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LI M U
Ngy nay, cng vi s pht trin mnh m ca Cng ngh sinh hc, cc ch phm enzyme c sn xut cng nhiu v c s dng trong hu ht trong cc lnh vc nh: ch bin thc phm, nng nghip, chn nui, y t Hng nm lng enzyme c sn xut trn th gii t khong trn 300.000 tn vi gi tr trn 500 triu USD, c phn phi trong cc lnh vc khc nhau [4]. 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 l enzyme c s dng nhiu nht hin nay trong mt s ngnh sn xut nh: ch bin thc phm (ng t sa lm pho mat, lm mm tht, b sung lm 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 san 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% [6]. V vy, vic nghin cu ci tin phng php tch v tinh ch enzyme nhm thu c ch phm c tinh khit cao rt cn thit. tch v tinh ch enzyme ni ring v protein ni chung thng c mt lot phng php ha-l v ha hc khc nhau. C th chia lm ba nhm phng php sau: - Phng php kt ta - Phng php sc k

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Protease phn b thc vt, ng vt, vi sinh vt. Tuy nhin ngun enzyme vi sinh vt phong ph nht, 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 ngh v i sng.

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Chng 1: TNG QUAN V ENZYM PROTEASE

1.1. Tnh hnh nghin cu enzyme. 1.1.1. Vn nghin cu enzyme nc ta Hu nh mi phn ng ho hc trong c th sng u cn phi c vai tr xc tc ca enzyme - cht xc tc sinh hc. Chnh v vy, cc nghin cu v enzyme thu ht s quan tm ca cc cn b ho sinh hc, sinh hc thc nghim v nhiu nh nghin cu cc lnh vc lin quan khc. Cc nghin cu nhm theo hng tch, tinh sch enzyme, to cc ch phm c sch khc nhau, nghin cu cu trc, mi lin quan gia cu trc v hot tnh sinh hc ca enzyme, kh nng ng dng enzyme trong thc t. Nghin cu v cng ngh enzyme c tin hnh bi nhiu tc gi nh s dng ph tng ca l m sn xut pancrease, pepsin, trypsin... s dng mm m sn xut amylase. c nhng th nghim cng ngh nh sn xut amino acid t nhng tm bng protease, bt protein tht bng bromelain t t da, ln men ru bng enzyme c nh trn ct. Cng c nhng nghin cu s dng peroxydase, cyt-P450 trong ch to biosensor v thuc pht hin cht c... Trong lnh vc y dc, vic nghin cu su v c ch tc dng ca mt s enzyme nhm mc ch kin to nn mt s thuc dng iu tr mt s bnh c bit l to ra mt s ch phm thuc chng suy dinh dng tr em, ng thi tin hnh sn xut i tr. y l mt ng gp rt thit thc v kp thi trong vic phng chng suy dinh dng nc ta. 1.1.2. Cng ngh sn xut enzyme trn th gii. Trong nhng nm gn y, gi tr thng mi ca cc enzyme cng nghip trn ton th gii t khong 1 t USD, trong ch yu l cc enzyme thy phn (75%), v protease l mt trong ba nhm enzyme ln nht s dng trong cng nghip (60%) [4]. 1.2. Tng quan v enzyme Protease.

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Nhm enzyme protease (peptit hidrolase 3.4) xc tc qu trnh thu phn lin kt lin kt peptit (-CO-NH-)n trong phn t protein, polypeptit n sn phm cui cng l cc axit amin. Ngoi ra, nhiu protease cng c kh nng thu phn lin kt este v vn chuyn axit amin.

Hnh 1.1. M hnh enzyme Protease thy phn phn t Protein. 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 v 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.

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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 thu phn trit v a dng.

Hnh 1.2. Cu trc khng gian enzyme Protease. 1.2.2. Phn loi Protease Protease (peptidase) thuc phn lp 4 ca lp th 3 (E.C.3.4) .

