LI M U

Ngay t thi c xa, vi sinh vt c con ngi s dng ch bin v sn xut thc phm, n l mt b phn trong khu phn n ca con ngi v cc loi vt nui. Ngy nay vic sn xut protein t ngun vi sinh vt khng ch nhm mc ch s dng lm thc n, m sn xut protein t vi sinh vt c trong nhiu lnh vc khc nhau. Protease l enzyme hin nay c s dng nhiu nht trong mt s ngnh sn xut nh: Ch bin thc phm ( ng t sa dng lm phomat, 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 c ci thin ng k hiu qu sn xut v sn phm c to ra nhiu hn. Protease phn b thc vt, ng vt, vi sinh vt. Tuy nhin ngun enzyme vi sinh vt phong ph nht v c nhiu u im nht nh: c hu ht cc vi sinh vt nh vi khun, nm mc, x khun, c hot tnh mnh ch cn mt lng nh c th chuyn ha mt lng c cht ln, c cng sinh sn rt mnh v tng hp enzyme vi tc rt nhanh chng, trong mt thi gian ngn c th thu c mt lng enzyme ln.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. y cng chnh l l do em chon ti Nghin cu quy trnh sn xut enzyme protease t vi sinh vt

Chng I: TNG QUAN TI LIU 1.1. Tng quan v enzyme Enzyme l nhng protein c kh nng xc tc c hiu cho cc phn ng ha hc, l cht xc tc sinh hc c ngha c bit quan trng nh: cng lc xc tc ln, s chuyn ha c cht mnh. 1.1.2. Thnh phn cu to ca enzyme[2]co thanh nga Cng nh protein, enzyme c th l protein n gin hoc protein phc tp. Trn c s , ngi ta phn enzyme thnh hai nhm: Enzyme mt thnh phn (enzyme mt cu t) v enzyme hai thnh phn (enzyme hai cu t). Phn protein ca enzyme hai thnh phn c gi l apoprotein hay apoenzyme, cn phn khng phi protein gi l nhm ngoi hoc coenzyme. Phn khng phi protein thng l nhng cht hu c c hiu c th gn cht vo phn protein hoc ch c th lin kt lng lo v c th tch khi phn protein khi cho thm tch qua mng. Mt phc hp hon chnh gm c apoenzyme v coenzyme c gi l holoenzyme. Trong coenzyme trc tip tham gia phn ng xc tc, gi vai tr quyt nh kiu phn ng m enzyme xc tc v lm tng bn ca apoenzyme i vi cc yu t gy bin tnh. Cn apoenzyme c tc dng nng cao hot tnh xc tc ca coenzyme v quyt nh tnh c hiu ca enzyme. 1.1.3. Phn loi enzyme http://www.web-books.com/MoBio/Free/Ch2E1.htm Da trn nhng phn ng xc tc, y ban danh php ca Lin minh quc t v Ha sinh v Sinh hc phn t (IUBMB) ngh vic phn loi sau y. 1. Oxidoreductases xc tc cho mt lot cc phn ng oxy ha gim. Cc tn thng gp bao gm dehydrogenase, oxidase, reductase v catalase.

1.1.1. nh ngha enzyme[1] co thanh nga

2. Transferases xc tc chuyn giao ca cc nhm (acetyl, methyl, phosphate, vv). Cc tn thng gp bao gm acetyltransferase, protein kinase methylase, v polymerase. Ba lp con u tin ng vai tr quan trng trong quy nh ca qu trnh t bo. Phn ng ha hc ca h c th hin tronghnh 2-E-1 . Polymerase l iu cn thit cho s tng hp DNA v RNA. 3. Hydrolases xc tc phn ng thy phn mt phn t c chia thnh hai hoc nhiu phn t nh hn bng cch cho thm nc. V d ph bin c a ra di y. Protease chia tch cc phn t protein. V d: HIV protease v caspase . HIV protease l iu cn thit cho nhn bn HIV khng. Caspase ng mt vai tr quan trng trong qu trnh apoptosis. Nucleases chia tch cc axit nucleic (DNA v RNA) . Cn c vo loi cht nn, h c chia thnh RNase v DNase. RNase xc tc thy phn ca RNA v DNase hnh vi trn DNA. H cng c th c chia thnh exonuclease v endonuclease.Exonuclease dn dn tch ra khi nucleotide duy nht t mt u ca DNA hoc RNA. Endonuclease chia tch DNA hoc RNA ti cc trang web ni b. Phosphatase xc tc dephosphorylation (loi b cc nhm phosphate) . V d:calcineurin . Cc c ch min dch thuc FK506 v Cyclosporin A l nhng cht c ch calcineurin. 4. Lyases xc tc s phn tch ca CC, CO, CS v CN tri phiu bng cc phng tin khc hn so vi thy phn hoc qu trnh oxy ha. Tn gi thng thng bao gm decarboxylase, aldolase. 5. Isomerase xc tc ti sp xp nguyn t trong mt phn t . V d nh rotamase, isomerase disulfide protein (PDI), epimerase v racemase.

