 2 loi : endopeptidase v exopeptidase. Da vo v tr tc ng trn mch polipeptide, exopeptidase c phn chia thnh 2 loi : + Aminopeptidase: xc tc thy phn lin kt peptide u N t do ca chui polypeptide gii phng ra mt acid amin,mt dipeptide hoc tri peptide. + Carboxypeptide: xc tc thy phn lin kt peptide u C ca chui polypeptide v gii phng ra mt acid amin hoc mt dipeptide. * Da vo ng hc ca c ch xc tc ,endopeptidase c chia thnh 4 nhm:

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+ Serin proteinase: l nhng protein cha nhm OH ca gc serine trong trung tm hot ng v c vai tr c bit quan trng i vi hot ng xc tc ca enzyme. Nhm ny bao gm hai nhm nh: chymotrypsin v subtilisin.Nhm chymotrypsin bao gm cc enzyme ng vt nh chymotrypsin.trypsin,elastase.Nhm subtilisin bao gm hai loi enzyme vi khun nh subtilisin Carsberg, subtilisin BPN. Cc serine proteinase thng hot ng mnh vng kim tnh v th hin tnh c hiu c cht tng i rng. + Cysteine proteinase: Cc proteinase cha nhm SH trong trung tm hot ng. Cystein proteinase bao gm cc proteinase thc vt nh papayin, bromelin, mt vi protein ng vt v protein k sinh trng.Cc cystein proteinase thng hot ng vng pH trung tnh, c tnh c hiu c cht rng. + Aspatic proteinase: Hu ht cc aspartic proteinase thuc nhm pepsin. Nhm pepsin bao gm cc enzyme tiu ha nh: pepsin, chymosin, cathepsin, renin. Cc aspartic proteinase c cha nhm carboxyl trong trung tm hot ng v thng hot ng mnh pH trung tnh. + Metallo proteinase: Metallo proteinase l nhm proteinase c tm thy vi khun, nm mc cng nh cc vi sinh vt bc cao hn. Cc metallo proteinase thng hot ng vng pH trung tnh v hot gim mnh di tc dng ca EDTA. Ngoi ra, protease c phn loi mt cch n gin hn thnh 3 nhm: - Protease acid: pH 2-4 - Protease trung tnh: pH 7-8 - Protease kim: pH 9-11 1.2 c im v tnh cht ca protease vi sinh vt Cc cng trnh nghin cu protease vi sinh vt ngy cng nhiu. Cc kt qu nghin cu cho thy ngay c cc protease ca cng mt loi vi sinh vt cng c th khc nhau v nhiu tnh cht. Cn c vo c cht phn ng, pH hot ng

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thch hp,cc nh khoa hc phn loi cc protease vi sinh vt thnh 4 nhm sau: P-xerin, P-thiol, P-kim loi, P-acid Mt s tc gi khc chia protease ra 3 nhm, da vo hot ng pH ca chng bao gm: protease acid, protease trung tnh, protease kim. Trong 4 protease k trn, cc protease xerin v protease thiol c kh nng phn gii lin kt este v lin kt amide ca cc dn xut acid ca acid amin. Ngc li cc protease kim loi, protease acid thng khng c hot tnh esterase ca cc dn xut ca acid amin. Nhiu protease ngoi bo ca vi sinh vt c nghin ca tng i k v cu to phn t, mt s tnh cht ha l v c ch tc dng. Kt qu nghin cu cho thy trng lng phn t ca cc enzyme ny tng i b, nht l cc P-xerin. Cc P-xerin c trng lng phn t thp vo khong 20.000 27.000 dalton. Tuy nhin, cng c mt s p-xerin c trng lng phn t ln hn nh cc enzyme Pelicillium cyoneo-fulvum (44.000), Asp (52.000), Trng lng phn t ca cc protease kim loi ln hn so vi P-xerin ( vo khong 33.800 48.400). Protease thiol v nhiu protease acid cng c trng lng phn t vo khong 30.000 40.000 dalton. 1.3 Cu trc trung tm hot ng ( TTH) ca protease. Trong TTH ca protease vi sinh vt (VSV) ngoi gc aa c trng cho tng nhm cn c mt s gc aa khc. Cc kt qu nghin cu chung v TTH ca mt s protease VSV cho php rt ra mt s nhn xt chung nh sau: -TTH ca protease ln v bao gm mt s gc aa v trong mt s trng hp cn c c cc cofact kim loi. + Cc protease kim loi c TTH ln hn vo khong 21 A0 , c th phn bit thnh 6 phn di TTH ( subsite), mi phn di trung tm hot ng tng ng vi mi gc aa trong phn t c cht.

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+ i vi cc protease acid, theo nhiu nghin cu cu trc TTH ca cc tinh th protease acid ca Phizopus chinenis v endothia cho thy phn t cc phn t protease ny c 2 ht, gia chng c khe h vo khong 20 A0. Khe h ny l phn xc tc ca cc enzyme, cc gc asp-35 v asp-215 xp i din nhau trong khe y. - i vi cc protease khng cha cysteine, TTH ca chng c tnh mm do hn v cu trc khng gian ca chng khng c gi vng bi cc cu disulphide. Mc d TTH ca cc protease VSV c khc nhau nhng cc enzyme ny u xc tc cho phn ng thy phn lin kt peptid theo cng mt c ch chung nh sau: E+S enzyme S Enzyme-S + P1 enzyme + P2

Trong : E: Enzyme, S: c cht, Enzyme-S: phc cht enzyme-c cht, P1, P2: l sn phm u tin v th 2 ca phn ng.

Phn II Ngun thu nhn

Enzyme protease l protein c sinh vt tng hp trong t bo v l cht tham gia xc tc cho mi phn ng sinh hc. Chnh v th, mi vi sinh vt u c xem l ngun thu nhn sn xut enzyme. Nhng vn ch yu l 3 ngun chnh: ng vt, thc vt, vi sinh vt. 2.1 Ngun ng vt - Ty tng: y l ngun enzyme sm nht, lu di nht v c nhiu enzyme nht. D dy b: trong ngn th t ca d dy b c tn ti enzyme thuc nhm Protease tn l renin. Enzyme ny t lu c dng ph bin trong cng ngh phomat. Renin lm bin i cazein thnh paracazein c kh nng kt ta

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trong mi trng sa c nng Ca2+. y l qu trnh ng t sa rt in hnh, c nghin cu v ng dng y nht. Trong thc t nhiu ch phn renin b nhim pepxin ( trong trng hp thu ch phm renin b qu th. Khi d dy b pht trin y c kh nng tit ra pepxin) th kh nng ng t sa km i. Gn y c nhiu nghin cu sn xut protease t vi sinh vt c c tnh renin nh cc loi Edothia Parasitica v Mucor Purullus. Ngoi ra, ngi ta cn nghin cu thu nhn enzyme t rut c basa. Dch trch ly enzyme thu c t rut c basa c tng hot tnh cao nht l 15.78 UI/gCKNT trong iu kin trch ly : t l mu/dung mi: 1/1 (w/w), pH 9.5: nhit 35 0C: thi gian trich ly 10 pht. 2.2 Ngun thc vt C 3 loi protease thc vt nh Bromelain, Papain v Ficin. Papain thu c t nha c l, thn, qu u ( Carica papaya) cn Bromelain thu c t qu chi da, v da ( pineapple pant). Cc enzyme ny c s dng chng li hin tng ta trng ca Bia khi lm lnh ( chilling proofing) do kt ta protein. Nhng ng dng khc ca proease thc vt ny l trong cng ngh lm mm tht v trong mc tiu tiu ha. Ficin thu c t nha cy c ( Ficus carica). Enzyme c s dng thy phn protein t nhin.