Peptidase (Protease) (E.C.3.4)

Exopeptidase (E.C. 3.4.11-17)

Endopeptidase (E.C. 3.4.21-99) Serine proteinase

Aminopeptidase Cystein proteinase

Carboxypeptidase

Aspartic proteinase

Metallo proteinase

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Hnh 1.3. S phn loai protease Protease c phn chia thnh hai loi: endopeptidase v exopeptidase. * Da vo v tr tc ng trn mch polypeptide, exopeptidase c phn chia thanh hai loai: + Aminopeptidase: xc tc thy phn lin kt peptide u N t do ca chui polypeptide gii phng ra mt amino acid, mt dipeptide hoc mt tripeptide. + Carboxypeptidase: xc tc thy phn lin kt peptide u C ca chui polypeptide v gii phng ra mt amino acid hoc mt dipeptide. * Da vo ng hc ca c ch xc tc, endopeptidase c chia thnh bn nhm: + Serin proteinase: l nhng proteinase cha nhm OH ca gc serine trong trung tm hot ng va 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 Carlsberg, 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 proteinase k sinh trng. Cc cystein proteinase thng hot ng vng pH trung tnh, c tnh c hiu c cht rng. + Aspartic proteinase: Hu ht cc aspartic proteinase thuc nhm pepsin. Nhm pepsin bao gm cc enzyme tiu ha nh: pepsin, chymosin, cathepsin, renin.

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Cc aspartic proteinase c cha nhm carboxyl trong trung tm hot ng v thng hot ng mnh pH trung tnh. + Metallo proteinase: Metallo proteinase la 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 ba nhm: - Protease acid: pH 2-4 - Protease trung tnh: pH 7-8 - Protease kim: pH 9-11

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Chng 2: NGUN THU NHN ENZYM PROTEASE

2.1. Ngun ng vt: - Ty tng: y l ngun enzyme sm nht, lu di nht v c cha 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 s dng ph bin trong cng ngh phomat. Renin lm bin i cazein thnh paracazein c kh nng kt ta 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 phm 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 nghin cu sn xut protease t vi sinh vt c c tnh renin nh cc loi Eudothia Parasitica v Mucor Purillus. 2.2. Ngun thc vt: C 3 loi protease thc vt nh Bromelain, Papain v Ficin. Papain thu c t nha ca l, thn, qu u (Carica papaya) cn Bromelain thu t qu, chi da, v da (Pineapple plant). 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 protease thc vt ny l trong cng ngh lm mm tht v trong mc tiu tiu ho. Ficin thu c t nha cy c (Ficus carica). Enzyme c s dng thu phn protein t nhin.

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Hnh 2.1. Ngun thu enzyme Protease t thc vt 2.3. Ngun vi sinh vt: Enzyme Protease phn b ch yu vi khun, nm mc v x khungm nhiu loi thuc Aspergillus, Bacillus, Penicillium, Clotridium, Streptomyces v mt s v mt s loi nm men. Vi khun Lng protease sn xut t vi khun c c tnh vo khong 500 tn, chim 59% lng enzyme c s dng [6]. Protease ca ng vt hay thc vt ch cha mt trong hai loi endopeptidase hoc exopeptidase, ring vi khun c kh nng sinh ra c 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 [6]. Trong cc chng vi khun c kh nng tng hp mnh protease l Bacillus subtilis, B. mesentericus, B. thermorpoteoliticus v mt s ging thuc chi Clostridium. Trong , B. subtilis c kh nng tng hp protease mnh nht (Nguyn Trng Cn v cs, 1998). Cc vi khun thng tng hp cc protease hoat ng thch hp vng pH trung tnh v kim yu.

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Cc protease trung tnh ca vi khun hot ng khong pH hp (pH 5-8) 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 i 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 Clostridium.

Hnh 2.2. Clostridium Protease ca Bacillus a kim c im ng in bng 11, khi lng phn t t 20.000-30.000. n nh trong khong pH 6-12 v hot ng trong khong pH rng 7-12 .

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Hnh 2.3. Bacillus

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 oryzae, A. terricola, A. fumigatus, A. saitoi, Penicillium chysogenum) Cc loi nm mc ny c kh nng tng hp c ba loi protease: acid, kim v trung tnh. Nm mc en tng hp ch yu cc protease acid, c kh nng thy phn protein pH 2,5-3. Mt s nm mc khc nh: A. candidatus, P. cameberti, P. roqueforti cng c kh nng tng hp protease c kh nng ng t sa s dng trong sn xut pho mat.

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Hnh 2.4. Nm mc 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 c mt s chng c kh nng tng hp protease cao nh: Streptomyces grieus, S. fradiae, S. Trerimosus...