6. Ligases xc tc cho phn ng m tham gia hai phn t. Cc v d bao gm cc enzym tng hp peptide, aminoacyl-tRNA synthetase, DNA ligase v RNA ligase. 1.1.4. Trung tm hot ng ca enzyme[2]co thanh nga Trung tm hot ng ca enzyme l phn ca phn t enzyme trc tip kt hp vi c cht, tham gia trong vic to thnh v chuyn ha phc cht trung gian gia enzyme v c cht to thnh sn phm phn ng. Trung tm hot ng bao gm nhiu nhm chc nng khc nhau ca amino acid, phn t nc lin kt v nhiu khi c c cofactor hu c (coenzyme) v v c. Theo quan nim ca Fisher th trung tm hot ng ca enzyme c hnh thnh sn vi mt cu to nht nh ch cho php c cht c cu to tng ng kt hp vo. Do c th v s tng ng nh kha vi cha kha. Cn theo Koshland th c im ca vng trung tm hot ng l rt mm do v linh hot , cc nhm chc nng ca trung tm hot ng ca enzyme t do cha t th sn sng hot ng, khi tip xc vi c cht, cc nhm chc nng trong phn trung tm hot ng ca phn t enzyme thay i v tr trong khng gian, to thnh hnh th khp vi hnh th ca c cht. Cng v vy, ngi ta gi m hnh ny l m hnh tip xc cm ng hoc khp cm ng. Gia c cht v trung tm hot ng to thnh nhiu tng tc yu, do c th d dng b ct t trong qu trnh phn ng gii phng ezyme v sn phm phn ng. 1.1.5. Tnh c hiu ca enzyme[2] co thnah nga Mi enzyme ch c kh nng xc tc cho s chuyn ha mt hay mt s cht nht nh theo mt kiu phn ng nht nh. c tnh tc dng la chn cao ny gi l tnh c hiu hoc tnh chuyn ha ca enzyme. Tnh c hiu l mt trong nhng c tnh c bn quan trng nht ca enzyme. C th phn bit hai kiu c hiu: c hiu kiu phn ng v c hiu c cht.

1.1.5.1.

c hiu kiu phn ng Phn nhiu mi enzyme u c tnh c hiu vi mt loi phn ng nht nh. Nhng cht c kh nng xy ra nhiu loi phn ng ha hc th mi loi phn ng y phi do mt enzyme c hiu xc tc. V d: Amino acid c kh nng xy ra phn ng kh carboxyl, phn ng kh amin bng cch oxy ha v phn ng vn chuyn nhm amin. V vy mi phn ng y cn c mt enzyme c hiu tng ng xc tc theo th t l decarboxylase, aminoacid oxydase v aminotransferase. c hiu kiu c cht Mi enzyme ch xc tc cho s chuyn ha mt hoc mt s cht nht nh. Mc c hiu c cht ca cc enzyme khc nhau khng ging nhau, ngi ta thng phn bit thnh cc mc nh sau: a) c hiu tuyt i Mt s enzyme hu nh ch xc tc cho phn ng chuyn ha mt c cht nht nh v ch xc tc cho phn ng y m thi. V d: Urease c th phn gii ure, ngoi ra n cn c th hydroxyure nhng tc thp hn 120 ln. b) c hiu nhm tuyt i Cc enzyme ny ch tc dng ln nhng cht c cng mt kiu cu trc phn t, mt kiu lin kt v c nhng yu cu xc nh i vi nhm nguyn t phn lin kt chu tc dng. V d:Maltase thuc nhm -glucosidase ch xc tc cho phn ng thy phn lin kt glucoside c to thnh t nhm OH glucoside ca glucose vi nhm OH ca mt monose khc. c) c hiu nhm tng i Mc c hiu ca cc enzyme thuc nhm ny hn nhm trn. Enzyme c kh nng tc dng ln mt kiu lin kt ha hc nht nh trong phn t c cht m khng ph thuc vo cu to ca cc phn tham gia to thnh mi lin kt .