Ngun protease thu t thc vt

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Thu nhn t ht cc ny mm: Malt l loi ht ha tho ny mm trong nhng iu kin nhn to( nhit , m, thi gian) xc nh gi l qui tc malt. Qu trnh sn xut malt bao gm cc khu sau: + Thu nhn, x l, lm sch, phn loa v bo qun ht. + Ra, st trng v ngm ht. + Um mm ta s thu c malt ti. + Sy malt ti. + X l v bo qun malt kh. Hot tnh protease trong ht ban u khng ng k nhng khi ny mm tng ln 4-8 ln, tng nhanh hn hot tnh amylase v t cc i vo khong ngy ny mm th 5. S thy phn protein bt u bng tc dng ca protienaza to thnh albumoza, polypeptit, pepton v sau di tc dng ca peptidaza to thnh cc acidamin. Tuy nhin s bin i ny thng khng hon ton v cc iu kin ny mm thng khng phi l iu kin ti thch cho hot ng ca h enzyme proteaza ca ht. Tuy nhin, v nhit ny mm tng i thp v thi gian ny mm tng i di s c s thy phn protein tng i su sc to thnh mt lng ng k cc polypeptit v cc acidamin. Tc ng ny ch din ra vi ni nh, trong khi protein lp ngn cch ni nh vi v v c phn v malt th khng b bin i c trong c qu trnh ch bin v sau. 2.3 Ngun vi sinh vt Vi sinh vt l ngun thu nhn enzyme khng l, enzyme protease phn b ch yu vi khun, nm mc v x khun,gm cc loi thuc Aspergillus, Bacillus, Penicillium, Clotridium, Streptomyces v mt s loi nm men. 2.3.1 Vi khun

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Lng protease sn xut t vi khun c c tnh vo khong 500 tn, chim 59% sn lng enzyme c s dng. Protease ca ng vt v thc vt ch cha mt trong hai loi endopeptidase hoc exopeptidase, ring vi khun c kh nng sinh ra hai loi trn, do protease ca vi khun c tnh c hiu c cht cao. Chng c kh nng phn hy ti 80% cc lin kt peptide trong phn t protein. Trong cc chng vi khun c kh nng tng hp mnh protease l Bacillus subtilis, B.mesentericus, B.thermorpoteoliticus v mt s chi Clotridium. Trong , B,subtilis c kh nng tng hp protease mnh nht. Cc vi khun thng tng hp c protease hot ng thch hp vng pH trung tnh v kim yu. Cc protease trung tnh ca vi khun hot ng trong khong pH hp ( pH 58) v c kh nng chu nhit thp. Cc protease trung tnh to ra dch thy phn protein thc phm t ng hn so vi protease ng vt v tng gi tr dinh dng. Cc protease trung tnh c kh nng i lc cao vi cc amino acid a bo v thm. Chng c sinh ra nhiu bi B.subtilis, B.mesentericus, B.thermorpoteoliticus v mt s ging thuc chi Clotridium.

Bacillus 2.3.2 Nm Nhiu loi nm mc c kh nng tng hp mt lng ln protease c ng dng trong cng nghip thc phm l cc chng: Aspergillus, A.terricola, A.saitoi, Cc nm mc ny c kh nng c ba loi protease: acid, kim v trung

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tnh. Nm mc en tng hp ch yu l cc protease acid, c kh nng thy phn protein pH 2.5-3. Mt s nm mc nhA.candidatus, P.camerberti, P.roqueforticng c kh nng tng hp protease c kh nng ng t sa s dng trong sn xut phomat.

Nm mc

2.3.3 X khun V phng din tng hp protease, x khun c nghin cu t hn vi khun v nm mc. Tuy nhin, ngi ta cng tm ra c mt s chng c kh nng tng hp protease cao nh: Streptomyces grieus, S.fradiae, S.Terimosus, X khun

Cc ch phm protease t x khun c bit nhiu l pronase (Nht) c chit tch t S.grieus, enzyme ny c tnh c hiu rng, c kh nng thy phn ti

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90% lin kt peptide ca nhiu protein thnh amina acid. Lin X (c) ngi ta cng tch c ch phm tng t t S,grieus c tn l protelin.

T S.fradiae cng c th tch chit c keratinase thy phn keratin. M, ch phm c sn xut c tn l M-zim dng trong sn xut Da. Protease t S.fradiae cng c hot tnh elastase cao, do chng c dng trong nghip ch bin tht. Hu ht cc protease phn ct protein cc lin kt c hiu, v th c th s dng cc enzyme ny theo chiu phn ng tng hp tng hp cc lin kt peptide inh trc. Yu t tng cng qu trnh tng hp bao gm pH, cc nhm carboxyl hoc nhm amine c la chn bo v, kh nng kt ta sn phm, phn ng trong h hai pha lng. C th ni vi sinh vt l ngun nguyn liu thch hp nht sn xut enzyme quy m ln dng trong cng ngh v i sng. Dng ngun vi sinh vt c nhng li ch chnh nh sau: Ch ng v nguyn liu nui cy v ging vi sinh vt. Chu k sinh trng ca vi sinh vt ngn: 16 100 gi nn c th thu hoch nhiu ln quanh nm. C th iu khin sinh tng hp enzyme d dng theo hng c li. Gi thnh tng i thp v mi trng tng i r, n gin, d t chc sn xut. Tuy nhin, trong mi trng hp cn lu kh nng sinh c t ( gy c, gy bnh) c bin php phng nga, x l. sn xut ch phm enzyme , ngi ta c th phn lp ging vi sinh vt c trong t nhin hoc ging t bin c la chn theo hng c li nht, ch tng hp u th mt loi enzyme nht nh cn thit no .

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Phn III Thu nhn v lm sch enzyme protease t vi simh vt v thc vt
3.1 Thu nhn v lm sch enzyme Protease t vi sinh vt. Ngun thu protease vi sinh vt ch yu l vi khun, nm mc v x khun. Qu trnh sn xut c ch phm enzyme vi sinh vt bao gm cc giai on ch yu: Tuyn chn v ci to ging vi sinh vt

Phng php bo qun ging vi sinh vt

Mi trng nui cy vi sinh tng hp enzyme

Tch v lm sch ch phm enzyme

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Cc cng on sn xut ch phm enzyme 3.1.1 Tuyn chn v ci to ging vi sinh vt cho enzyme c hot lc cao. chn ging vi sinh vt c kh nng sinh tng hp enzyme cao, ngi ta c th phn lp t mi trng t nhin hoc c th dng cc tc nhn gy t bin tc ng ln b my di truyn hoc lm thay i c tnh di truyn to thnh cc bin chng c kh nng tng hp c bit hu hiu mt loi enzyme no , cao hn hn chng gc ban u. 3.1.1.1 Phng php gy t bin. y l phng php hay c dng nht nhm : -To nhng t bin tng hp enzyme c cu trc bc 1 thay i do c th gim thay i vi kiu km hm theo c ch lin h ngc. Nu s thay i cu trc bc 1 xy ra vng trung tm hot ng hoc gn th c th lm thay i r rt hot tnh ca enzyme. Gy t bin on gene hot ho promotor lm tng p lc ca n i vi ARN-polymerase do lm tng tc sao chp m Dng bin php ny c th lm tng lng glucoza-6-phosphatdehydrogenaza ln 6 ln. Hin tng t bin thng lin h vi s thay i mt gene, chng hn b li mt bazo khi ti to phn t AND. to mt t bin gene c th dng tc nhn vt l (tia t ngoi, tia phng x) hay ho hc (cc ho cht) tc dng ln t bo sinh vt. 3.1.1.2 Phng php bin np. L s bin i tnh trng di truyn ca mt ni sinh vt di nh hng ca AND trong dch chit nhn c t t bo ca sinh vt khc. y yu t bin np l AND. S chuyn vt liu di truyn (AND) t t bo cho n t bo nhn c th xy ra trong ng nghim (invitro) khi cho t bo nhn tip xc vi dch chit t t bo cho m khng c s tip xc gia cc t bo.