Hnh 2.5. X khun Cc ch phm protease t x khun c bit nhiu l pronase (Nht) c tch chit t S. grieus, enzyme ny c c tnh c hiu rng, c kh nng thy SVTH: Trn Giang V Vi Trang: 12

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phn ti 90% lin kt peptide ca nhiu protein thnh amino 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 karetin. M, ch phm c sn xut c tn l M-Zim dng trong sn xut da. Protease t S. fradiae cng c hoat tnh elastase cao, do chng c dng trong cng 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 nh 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 vi sinh vt v ging vi sinh vt. Chu k sinh trng ca vi sinh vt ngn: 16 100 gi nn c th thu hoc nhiu ln quanh nm. C th iu khin sinh tng hp enzyme d dng theo hng c li (nh hng s dng v tng hiu sut tng thu hi). 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 thch hp. sn xut ch phm enzyme, ngi ta c th phn lp cc ging vi sinh vt c trong t nhin hoc cc 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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Chng 3: THU NHN V LM SCH ENZYM PROTEASE T VI SINH 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 cc 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 vt sinh tng hp enzim

Tch v lm sch ch phm enzim

Hnh 3.1. Cc cng on sn xut ch phm enzyme SVTH: Trn Giang V Vi Trang: 14

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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 b gim kh nng sinh tng hp repressor hoc tng hp repressor c i lc thp vi gene opertor. 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 ha promotor lm tng p lc ca n i vi ARN-polymerase do lm tng tc sao chp mDng 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 ADN. to mt t bin gene c th dng tc nhn vt l (tia t ngoi, tia phng x) hay ha hc (cc ha 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 vi sinh vt di nh hng ca ADN trong dch chit nhn nhn c t t bo ca vi sinh vt khc. y yu t bin np l AND. S chuyn vt liu di truyn (ADN) 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 ADN no ch khng i hi phi l ADN t cc ging h hng. Tuy nhin t bo ch c th nhn mt s on ADN nht nh, thng khng qu 10 on. Cc on ADN c di truyn 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 truyn 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 (ADN) 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 lu 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 iu kin nui cy thch hp cho ging vi sinh vt . Sau khi ging mc tt cn bo qun nhit lnh 3-40C 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 SVTH: Trn Giang V Vi Trang: 16

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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 chn 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.

Hnh 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. Phg php ny rt hay c s dng bo qun nm mc, x khun, mt vi loi nm men, vi khun thi gian giu ging c th c 1 nm.

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Phng php lm kh cng thc hin n gin, khng cn dng c t tin. Tuy nhin ging nh phng php cy chuyn thi gian bo qun tng i ngn. Phng php ng kh

Hnh 3.3. ng kh vi sinh vt. 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 c lm 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 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 (-196C -> -80 C). SVTH: Trn Giang V Vi Trang: 18

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Hnh 3.4. Bo qun lnh su 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

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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 -20 C, -30 C, -40 C, -70 C, -140C v -196 C. Ni chung mc nhit cao hn -30 C 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 vi 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 c 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 nh 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.

Hnh 3.5. Bo qun trong nit lng.

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3.1.3. Mi trng nui cy vi sinh vt tng hp enzyme Protease. Cn phi chn mi trng v thnh phn mi trng dinh dng c nh hng trc tip n s sinh trng v tng hp enzyme ca vi sinh vt. Trong thnh phn mi trng phi c cc cht m bo 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. 3.1.3.1. Ngun cacbon. Thng l hp cht hu c trong ch yu l gluxit, ty thuc vo c tnh ca enzyme v ni vi 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 ca 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 arabinoza manoza galactoza lactoza. i vi Asp. oryzae 79: fructoza sacaroza maltoza glucoza manit arabinoza 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 a nhit, trong Micromonospora vulgaris 42, mc tt v sinh tng hp protease cao mi trng c tinh bt. Tng nng tinh bt t 0,251,5% sinh khi cng tng ng thi vi hiu xut 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 khng ch ng ho c c hydrocacbon bo m cn ng ho c hydrocacbon thm. 3.1.3.2. Ngun nito. Ngun nito s dng rt phong ph, bao gm 2 nhm: v c v hu c. i vi mt s loi 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. Trn mi trng kzapek NaNO3 c thay bng c cazein hot lc protease ca nm mc c tng ln 3,5 ln, cn khi thay tinh bt bng glucoza v b sung pepton hot lc enzyme tng 6 ln. 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 trng s to iu kin thun li cho sinh tng hp protease. Trong s cc ngun nit v c th NH4, SVTH: Trn Giang V Vi Trang: 22