1.1.5.2.

V d: Lipase c kh nng thy phn c tt c cc mi lin kt este. d) c hiu quang hc (c hiu lp th) Hu nh tt c cc enzyme u c tnh c hiu khng gian rt cht ch, ngha l enzyme ch tc dng vi mt trong hai dng ng phn khng gian ca c cht. V d: Phn ng kh nc ca malic acid to thnh fumaric acid di tc dng ca fumarathydratase ch xy ra i vi L-malic acid m khng tc dng ln D-malic acid. 1.2. Tng quan v enzyme protease 1.2.1.Gii thiu chung v protease 1.2.1.1. nh ngha v enzyme protease Protease l enzyme thy phn cc lin kt peptid (-CO-NH-)n trong phn t protein gii phng cc acid amin, pepton hoc ditripepton.

Hnh 1.2. Cu trc khng gian enzyme protease 1.2.1.2. Cu trc trung tm hot ng (TTH) ca Protease [7]

Trong TTH ca Protease vi sinh vt ngoi gc acid amin c trng cho tng nhm cn c mt s gc acid amin khc. Cc kt qu nghin cu chung v TTH ca mt s Protease vi sinh vt 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 cofact kim loi. + Cc Protease kim loi c TTH ln hn vo khong 210A, c th phn bit thnh su phn di TTH (subsite), mi phn di TTH tng ng vi mi gc aa trong phn t c cht. + i vi cc Protease acid, theo nhiu nghin cu cu trc TTH ca cc tinh th Protease acid ca Phizopus chinenis v Endothia parasilica cho thy phn t cc Protease ny gm c hai ht, gia chng c khe h vo khong 200A. Khe h ny l phn xc tc ca cc E, 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 vi sinh vt c khc nhau nhng cc enzyme ny u xc tc cho phn ng thy phn lin kt peptide theo cng mt c ch chung nh sau:
E+S enzyme S enzyme S + P1 enzyme + P2

Hnh 1.4 C ch xc tc TTH Enxyme Trong : E: enzyme, S: c cht, enzyme - S: Phc cht enzym- c cht, P1: L sn phm u tin ca phn ng, P2: L sn phm th hai ca phn ng. 1.2.2. Phn loi protease http://www.scribd.com S phn loi enzyme protease:

Peptidase ( protease)

Exopeptidase

Endopeptidase

Aminopeptidase

Serine proteinase

Carboxypeptidase

Cystein proteinase

Aspartic proteinase

Metallo proteinase Hnh 1.3. S phn loi protease Protease c phn chia thnh hai loi: endopeptidase v exopeptidase. * Da vo v tr tc ng trn mch polypeptide, exopeptidase c phn chia thnh hai loi: + 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 v c vai tr c bit quan trng i vi hot ng xc tc ca enzyme.

+ Cysteine proteinase: Cc proteinase cha nhm SH trong trung tm hot ng. Nh: papayin, bromelin, mt vi protein ng vt v proteinase k sinh trng. + Aspartic proteinase: Hu ht cc aspartic proteinase thuc nhm pepsin. C cha nhm carboxyl trong trung tm hot ng v thng hot ng mnh pH trung tnh. + 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 ba nhm: - Protease acid: pH 2-4 c nhiu t bo ng vt, nm men, nhng t thy vi khun. - Protease trung tnh c pH 7-8 nh papain t qu u , bromelain t qu da. - Protease kim c pH 9-11. * Ngoi ra c th phn thnh 2 loi khc: - Enzyme protease ni bo l nhng enzyme c tit ra t bn ngoi hoc ngoi bin mng protein v c trch ly vo mi trng bng k thut trch ly - Enzyme protease ngoi bo c thu nhn t qu trnh ln men, nh qu trnh ln men trn mi trng rn. Enzyme c thu nhn khi qu trnh ln men hon tt hoc ngay khi qu trnh ln men ang din ra. 1.2.3. Tnh hnh nghin cu enzyme protease http://bookluanvan.vn/f52/ung-dung-cuaenzyme-protease-trong-san-xuat-bia-30369/?langid=1