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Cc t bo c th nhn bt k loi AND no ch khng i hi phi l AND t cc ging h hng. Tuy nhin t bo ch c th nhn mt s on AND nht nh, thng khng qu 10 on. Cc on AND c di trun trong bin np c M=106-107 v phi c cu trc xon kp. Hin tng bin np ph bin nhiu loi vi khun nh: Diplococus, Staphylocus, Hemophilus, Agrobacterium, Rhizobium, Bacillus, Xantomonas. 3.1.1.3 Phng php tip hp gene. Khc vi bin np, y vt liu di truyn ch c t t bo cho n t bo nhn khi hai t bo tip xc vi nhau. Do vy cc vi sinh vt c kh nng bin np th s khng c kh nng tham gia tip hp gene na. Hin nay qu trnh tip hp gene c nghin cu mt s loi vi khun nh E.coli, Salmonella, Pseudomonas aeruginosa. 3.1.1.4. Phng php ti np Vt liu di truyn (AND) c chuyn t t bo cho sang t bo nhn nh vai tr trung gian ca thc khun th (phage). Trong qu trnh ti np, cc on AND c chuyn t t bo cho n t bo tip hp vi AND ca t bo nhn. Do lm bin i tnh cht di truyn ca t bo nhn. 3.1.2. Phng php bo qun ging vi sinh vt. Khi s dng vi sinh vt sn xut enzyme cn chn ging thun chng, c kim tra y v cc c tnh ha sinh, vi sinh, nui cy v cn c bit l n iu kin bo qun ging. Thc t khi bo qun ging gc trong mt thi gian c th to ra cc bin d ngu nhin khng mong mun do nh k phi cy chuyn v kim tra li cc c tnh ban u. Phng php cy chuyn. y l phng php ph bin nht d thc hin bng cch gi ging trn mi trng thch (thch nghing, hp petri,) vi thnh phn mi trng nui cy v

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iu kin nui cy thch hp cho ging vi sinh vt . Sau khi ging mc tt cn bo qun nhit lnh 3-400C v sau mi tun phi cy chuyn li. Khi cy chuyn ch ly bo t hoc khun lc m khng nn ly c mi trng dinh dng m bo khng chuyn cc sn phm trao i cht vo mi trng mi (c th gy bin i bt li khng th lng ht c). Nu l x khun th khng nn bo qun ging trn mi trng thch m nn gi trong t kh trng. ko di thi gian bo qun ging t hng thng n 1 nm, ngi ta ph 1 lp paraphin lng tit trng trn b mt ging hn ch s pht trin ca n. Cn lu ch ph lp du sau khi cy vi sinh vt t n chin sinh l. Phng php cy chuyn rt c hiu qu bo qun cc ging nm men, vi khun v rt hu hiu, d dng trin khai ging ra sn xut ln, hn ch cc tai bin c th dn n h hng ging gc. 3.2. Bo qun bng phng php cy truyn trn mi trng thch Phng php lm kh Bng cch gi ging trn ct, t, silicagen trong iu kin kh ro (tt c u c kh trng cn thn). Trong iu kin nh vy s hn ch s pht trin tip tc ca ging khi bo qun. Phng php ny rt hay c s dng bo qun nm mc, x khun, mt vi loi nm men, vi khn thi gian gi ging c th c mt n. Phng php lm kh cng thc hin n gin, khng cn dng c t tin. Tuiy nhin ging nh phng php cy chuyn thi gian bo qun tng i ngn. Phng php ng kh ng kh l qu trnh m nc c ly ra khi mu khi cc mu ang trng thi lnh su. y vi sinh vt c huyn ph trong mi trng thch hp v clm lnh trong mi trng chn khng. Thit b ng kh s ht nc v cui cng mu c lm kh n mc nht nh. Mu c hn kn cho mi trng cha mu l chn khng. y l phng php ph bin c hiu qu cao cho bo qun cc i tng vi sinh vt khc nhau nh nm si, nm men, vi khun v mt

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s virut. Tuy nhin phng php ny t c ng dng i vi to, ng vt nguyn sinh v t bo ng vt. Phng php bo qun lnh su i vi phng php bo qun lnh su th vi sinh vt c bo qun trong mi trng dch th v nc cn cho hot ng sng ca vi sinh vt b bt hot nhit lnh su (-196oC -> -80oC). Vi phng php ny, t bo c th b v trong qu trnh lm lnh v lm tan mu. Mt nguyn nhn dn n lm v t bo l vic tch lu cc cht in gii trong mu bo qun v hnh thnh cc tinh th nc trong t bo. khc phc nhc im ny ngi ta b sung cc cht lm hn ch tc lnh su v lm tan nhanh nh glycerol, DMSO (dimethyl sulfoxide). Vic bo qun theo phng php lnh su ny c thc hin cc thang nhit khc nhau nh -20oC, -30oC, -40oC,-70oC, -140oC v -196oC. Ni chung mc nhit cao hn -30oC cho hiu qu thp do t bo chu nng mui cao sinh ra t cc cht in gii. Phng php bo qun ny c hiu qu vi nhiu nhm sinh vt khc nhau nh nm si, nm men, vi khun, x khun v virut. c bit vi phng php bo qun lnh su trong nit lng l phng php vn nng hn c. Phng php ny thch hp vi nhiu i tng vi sinh vt khc nhau nh, vi khun, nm si, nm men, virut, to v cc dng t bo ng vt. Tuy nhin, phng php ny cng bc l mt s nhc im nh u t kinh ph cho thit b v in, nit lng hoc ri ro nhue chy n c bit phng php ny khng thch hp vi cc chng vi sinh vt thng xuyn dng n. Ni chung phng php ny thng c dng vi cc chng vi sinh vt c nhng c tnh qu m khng thch hp vi phng php ng kh. 3.3. Mi trng nui cy vi sinh vt tng hp enzyme Protease. Cn phi chn mi trng v thnh phn mi trng dinh dng cso nh hng trc tip n s sinh trng v tng hp enzyme ca vi sinh vt. trong thnh

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phn mi trng phi c cc cht bo qun c s sinh trng bnh thng ca vi sinh vt v tng hp enzyme. c bit lu l tng s tng hp enzyme ngi ta thng da vo hin tng cm ng. V nu nh trong thnh phn mi trng c cc cht cm ng th cht hay sn phm phn gii ca n s km hm hoc lm yu tc dng km to ca cht km hm nhm bo m kh nng sinh tng hp enzyme cho khng b cn tr.

Cht cm ng tng hp enzyme cho thm vo mi trng nui thng l c cht tng ng ca enzyme cn tng hp. Thnh phn chnh ca mi trng: C, N, H, O. Ngoi ra cc cht v c: Mn, Ca, P, S, Fe, K v cc cht vi lng khc. 3.3.1. Ngun cacbon. Thng l hp cht hu c trong ch yu l gluxit, tu thuc vo c tnh ca enzyme v ni sinh vt m ngi ta la chn cho thch hp. C nhiu cht hydratcacbon v cc hp cht khc l ngun cacbon thch hp i vi nm mc sinh ra enzyme protease c hot lc cao. Cc ngun cacbon c tc dng n sinh tng hp proteinase cu nm mc c th theo th t: i vi Asp.flavus 74: fructoza glucoza sacaroza ramnoza manoza galactoza arabinora lactoza. i vi Asp. Awamori 200: fructoza manit sacaroza arabinora manoza galactoza lactoza. i vi Asp. Oryzae 79: fructoza sacaroza maltoza glucoza manit arabinora galactoza. Tinh bt l ngun cacbon ca nhiu chng vi khun sinh tng hp enzyme Protease. V d: VI khun Bac. Subtilis c kh nng sinh tng hp protease mi trng tinh bt >8%.