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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 a nhit Actynomyces Vulgaris U2 th pepton 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. Glyxin, alanin, metionin v lxin c tc dng lm tng hot lc protease ca chng t bin A. oryzae 251- 90 n 6- 9% v nguyn chng A. oryzae 132- 63 ti 7- 24%. Nhiu axit amin c tc dng c ch n sinh tng hp enzyme, Valin, axit glutamic, izolxin v valin c ch tng hp enzyme B. megaterium 60%. Cn i vi B. subtilis cc axit amin c ch trong qu trnh ny l glyxin, metionin, axit glutamic, alanin, 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 ng k sinh tng hp proteinza vi sinh vt. 3.1.3.3. 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+ c tc dng sinh tng hp v n nh cc enzyme c hot tnh nhit cao. C nhiu yu t nh hng i vi mi trng nui cy. Nhit nui cy thng thng t 25 - 300C. Tr s pH ban u ca 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. 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

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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 cho 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 ha quy hoch thc nghim ton phn. Phng php ton hc m hnh ha thc nghim. 3.1.4. 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 cho mc 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 ch 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 trng cn ch n cc cht c tc dng iu ho sinh tng hp enzyme, c bit cc cht cm ng. C nhiu yu t nh hng i vi mi trng nui cy. Nhit nui cy thng thng t 25 - 300C. Tr s pH ban u ca 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. 3.1.5. Cc phng php nui cy vi sinh vt. Nui cy b mt:

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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 c 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 b ru. Quy trnh cng ngh: Nguyn liu Trn Lm m Thanh trng bng nhit Lam ngui, ti Gieo ging vsv Chuyn vo dng c nui cy Nui cy, theo di, x 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 100lit nm mc chm lc b v t bo vi sinh vt. 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 nu 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 ln n 20T/ngy.

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Nui cy trong iu kin v trng tuyt i v trong qu trnh nui cy nu c nhim trng phn no, khu vc no th ch cn loi b canh trng phn . Tuy nhin phng php b mt c 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 nui 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 tuyt 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 ca 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 c 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 bao iu kin v sinh, v trng. 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. 3.1.6. Tch v lm sch ch phm enzyme. Mt iu cn ni thm na l enzyme thng cha cc t bo sinh vt gi l cc enzyme trong t bo (intracellular), nhng n cng c th c cc sinh vt

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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 ca 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 ct thch anh, lm ng ha bng thit b ng ha (homogenizator). 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 butanol, 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 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 chit rt v tin hnh kt ta enzyme nhit thp (t 3 n 50C). Cc thao tc phi nhanh. Mt s cht in ly 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. 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.

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loi b cc protein tp (protein cu trc, protein tr) v cc cht c phn t lng cao khc ngi ta hay dng kt hp cc phng php khc nhau: phng php bin tch chn lc nh tc dng ca nhit hoc pH ca mi trng, phng php 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 quy 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 b 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 thch hp khng c mt cc cht gy bin hnh enzyme. 3.1.6.1. Phng php kt ta

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Phng php kt ta phn on bng (NH4)2 SO4 da trn c s s khc nhau v kh nng kt ta ca cc protein enzyme mt nng mui (tnh theo phn % nng bo ha) xc nh c dng ph bin loi b bc u protein tp ca cc dch enzyme. Cc loi mui c th c dng l (NH4)2 SO4, Na2 SO4, MgSO4 ... ngi ta nhn thy mui (NH4)2 SO4 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 ha 767g/l 250C). 3.1.6.2. 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 cht 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 v 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 yu 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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coarse), ht trung bnh (medium), ht mn (fine), ht siu mn, rt mn (superfine).

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Phng php sc k trao i ion da vo s khc nhau v in tch tng s ca cc protein enzyme. Hay ni cch khc, phng php ny c 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 i 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 polystirol. Cht gi th ny thng kt hp vi cc nhm ion ha. Cc cht trao i 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

y l cht trao i anion Nu cht gi l sephadex th chng ta c cht trao i ion sephadex. l cc loi DEAE - sephadex v CM - sephadex. u im ca loi ny l va tch c

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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-

y l cht trao i cation. Ngoi ra c cc phng php : - Phng php dng cht hp ph - Phng php dng cht hp ph c hiu sinh hc hay l phng php sc k i lc (affinity chromatography)

3.1.6.3. 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 polymer/mui nh PEG v potassium phosphate, sodium sulphate, sodium phosphate,... Cc protein v mnh v t bo c kh nng ha tan khc nhau gia hai pha, v vy phng 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%), t tinh sch cao, hiu sut cao, d dng s dng quy m ln v c bit polymer c ti s dng.