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Nhiu x khun u nhit, trong Micromonospora vulgaris 42, mc tt v sinh tng hp protease cao mi trng c tinh bt. Tng nng tinh bt t 0,25 1,5% sinh khi cng tng ng thi vi hiu sut tng hp enzyme. Nu tng nng tinh bt hn na s khng thu c kt qu dng tnh. T s gia cacbon v nit cn phi l 4:1. trn mi trng c maltoza (0,5 2%) vi sinh vt pht trin bnh thng, nhng tng hp protease ngoi bo b c ch. X khun khng pht trin c trn mi trng c cc ngun cacbon duy nht l sacaroza, lactoza hoc arabinoza. Glucoza, manoza,fructoza, xyloza c trong mi trng (0,2 5%) lm c ch sinh trng ca x khun v sinh tng hp enzyme. Ngoi ra, mt s loi hydrocacbon cng c ngun cacbon c 125 chng vi sinh vt. Chng hn chng Ps. Seruginosa c th ng ho hydrocacbon v c kh nng sinh tng hp protease c hot lc cao trn mi trng n-parafin vi 12, 14 v 16 nguyn t cacbon, du nng hoc propylenglycol. Chng khgn ch ng ho ch c hydrocacbon bo m cn ng ho c hydrocacbon thm. 3.3.2. Ngun nit. Ngun nit s dng rt phong ph, bao gm 2 nhm: v c v hu c. i vi mt s lai nm mc thuc h (A. oryzae, A. awamori, A. niger, A. flavus) trn mi trng c cc ngun nit hu c sinh tng hp protease axit cao. Tren mi trng kzapek NaNO3 wocj thay bng c cazein v b sung pepton hot lc enzyme tng 6ln. Nhiu kt qu nghin cu cho thy sinh tng hp enzyme c nng cao khi trong mi trng c ng thi c ngun nit hu c v nit v c. Cho thm vo mi trng c cm m, bt u tng tch cht bo, cc ngun nit v hu c hot lc enzyme protease tng 22 74%. Cn trng hp dng cc ngun nit v c duy nht trong mi trng s dn n ngng sinh tng hp protease ni chung. Trong qu trnh nui cy vi khun, trong c B. subtilis v B. mesentericus, cc hp cht nit v c v hu c c dng phi hp trong mi

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trng s to iu kin thun li cho sinh tng hp protease. Trong s cc ngun nit v c th NH4, H2PO4 l tt hn c. Nhng mui khc NH4Cl, (NH4)2SO4, NH4NO3, NaNO3, KNO3. Ca(NO3)2 lm gim hot lc protease ti 30-50%, cn amylaza gim 7-10 ln. Cn trong mi trng ch c ngun nit hu c hot lc protease v amylaza cng thp hn trong mi trng i chng (NH4)2HPO4 v nc chit u tng. i vi x khun u nhit Actynomyces Vulgaris U2 th peptin l cht cm ng sinh tng hp enzyme protease l tt nht. Cc axit amin c nh hng r rt ti sinh tng hp enzyme bng vi sinh vt. Glin, alanin, metionin v lxin cso tc dng lm tng hot lc protease cu chng t bin A. oryzae 251-90 n 6-9% v nguyn chng A. oryzae 132-63 ti 7-24%. Nhiu axit amin c tcs dgn c ch n sinh tng hp enzyme, Valin, Axit glutamic, izolin v valin c ch tng hp enzyme B. megaterium 60%. Cn i vi B. subtilis cc axt amin c ch trong qu trnh ny l glyxin, metinonin, axit glutamic, alamin, lxin, Ngoi ra, cc baz purin nh A (adenin), G (guanin) v cc dn xut ca chng, ARN v cc sn phm thu phn cng lm tng ang k sinh tng hp proteinza vi sinh vt. 3.3.3. Ngun cc nguyn t khong v cc yu t (cht) kch thch sinh trng. Mui khong rt cn thit cho hot ng vi sinh vt. Ion Mg2+ cso tc dng sinh tng hp v n nh cc enzyme cso hot tnh nhit cao. C nhiu yu t nh hng i vi mi trng nui cy. Nhit nui cy thng thng t 25-30oC. Tr s pH ban u cu mi trng (ch yu mi trng nc) cng c th gy nh hng no n s to thnh enzyme, nhng khi cng cn tnh n kh nng bin i nhanh chng ch s bi vi sinh vt.

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thng kh cng rt cn thit cho vic sinh tng hp enzyme. V vy mi trng b mt ngi ta thng thm cht xp nh tru vo, cn mi trng b su (mi trng dch th), th ngi ta thng lc (nu enzyme cn lc th vic ny cc k quan trng). m cng rt quan trng (ch c tc dng nui cy b mt), ph thuc vo thnh phn mi trng b mt. Khi la chn mi trng cn ch n c thnh phn nh tnh v nh lng sao chi qu trnh sinh tng hp enzyme mong mun l cao nht. Mun vy ngi ta c th s dng mt s phng php:

Phng php ti u ho quy hoch thc hin ton phn. Phng php ton hc mhnh ho thc nghim. 3.4. Cc loi mi trng nui cy vi sinh vt tng hp enzyme. C th chia lm 2 loi: mi trng tng hp v mi trng t nhin. Mi trng tng hp: l mi trng bao gm cc cht vi liu lng xc nh (qua tm hiu, nghin cu), chng hn ngun cacbon c th l tinh bt, xenluloza, ng, axit, ru, ngun nit v c v hu c. Loi mi trng ny c s dng chomc ch nghin cu. Mi trng t nhin: thng dng cc loi ph liu, nguyn liu c cha cc ngun cacbon, nit, khong, cc yu t sinh tng hp trng. Mt khc, cc nguyn liu ny c sn, r tin nn c s dng rt nhiu trong cng nghip sn xut cc ph phm enzyme t vi sinh vt. Cc nguyn liu chun b lm mi trng t nhin bao gm: cm v bt ht cc, nc chit ng, dch p hoa qu, rau, kh du, b ru, r ng, sn phm phn hu nm, men bia, tru, li ng. Khi la chn s dng mi trung cn ch n cc cht cso tc dng iu ho sinh tng hp enzyme, c bit cc cht cm ng.

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C nhiu yu t nh hng i vi mi trng nui cy. Nhit nui cy thng thng t 25-30oC. Tr s pH ban u cu mi trng (ch yu mi trng nc) cng c th gy nh hng no n s to thnh enzyme, nhng khi cng cn tnh n kh nng bin i nhanh chng ch s bi vi sinh vt. 3.5. Cc phng php nui cy vi sinh vt. Nui cy b mt: Phng php ny rt thch hp nui cy cc loi nm mc do kh nng pht trin nhanh, mnh, nn t b tp nhim. Khi nui nm mc pht trin bao ph b mt ht cht dinh dng rn, cc khun ty cng pht trin m su vo lng mi trng c tit trng, lm m. i vi mt s mc ch c bit, ngi ta nui vi sinh vt trc tip trn b mt ht go (sn xut tng), ht u tng (u tng ln men-mis) c nu chn trn ht cc cn sng (lm men thuc bc, men dn tc, lm tng). Ngi ta thng dng cm m, cm go, ng mnh cso cht ph gia l tru. Cm, tru, c b mt tip xc ln, mng, to c xp nhiu, khng c nhng cht gy nh hng xu n s pht trin ca nm mc. T l cc cht ph gia phi bo m sao cho hm lng tinh bt trong khi nguyn liu khng c thp hn 20%, c th b sung thm ngun nit v c ((NH4)2SO4, (NH4)2CO), photpho, nit hu c v cc cht kch thch sinh trng nh malt, nc chit ng, nc lc ru. Quy trnh cng ngh: Lm ngui, ti. Thanh trng bng nhit . Lm m. trn nguyn liu. Nui cy, theo di, x l. Chuyn vo dng c nui cy. Gieo trng vsv l. u nhc im: Nng enzyme to thnh cao hn nhiu ln so vi dch nui cy chm sau khi tch t bo vi sinh vt. Trong cng nghip ru mun ng ho 100kg tinh bt ch cn 5kg ch phm nm mc b mt nhng phi cn n 100lt nm mc chm lc b v t bo vi sinh vt.

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Ch phm d dng sy kh m khng lm gim ng k hot tnh enzyme, ch phm kh, d bo qun, vn chuyn, nghin nh hoc s dng trc tip m khng cn khu tch v lm sch enzyme. Tn t nng lng, thit b, dng c nui cy n gin, c th thc hin qui m gia nh, trang tri cng nh qui m n 20T/ngy. Nui cy trong iu kin v cng tuyt i v trong qu trnh nui cy u c nhim trng phn no, khu vc no th ch cn loi b canh trng phn . Tuy nhin phng php b mt cso nng sut thp, kh c kh ho, t ng ho, cn din tch nui ln, cht lng ch phm cc m khng ng u. Nui cy chm

Vi sinh vt c nuoi cy trong mi trng lng vi c cht ch yu trong a s trng hp l tinh bt. Ch c mt s t ging vsv dng ngun c cht cacbon l ng glucoza, saccharoza. Thc t, trong mt s trng hp ng ho s b tinh bt trc khi thanh trng. Khi ng maltoza c to thnh l cht cm ng tt, mi trng thng gim nht nn d dng cho qu trnh khuy trn v sc kh. Phng php nui cy b su i hi phi c v trng tuet i cc khu v sinh tng hp, thanh trng mi trng dinh dng, thao tc nui cy, khng kh cung cp cho qu trnh nui cy. Cc giai on chnh cu qu trnh nui cy chm 1 bc gm: chun b mi trng nui cy, nui cy nm men ging, nui cy nm mc sn xut. u nhc im Phng php nui cy hin i d c kh ho, t ng ho, nng sut cao, d t chc sn xut. C th nui cy d dng cc chng vi sinh vt t bin cso kh nng sinh tng hp enzyme cao v la chn ti u thnh phn mi trng, cc iu kin nui cy, enzyme thu c tinh khit hn, m bo iu kin v sinh, v trng.

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Tuy nhin do thu c canh trng v nng enzyme thp nn khi tch thu hi enzyme s c gi thnh cao. Tn in nng cho khuy trn, nu khng bo m v trng s b nhim hng lot, ton b gy tn thng ln. 3.6 Tch v lm sch ch phm enzyme. Mt iu cn ni thm na l enzyme thng cha cc t bo vi sinh vt gi l cc enzyme trong t bo (intracellular), nhuwnh n cng c th c cc vi sinh vt tit ra mi trng sng. l cc enzyme ngoi t bo (extracellular). Enzyme vi sinh vt thng chit l enzyme ngoi bo. Cc phn t enzyme khng c kh nng i qua mng t bo v mng ca cc cu t ca t bo. Do c th chit rt cc enzyme ni bo, bc u tin l phi ph v cu trc ca cc t bo c cha enzyme v chuyn chng vo dung dch. C th ph v cu trc ca cc t bo bng cc bin php c hc nh nghin vi bt thy tinh hoc cat thch anh, lm ng ha bng thit b ng ha (homogenzizator). Mun tch c cc enzyme trong cc cu t ca t bo, ngi ta cn phi dng cc yu t vt l v ha hc khc nhau nh sng siu m. Dng cc dung mi hu c nh buthanol, aceton, glycerin, ethyl acetate v cht detergent. Cc ha cht c tc dng tt cho vic ph v cc cu t ca t bo v trong cc c quan ny thng cha m. Sau khi ph v cc cu trc ca t bo, enzyme c chit bng nc ct, bng cc dung dch m thch hp hoc bng cc dung dch mui trung tnh. C mt s yu t nh hng n qu trnh chit rt cn lu . Trc ht l nhit . trnh mt hot tnh hoc thm ch v hot, cn thit rt v tin hnh kt ta enzyme nhit thp (t 3 n 5 oC). Cc thao tc phi nhanh. Mt s cht in li lm tng qu trnh chit rt enzyme nh NaCl, ZnCl2, CaCl2. Tc dng ca chng cn ph thuc vo phng php dng khi chit rt.

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loi b mui khong v cc loi ng l cc tp cht c phn t lng thp, ngi ta thng dng phng php thm tch (dialysis) i nc hay i cc dung dch m long hoc bng cch lc qua gel sephadex.

Thm tch loi mui (NH4)2SO4 trong kt ta protein loi b cc protein tp (protein cu trc, protein tr) v cc cht c phn t lng cao khc ngi ta hay dung kt hp cc phng php khc nhau: phng php bin tch chn lc nh tc dng ca nhit hoc pH ca mi trng, phng kt ta phn on bng mui trung tnh hoc cc dung mi hu c, cc phng php sc k (sc k hp ph, sc k trao i ion) in di, phng php lc gel. Mc ch yu cu: Cc ch phm enzyme c s dng cc dng khc nhau theo mc tinh khit (hot ring). Trong mt s trng hp, canh trng nui cy vi sinh vt c cha enzyme c s dng trc tip di dng th khng cn tch tp cht nu chng khng gy nh hng ng k n sn phm v qui trnh cng ngh sau ny( v d: sn xut ru, nc chm thc vt, da). Cng c khi ngi ta cn s dng ch phm enzyme tinh khit trong cng nghip dt, cng nghip mch nha, y hc, nghin cu khoa hc. Enzyme ni chung rt d gim hot tnh di tc dng ca cc tc nhn bn ngoi do khi tch v tinh ch enzyme trnh s bin hnh protein nh hng ln n hot tnh enzyme cn tin hnh nhanh chng nhit thp, pH thp khng c mt cc cht gy bin hnh enzyme.

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3.6.1 Phng php kt ta Phng php kt ta phn on bng (NH4)2SO4 da trn c s s khc nhau v kh nng kt ta ca cc protein enzyme mt nng mui (tnh theo % nng bo ha) xc nh c dng ph bin loi b bc u protein tp ca cc dung dch enzyme. Cc loi mui c th c dng l (NH4)2SO4, Na2SO4, MgSO4 ngi ta nhn thy mui (NH4)2SO4 l tt nht v n khng lm hi m lm n nh (lm bn) hu ht cc loi enzyme. Loi mui ny li r v ph bin. ha tan ca n li rt ln (bo ho 767 g/l 25 0C). 3.6.2 Phng php sc k *Cc k thut sc k ct Dch chit enzyme c loi b phn ln cc protein tp nhng vn cha m bo ng nht cn thit. Do dch chit enzyme c tip tc lm sch bng phng php sc k ct. - Phng php dng ch ry phn t (lc gel gel filtration) C s ca phng php lc gel l da vo s khc nhau v kch thc hnh dng phn t lng ca enzyme c trong hn hp tch chng ra. m bo cho vic tch enzyme c tt, cht ry phn t phi l cht tr, khng phn ng vi protein enzyme. Cht ny cng khng ha tan v tng i bn vi cc yeus t v c hc cng nh sinh hc. Ngoi ra cht c s dng cho mc ch lc phn t phi l cht khng c tnh n hi (khng co) v phi l cht a nc (hydrophyl).