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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...
Protease v tp cht

PEG PPB
Trn u Sau khi phn tch

PEG: polyethylene glycol PPB: potassium phosphate buffer

tp cht

Hnh 3.9. Phng php tch h hai pha nc 3.2. Thu nhn enzyme t ht cc ny mm. Malt l loi ht ho 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 loi v bo qun ht. Ra, st trng v ngm ht. m mm ta s thu c malt ti. Sy malt ti. X l v bo qun malt kh.

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Hot tnh proteaza trong ht ban u khng ng k nhng khi ny mm tng ln 48 ln, tng nhanh hn hot tinh amylaza v t cc i vo khong ngy ny mm th 5. S thu phn protein bt u bng tc dng ca proteinaza to thnh albumoza, polypeptit, pepton v sau di tc dng ca peptidaza to thnh cc axit amin. 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 thu phn protein tng i su sc to thnh mt lng ng k cc polypeptit v axitamin. 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. 3.3. Thu nhn enzyme protease t thc vt Bao gm tt c cc ni da trng ly qu, ly si (k c cc ni da di). Cc nghin cu ch ra rng cc nguyn liu ly kh nhit 4000C s gi c hot tnh enzyme tt hn so vi nguyn liu c bo qun lnh nhit 40C.

3.3.1. H da (Bromalaceae)

Nguyn tc chung thu nhn Bromelain.

Ph liu da lm dp chit lc ly tm kt ta ly tm lc ch phm k thut sc k trao i ion sy thng hoa sn phm tinh khit. Thu nhn Bromelain bng phng php nhanh s dng CMC CMC c tm t bng dung dch m photphat 0,05M, pH= 6,1. Cho dch chi da vo, thnh thong khuy trn. Enzyme ny ln b mt ca CMC. Sau 2 gi ly ra vt nc loi b cn bn bm vo CMC, ra tri cc protein khng phi enzyme bng m photphat pH= 6,5. Sau cho phn hp th ln 1. Dung dch phn hp th l m photphat pH= 7,1, NaCl 0,5 N khuy trn. Sau 2 gi ly ra vt c dung dch m c cha Bromelain. Tip tc phn hp th SVTH: Trn Giang V Vi Trang: 34

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nh ln th 2 ri gp chung dch chit ca c hai ln kt ta enzyme bng axeton hay cn lnh. ng dng Thy phn gan b Lm ng sa S dng Bromelain thu nhn cc cht c ch proteaza. 3.3.2. Nha u (Carica Papaya. L) y l loi cy n qu ph bin cc nc nhit i. T qu ti hoc thn thu c nha (latex) chnh l ch phm papain th t tinh ch thnh papain thng phm. Hin nay ngi ta to ra c cc ging u c sn lng m v hot tnh papain cao khai thc c hiu qu ngun enzyme ny. Papain c M= 20.700, Top= 800C, pHop= 5 5,5. B c ch v mt hot tnh bi H 2O2, Iodoaxetat, I2, fericianua. c hot ho bi CN, cystein, H2S v glutation.

Thu nhn papain th: Dng cc loi qu u cn non, u gi (cha chn), dng khn lau sch v, ly dao co sch nhng ng khng qu su, hng nha vo cc ri lm kh. Kt qu cho thy hot tnh papain sau khi chit tch cao hn khi 3 thng.

Thu nhn papain thng phm: Ngm papain th ha tan nha ti (catex) trong nc ct c b sung glyxerin tng ha tan, lc qua vi mn. Kt ta bng axeton lnh vi t l 2:1 so vi th tch dch lc. Ly tm lnh ly kt ta, sy 45- 500C, em nghin thnh bt.

ng dng: lm ng sa, thu phn protein nh sn xut bt c thc phm, sn xut nc mm ngn ngy, lm mm tht.