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- Phng php sc k trao i ion Phng php sc k trao i ion da vo s khc nhau in tch tng s ca cc protein enzyme. Hay ni cch khc, phng php ny da trn c s ca phn ng trao i ion gia protein c tan trong nc hoc dung dch m long v cc tc nhn trao di ion. Tc nhn (hay nguyn liu) trao i ion c th l cht nha c tch nhm sinh ion hoc l cht ionit. y l nhng cht gi tr, khng tan trong nc, c bn cht l cellulose hoc cht gel dextran c li phn nhnh (Sephadex, Molselect) hoc l cht nha polystiron. Cht gi th ny thng kt hp vi cc nhm ion ha. Cc cht trao di ion c cht gi l cellulose, sephadex, molselect thng thng c dng tch protein enzyme, cn cc cht trao i ion c cht gi l polystirol (v d nh Dowex, Amberlite) ch dng tch cc peptid c trng lng phn t nh hn. Anionit DEAE cellulose (diethylamino ethyl cellulose) l dn xut este ca cellulose. C2H5 C2H5 Cellulose O CH2 N C2H5

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Trong H2O n c phn li: Cellulose O C2H4 N (C2H5) + H2O C2H5 Cellulose O C2H4 N+ C2H5 H y l ch trao i anion Nu cht gi l sephadex th chng ta c cht trao i ion sephadex. l cacs loi DEAE sephadex v CM sephadex. u im ca loi ny l va tch c protein enzyme v kch thc v v in tch tng s ca cc protein enzyme. Trng hp CM sephadex trn cht gi sephadex c gn nhm COO- O CH2 COOH Phn ly COO (mang in tch) y l cht trao i cation. Ngoi ra, c cc phng php : - Phng php dng chy ph - Phng php dng cht ph c hiu sinh hc hay l phng php sc k i lc (afinity chromatography). 3.6.3 Phng php tch h hai pha nc C s k thut ny da vo s phn b khc nhau ca hn hp trong dung dch hai pha khng ha tan vo nhau. Cc h hai pha c trng bng cch trn cc h dung mi vo nhau to thnh hai pha ring bit. H dung mi c s dng trong phng php ny c th l polymer/polymer nh: polyethylene glycol (PEG) v dextran hoc heejpolymer/mui nh PEG v potasium phosphate, sodium + OH

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phosphate , sodium sulphate Cc protein v mnh v t bo c kh nng ha tan khc nhau gia hai pha, v vy phng php php ny c th c dng cho c hai trng hp: phn tch protein khi mnh v t bo v phn chia cc enzyme trong sut qu trnh tinh sch protein. H hai pha nc c xem l phng php tt cho vic phn tch v tinh sch cc hn hp, v phn tch cht lng c mt khc nhau d dng hn phn tch cc cht rn ra khi cc cht lng. So vi cc phng php tinh sch khc th h hai pha nc c mt s u im hn nh: c ha tan trong nc ca hai pha ln (70 80 5), t tinh sch cao, hiu sut cao, d dng s dng quy m ln v c bit polymer c ti s dng. S phn tch t hiu qu cao ty thuc vo cc thng s nh khi lng phn t v in tch ca i tng nghin cu, nng v khi lng phn t ca cc polymer, nhit , pH, thi gian, lc ion ca hn hp v s hin din ca cc loi mui a ha tr nh phosphaste hoc sulphate PHN IV. CNG NGH SN XUT ENZYME PROTEASE 4.1 Quy trnh cng ngh ln men to enzyme protease 4.1.1 Gii thiu chung Cui th k 19 u th k 20, vic nui cy vi sinh vt thng c thc hin theo phng php b mt. Phng php ny c pht trin rt rng ri, khng ch thu nhn ch phm enzyme ma trc tin l phng php thu nhn khng sinh v nt s qu trnh ln men truyn thng. 4.1.2 u v nhc im ca phng php nui cy b mt. Phng php to iu kin cho vi sinh vt pht trin trn b mt mi trng. Nhng u im ca phng php nui cy b mt l: - Nui cy b mt rt d thc hin. Quy trnh cng ngh thng khng phc tp.

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- Lng enzyme c to thnh t nui cy b mt cao hn rt nhiu so vi nui cy chm. y l c im u vit rt quan trng trong gii thch ti sao phng php nui cy b mt hin nay pht trin mnh. - Ch phm enzyme th ( bao gm thnh phn mi trng sinh khi vi sinh vt, enzyme v nc). Sau khi thu nhn rt d sy kh v bo qun. - Nui cy b mt khng cn s dng nhiu thit b phc tp, do vic vn hnh cng ngh cng nh vic u t v n gin va khng tn km. - Trong trng hp b nhim cc vi sinh vt l, ta rt d dng x l. Mi trng c l mi trng tnh, khng c s xo trn nn khu vc no b nhim ta ch cn loi b khu vc o ra khi ton b khi nui cy, nhng khu vc khc s hon ton c an ton. Phng php nui cy b mt cng c nhng khuyt im cn c quan tm v khc phc hon thin phng php ny. Nhc im ln nht v d nhn thy nht l phng php ny kh tn din tch cho nui cy. Trong phng php ny vi sinh vt pht trin trn b mt mi trng nn rt cn din tch. a. Mi trng lng. mi trng lng, vi sinh vt s pht trin trn b mt mi trng, to thnh khun lc ngn cch pha lng ( mi trng) v pha kh ( khng kh). y, vi sinh vt s s dng cht dinh dng t dung dch mi trng, oxy t khng kh, tin hnh qu trnh tng hp enzyme. Enzyme ngoi bo s c tch ra t sinh khi v ha tan vo dung dch mi trng. Enzyme ni bo s nm trong sinh khi vi sinh vt. Nui cy vi sinh vt thu nhn enzyme trn mi trng lng theo phng php cy b mt thng c tin hnh trong cc khay c chiu cao khong 12-15cm, chiu rng v chiu di c thit k ty theo kch thc phng nui sao cho thun tin trong thao tc. y ngi ta quan tm nhiu n chiu cao mi trng lng. Nu chiu cao mi trng lng qu ln, vi sinh vt s khng c kh nng ng ha ht cc cht

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dinh dng pha y khay nui cy. Nu chiu cao mi trng nh, s thiu thnh phn cht dinh dng, hiu sut thu nhn enzyme s khng cao. Trong nhiu nh my, ngi ta thng to mi trng trong khay nui cy c chiu cao mi trng t 5-7cm l hp l. b. Mi trng c Phn ln cc nh my sn xut enzyme khi nui cy vi sinh vt thu nhan enzyme ngi ta thng s dng mi trng c. tng kh nng xm nhp ca khng kh vo trong lng mi trng, ngi ta thng s dng cm, tru, ht ng cc lm mi trng. Trong trng hp ny, vi sinh vt pht trin trn b mt mi trng, nhn cht dinh dng t ht mi trng v sinh tng hp ra enzyme ni bo v ngoi bo. Cc enzyme ngoi bo s thm thu vo trong cc ht mi trng cn cc enzyme ni bo nm trong sinh khi vi sinh vt. Vi sinh vt khng ch pht trin trn b mt mi trng, ni ngn cch pha rn ( mi trng) v pha kh ( khng kh) m cn pht trin trn b mt ca cc ht mi trng nm hn trong lng mi trng. Mi trng nui cy va c xp cao va phi c m thch hp. Nu m qu cao s lm bt mi trng li, khng kh khng th xm nhp vo lng mi trng, nu c m thp qu se khng thun li cho vi sinh vt pht trin, thng thng ngi ta thng to m khong 55-65% W l hp l. Nu s dng cm l nguyn liu chnh nui cy vi sinh vt thu nhn enzyme, ngi ta phi cho thm 20-25% tru lm xp mooi trng, to iu kin thun li khng kh d xm nhp vo lng mi trng. Phng php nui cy b mt bn rn ( mi trng c) ny rt thch hp cho ln men nm mc Asporyzae. 4.1.3 Sn xut nm mc ging nui cy to sinh khi. a. Mi trng cho sn xut mc ging. Dng mi trng tng hp

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Mi trng Benhamin. Bt ng Glucose Pepton Cao nm men Nc 20g 10g 10g 4g 1000ml

b. Chun b mc ging. Nui cy ging bao gm: - Trong ng thch nghing hay gi ng trong ng nghim. - Trong bnh tam gic ( nhn ging nh). - Trn sng, khay ( nhn ging ln). c. Nui cy to sinh khi. Sau khi phn lp thnh cng trn mi trng chn lc t a Petri cy chuyn nhng mc si sam ( hay ly nm mc t ng ging) ng thch khc. Yu cu ng ging phi tuyt i m bo thun khit, khng c ln ln bt k mt loi vi sinh vt no khc. Mi trng thch nghip phi m bo y dinh dng. Tip tc nhn ging v nui cy cho n khi t c sinh khi theo mong mun. 4.2 Thu nhn enzyme protease t nm mc Aspergillus oryzae a. Sinh trng v sinh tng hp protease t nm Khi nui vi sinh vt to protease c hai qu trnh lin quan mt thit vi nhau. Qu trnh tng hp sinh khi vi sinh vt v qu trnh tch t enzyme trong t bo hoc ngoi mi trng. mt s vi sinh vt, qu trnh sinh tng hp protease tin hnh song song vi qu trnh sinh trng, ngha l s tch t enzyme ph thuc tuyn tnh v s tng khi. Trong trng hp ny, sinh tng hp enzyme protease kt thc pha logarit

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cng ng thi vi s ngng sinh trng v s bt u pha pht trin n nh tip theo sau. Tuy nhin theo kin ca nhiu tc gi, s to thnh protease cc i thng xy ra sau khi qun th t bo vi sinh vt t im sinh trng. Trong trng hp ny, sinh trng ca vi sinh vt hu nh khng km theo s tch ly enzyme protease trong canh trng, ch sau khi kt thc pha sinh trng mi xy ra s tng hp enzyme cc ln.

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b. Quy trnh thu nhn enzyme protease Nui cy Thu nhn ph phm Enzyme th ch phm enzyme th em tinh ch nghin mn trch ly Lc kt ta enzyme thu nhn kt ta sy kt ta tinh cht kt ta thu nhn ch phm enzyme tinh khit enzyme kt tinh s dng ch phm enzyme tinh sch b cn hoc sulfat amon dng trong chn nui ch phm enzyme th em s dng

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4.3 Quy trnh thu nhn enzyme protease t nm mc Asp.oryzae Nc Nguyn liu dinh dng (bt bp, nm men, pepton) H p thanh trng
Lm ngu i n 300C

Khong hn hp

ln khay Ging Asporyzae Nui cy nhit phng Thu nhn sinh khi (enzyme th) Ch phm enzyme th em tinh ch sy Nghin mn Trch ly Lc Kt ta enzyme Thu nhn kt ta Sy Ch phm enzyme k thut. B dng trong chn nui Ch phm enzyme th em s dng

Tinh ch

Thu nhn ch phm enzyme tinh khit

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* Thuyt minh quy trnh cng ngh a. L do chn chng nm mc Asp.oryzae Asp.ryzae c c th sinh trng ca n bao gm nhiu si rt mnh, chiu ngang 5-7m, phn nhnh v c rt nhiu vch ngang, chia si thnh nhiu t bo. c im ca ging Asp.oryzae rt giu cc enzyme thy phn ni bo v ngoi bo ( amylase, protease, pectinase,), ta rt hay gp chng trong cc kho nguyn liu, trong cc thng cha bt, go ht nhng khng c ra sch, cn b bia, b ru, li ng, b sn Chng mc v pht trin c khi thnh lp mc, c mu sc en, vng Mu do cc bo t gi c mu sc. Cc bo t ny d b gi cun bay xa v ri vo u khi gp iu kin thun li s mc thnh nm mc mi. b. K thut nui cy Sau khi trn ging, mi trng c tri u ra cc khay vi chiu dy 2-3cm, ri c a v mi trng nui cy, t trn nhng gi . Cc gi ny c thit k sao cho lng khng kh c lu thng thng xuyn. Phng nui cy phi c h thng iu chnh nhit v m khng kh. Nhit thch hp cho nm si pht trin l 28-320C. Nhit thp qu hoc cao qua u nh hng khng tt cho nm si pht trin. Trong qu trnh nui cy, ta hon ton khng cn iu chnh pH. Mi trng bn rn l mi trng tnh nn s thay i pH mt vng no t khi nh hng n ton b khi mi trng. Thi gian nui nm si thu nhn enzyme vo khong 30-60 gi. iu ny cn ph thuc vo chng nm mc Asporyzae v iu kin mi trng cng nh ph thuc vo mi trng nui cy. Qu trnh pht trin ca nm mc trong mi trng bn rn khi nui cy bng phng php b mt ny tri qua cc giai on sau:

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Giai on 1: Giai on ny ko di 10-14 gi k t khi bt u nui cy. Enzyme mi bt u c hnh thnh. Trong giai on ny phi c bit quan tm n ch nhit . Tuyt i khng a nhit cao qu 300C v thi k u ny ging rt mn cm vi nhit . Giai on 2: Giai on ny ko di 14-18 gi. Enzyme proteasen c tng hp mnh. Lng O2 trong khng kh gim v lng CO2 tng dn, do trong giai on ny phi thng kh mnh v nhit c gng duy tr trong khong 29300C l tt nht. Giai on 3: Giai on ny ko di 10-20 gi. Qu trnh trao i cht yu dn, do mc gim cht dinh dng s chm li. Nhit ca khi mi trng gim, do lm gim lng khng kh mi trng xung 20-25 th tch khng kh/th tch png nui cy/1 gi. Nhit nui duy tr 300C, trong giai on ny bo t c hnh thnh nhiu do lng enzyme protease to ra s gim xung. Chnh v th vic xc nh thi im cn thit thu nhn enzyme rt cn thit. c. Thu nhn sn phm Kt thc qu trnh nui cy ta thu nhn c ch phm enzyme protease, ch phm ny c gi l enzyme th ( v ngoi thnh phn enzyme ra cn cha sinh khi vi sinh vt, thnh phn mi trng v nc trong mi trng). m bo ch phm enzyme protease khng b mt hot tnh nhanh ngi ta thng sy kh ch phm enzyme n mt m thp ( thit b sy thng dng y l my sy chn khng). m cn t c sau khi qu trnh sy kt thc l nh hn 10% m. m bo hot tnh enzyme khng thay i ngi ta thng sy nhit 3-400C. Enzyme protease nm mc Asporyzae s b mt hot tnh nu nhit ln n 60-700C. Ty theo mc ch s dng ta c th dng ch phm th khng cn phi qu trnh tinh sch. Trong nhng trng hp cn thit khc, ta cn phi tin hnh lm sch enzyme. sn xut enzyme tinh khit ngi ta phi tin hnh nh sau:

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Ton b khi lng enzyme th protease c em i nghin nh. Mc ch ca qu trnh nghin l va ph v thnh t bo va lm nh cc thnh phn ca ch phm th. Khi thnh t bo c ph v, cc enzyme ni bo cha thot ra khi t bo s angf thot ra khi t bo. Phn ln enzyme protease ngoi bo khi c tng hp v thot ra khi t bo ngay lp tc thm vo thnh phn mi trng. Khi ta nghin nh, enzyme thot ra khi thnh phn ny d dng hn. Trong khi nghin ngi ta thng s dng nhng cht tr nghin trong trng hp ny c dng l ct thch anh v bt thy tinh. Cc cht ny l nhng cht v c khng tham gia vo phn ng v kh nng tng mc ma st trc khi s dng ct thch anh v bt thu tinh phi c ra sch, sy kh nhit ln hn 100oC loi b nc v tiu dit vi sinh vt. Trch ly: Sau khi nghin mn ngi ta cho nc vo trch ly enzym protease. Cc loi enzyme thy phn c kh nng tan trong nc nn ngi ta thng dng nc nh mt dung mi ha tan. C mt phn ch phm enzym th, ngi ta cho45 phn nc, khuy nh v sau lc ly dch, phn b thu ring dng lm thc phm gia sc(ch cn loi b cc thch anh v bt thy tinh ra khi hn hp b ri mi cho gia sc n) Dch thu nhn c vn dng ch phm enzym th v trong cha nc v cc cht ha tan khc t khi lng mi trng nui cy. vic tip theo l lm sao tch enzym ra khi vt cht ny. Qu trnh kt ta enzym protease: lm vic trn ngi ta tin hnh kt ta enzym nh nhng tc nhn gy ta. Trong cng ngh tinh ch enzym, ngi ta thng dng cn v sunfat amon. Hai tc nhn kt ta ny d tm kim v gi r so vi nhng tc nhn gy ta khc. Trong khi tin hnh kt ta , ngi ta phi lm lnh c dung dch enzym th v c nhng tc nhn kt ta trnh lm mt hot tnh enzym. Khi cht lm kt tuarenzym vo dung dch enzym th phi ht sc t t trnh hin tng bin tnh.