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Chng 4: NG DNG ENZYM PROTEASE

Protease c s dng rng ri trong nhiu ngnh cng nghip nh cng nghip thc phm, cng nghip nh, cng nghip dc phm v nng nghip 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. (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 (enzyme, mui, bt ngt). Tim dung dch enzyme vo tht; tim dung dch enzyme vo con vt trc khi git m). S dng protease sn xut dch m: t Streptomyces fradiae tch c ch phm keratineza thu phn c keratin rt c gi tr sn xut dch m t da, lng v. Nu dng axit thu phn s mt i hon ton cc axit amin cha lu hunh, nu dng kim thu phn s b raxemic ho (chuyn dng L sang D lm gim gi tr sinh hc ca axit amin). thu phn su sc v trit protein (trong nghin cu, ch to dch truyn m y t) cn dng cc protease c tnh c hiu cao v tc dng rng, mun vy ngi ta thng dng phi hp c 3 loi protease ca 3 loi: vi khun, nm mc, thc vt vi t l tng cng 1- 2% khi lng protein cn thu phn. u im ca vic thu phn protease bi enzyme l bo ton c vitamin ca nguyn liu, khng to ra cc sn phm ph, khng lm sm mu dch thu phn.

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Trong ch bin thu sn: Khi sn xut nc mm (v mt s loi mm) thng thi gian ch bin thng l di nht, hiu sut thu 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 thc vt (bromelain v papain) v vi sinh vt rt ngn thi gian lm v ci thin hng v ca nc mm. Tuy nhin vn cn mt s tn ti cn phi hon thin thm v cng ngh. Trong cng nghip sa, protease c dng trong sn xut phomat nh hot tnh lm ng t sa ca chng. Protease t mt s vi sinh vt nh A. candidus, P. roquerti, B. mesentericus, c dng trong sn xut pho mat. 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. 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 A. oryzae c dung thy phn protein trong ht ng cc, to iu kin x l bia tt hn. Trong cng nghip da, protease c s dng lm mm da nh s thy phn mt phn protein ca da, ch yu l collagen, 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. griseus, S. rimosus...) vo cng nghip thuc da em li nhiu kt qu v dn dn chim mt v tr quan trng. Trong cng nghip dt: Proteinase vi sinh vt c s dng lm sch t tm, ty t nhn to (cc si nhn to c bng cc dung dch cazein, gelatin) si c c bng, d nhum. Protease c tc dng thy phn lp protein serisin

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lm dnh bt cc si t t nhin, lm bong v tch ri cc loi t tm, do lm gim lng ho cht ty trng. 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 amylase to thnh hn hp enzyme dng 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,

Hnh 4.1. Sn xut thuc - Sn xut keo ng vt, cht git tng hp git ty cc cht bn protein, sn xut m phm,

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Hnh 4.2. Sn xut x phng

KT LUN
Trong qu trnh thc hin n Cng ngh 1: S phn b, thu nhn v ng dng enzyme Protease ti t c cc kt qu sau: - Hiu ngha,vai tr ca enzyme Protease trong sn xut v i sng x hi, c bit trong tng lai khi cng ngh sinh hc pht trin. - Bit s phn b v nm c mt s phng php thu nhn enzyme. - Bit c mt s nhm vi sinh vt chnh c th sn xut enzyme Protease. - Nm c mt vi c im sinh l ca vi sinh vt.

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TI LIU THAM KHO

TING VIT 1. Trn Xun Ngch (2007), Cng ngh enzym, Trng i Hc Bch Khoa Nng. 2. Nguyn Trng Cn, Nguyn Th Hin, Th Giang, Trn Th Luyn (1998), Cng ngh enzyme, Nh xut bn nng nghip thnh ph H Ch Minh. 3. Lng c Phm (2004), Cng ngh vi sinh vt, Nh xut bn Nng nghip, H Ni. 4. Lu Th Nguyt Minh(2007), Phn tch Protease ca Bacillus Subtilis bng h hai pha Polyethylene glycol/ Potassium phosphate, n tt nghip. 5. L Xun Phng (2001), Vi sinh vt cng nghip, NXB Xy dng, H Ni. TI LIU MNG 1. http://www.zuova.cz/informace/nrlpab23.php SVTH: Trn Giang V Vi Trang: 40

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3.http://vietsciences2.free.fr/khaocuu/nguyenlandung/images/xakhuan_baotu00 3.jpg 4.http://images.google.com.vn/imgres? imgurl=http://www.chf.de/eduthek/projektarbeiten/abb/waschmittelvergl 5. http://faculty.ircc.edu/faculty/tfischer/images/transformation.jpg

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