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Trong qu trnh kt ta ngi ta dng cn hoc sunfat amon vi liu lng nh sau: C mt phn dung dch enzym th ngi ta cho 2 n 2.5 ln cn hoc sulfat amon. Khi cho cht kt ta vo dung dch enzym th, ngi ta tin hnh khuy nh sau yn trong i kin nhit lnh (thng l 4 n 7 oC) theo thi gian cc enzym s c to kt ta v lng xung y, ngi ta tin hnh gn v lc thu nhn kt ta dng paste( m ln hn 70%W) trng thi ny enzym rt d b bin tnh v cn nhiu nc d bo qun ngi tta sy kt ta enzym ptotease 40 oC cho n khi m cui cng t 5-8%W( thit b sy thng dng lm my sy phun sng- c th xem phn ph lc) Trong nhiu trng hp ch phm enzym ptotease dng kt ta vn hon ton cha sch v mt ha hc v trong cn cha 1 s enzym ta quan tm. PHN V. NG DNG CA ENZYME PROTEASE Protease khng nhng c ng dng nhiu trong y dc, ha hc, trong nng nghip m trong cng nghip protease chim vai tr quan trng, vic s dng enzyme trong cng nghip l a dng, phong ph v t c nhiu kt qu to ln. Th nhn thng k s b sau y v cc lnh vc dng protease ta c th thy c s a dng: cng nghip tht, cng nghip ch bin c, cng nghip ch bin sa, cng nghip bnh m, bnh ko, cng nghip bia, cng nghip sn xut sa kh v bt trng, cng nghip hng phm v m phm, cng nghip dt, cng nghip da, cng nghip phim nh, cng nghip y hc, Sau y l nhng ng dng m protease mang li trong cng nghip thc phm:

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5.1 Trong cng nghip ch bin tht.
- Protease c dng lm mm tht nh s thy phn mt phn protein trong tht, kt qu lm cho tht c mt mm thch hp v c v tt hn. Protease c s dng lm mm tht v tng hng v tht. C th ngm tht vo dinh dng protease pH v nhit xc nh phng php ny ph bin v thun li nht; tm hn hp lm mm tht ( E, mui, bt ngt ), tim dung dch enzyme vo tht ( tim dung dch enzyme vo con vt trc khi git m ).

5.2 Trong ch bin thy sn

- Khi sn xut nc mm th thi gian ch bin thng l di nht, hiu sut thy phn ( m ) li ph thuc rt nhiu a phng, phng php gi nn, nguyn liu c. Nn hin nay quy trnh sn xut nc mm ngn ngy c hon thin trong s dng ch phm enzyme protease rt ngn thi gian lm v ci thin hng v ca nc mm. Phng php s dng protease trong sn xut nc mm: + Nc mm truyn thng l kt qu ca qu trnh thy phn protein tht c di tc ng ca h protease ni ti trong iu kin c mui theo c ch: Protease Protein Protease polipeptid Protease peptid amino - acid

Trong 24h u tin, di tc ng ca cc h protease ni ti ca c mt phn protein tht c b thy phn thnh peptid, polipeptid ha tan trong nc bi. Sau protein tht c, polipeptid v cc peptid c to thnh ny tip tc b thy phn mnh m bi cc protease kim loi to thnh cc acid amin t do, cc peptid ngn v c s hnh thnh hng v ca nc mm.

5.3 Trong cng nghip sa

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- Protease c dng trong sn xut phomat nh hot tnh lm ng t sa ca chng. Protease t mt s sinh vt nh A.candidus, P.roquerti, B.mesentericus, .c dng trong sn xut phomat. Trong cng nghip sn xut bnh m, bnh quy,protease lm gim thi gian trn, tng do v lm nhuyn bt, to xp v n tt hn.

5.4 Trong sn xut Bia

- Ch phm protease c ngha quan trng trong vic lm tng bn ca Bia v rt ngn thi gian lc.Protease ca Aps.oryzae c dng thy phn protein trong ht ng cc, to iu kin x l Bia tt hn. ng dng enzyme protease lm trong v n nh cht lng nc qu v ru vang mt trong nhng nguyn nhn chnh gy kh khn cho vic lm trong nc qu v gy c nc qu l protein.

5.5 Trong cng nghip Da

- Trong cng nghip da, protease c s dng lm mm da nh s thy phn mt phn protein ca da, ch yu l collagel, thnh phn chnh lm cho da b cng. Kt qu loi b khi da cc cht nht v lm cho da c mm do nht nh, tnh cht c hon thin hn sau khi thuc da. Trc y, lm mm da ngi ta dng protease c phn lp t c quan tiu ha ca ng vt. Hin nay, vic a cc protease tch t vi khun (B.mesentericus, B.subtilis ), nm mc ( A.oryzae, A.flavus ) v x khun (S.fradiae, S.grieus, S.rimosus, ) vo cng nghip thuc da em li nhiu kt qu v dn chim mt v tr quan trng.

5.6 Trong cng nghip dt.

- Protease vi sinh vt c s dng lm sch t tm, ty t nhn to ( cc s nhn to c bng cc dung dung dch cazein, getalin ) si c bng, d nhum. Protease c tc dng thy phn lp protein serisin lm dnh bt cc si t t nhin, lm bong v tch ri cc loi t tm, do lm gim lng ha cht ty trng.

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Ngoi ra, protease cn c ng dng rng ri trong rt nhiu ngnh khc nh: - iu ch dch m thy phn dng lm cht dinh dng, cht tng v trong thc phm v sn xut mt s thc n king. - Protease ca nm mc v vi khun phi hp vi amilaza to thnh hn hp enzyme lm thc n gia sc c tiu ha cao, c ngha ln trong chn nui gia sc v gia cm. - iu ch mi trng dinh dng ca vi sinh vt sn xut vaccine, khng sinh,.

Sn xut thuc
- Sn xut keo ng vt, cht git tng hp git ty cc cht bn protein, sn xut m phm

Sn xut x phng

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Kt lun
Tm li, c th ni rng , vic nghin cu ng dng cc ch phm enzym cng c ch trng cc lnh vc khc nhau. Trong 20 nm cui th k XX v cc nm u th k XXI cc enzym khc nhau c ng dng Vit Nam bc u c nhiu nghin cu ng dng cc enzym trong ch bin nng sn, thc phm, nht l trong lnh vc sn xut bia, ru, ch bin bt ( vin cng nghip thc phm,Vin cng ngh sinh hc cng ngh thc phm, i hc Bch Khoa H Ni)Vic nghin cu cc enzyme phc v nng nghip cng nghip cng c quan tm v c kt qu ng k. V d, ch phm enzym mi ra i phc v nng nghip E 2001 c tc dng tng ph nhiu t, tng nng sut cy trng. c cc nghin cu ng dng protease trong sn xut ru, bia rt ngn thi k ln men cng nh sn xut nc mm ngn ngy bng cng ngh enzym protease./.

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