1.2.3.1. Tnh hnh nghin cu enzyme trong nc

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 enayme, kh nng ng dng enzyme trong thc t [3]. V gn y nht l nhng sng to mi m, mang tnh ng dng ln c s dng rng ri nh: + Trn Quc Hin, L Vn Vit Mn, Trung tm Cng ngh sau thu hoch, Vin Nghin cu nui trng thy sn II, Trng i hc Bch Khoa, i hc Quc gia H Ch Minh Nghin cu thu nhn ch phm Protease t rut c Basa (pangasius bocourti) thc hin nm 2006. Nghin cu ny kho st qu trnh trch ly v tinh sch enzyme Protease t rut c Basa (Pangasius bocourti). Dch chit protease kim thu c t rut c tng hot tnh cao nht l 15,79UI/gCKNT (cht kh ni tng) trong iu kin chit: t l mu/dung mi 1/1(w/w); pH 9,5; nhit 35oC; thi gian chit 10 pht. [14]. + Ti u mt s iu kin nui cy chng vi sinh vt bin Acinetobacter sp. QN6 sinh tng hp Prtease c thc hin ca Quyn nh Thi, Trn Th Qunh Anh, Nguyn Th Tho (2007), vin CNSH chng minh cc iu kin thun li nhm nui cy vi hiu sut cao nht i vi chng vi sinh vt bin Acinetobacter sp. QN6 sinh tng hp Prtease [15]. + Phan Th Bch Trm, Dng Th Hng Giang, H Thanh Ton, Phm Th nh Hng, i hc Cn Th, Trng HKH T nhin, H Quc Gia TP H Ch Minh tin hnh Tinh sch v kho st c im ca cc Serine Protease t Trn Qu ( 2007). Bc u kho st h enzyme Protease t trn qu (Perionyx excavatus) cho thy phn ln cc protease trong dch chit enzyme th c th tinh sch s b bng ta phn on vi ammonium sulfat nng trong khong 30-80%. Nhit v pH ti u cho enzyme th hot ng trn c cht casein l 550C v pH trong khong kim 10 -12. [16],[4].

+ Nghin cu ng dng Protease bacillus subtilis trong sn xut bt m thy phn t c Mi, V Ngc Bi, trng i hc thy sn Nha Trang. Qua nghin cu cho thy protease B. subtilis c th thy phn mnh m c tht c mi v hon ton c th s dng enzyme ny trong sn xut bt m thy phn. Khi b sung protease B. subtilis vi nng enzyme 0,3% vo hn hp c tht c mi v thy phn 500C. + Nghin cu ng dng Protease trong sn xut Bia, thc hin trong cc nm 1993-1994, Thc hin: TS.Trng Th Ha v cc cng tc vin Vin Cng nghip thc phm. Protease ca Aps. oryzae c dng thy phn protein trong ht ng cc, to iu kin x l bia tt hn. [8] 1.2.3.2. Tnh hnh sn xut enzyme Protease trn th gii Trong cc Protease, cc enzyme ca h tiu ha c nghin cu sm hn c. Nm 1857, Corvisart tch c tripxin t dch ty, l Protease u tin nhn c dng ch phm. Nm 1861 Brucke cng tch c Pepxin t dch d dy Ch dng tng i tinh khit. Ngoi cc enzyme ca h tiu ha, ngi ta cng quan st u tin v cc Protease trong mu [11]. Cc Protease thc vt c pht hin mun hn. Nm 1879 Wurtz c xem l ngi u tin tch c Protease thc vt. n nay ngi ta nghin cu c kh y v cu trc phn t ca nhiu Protease nh: papain, tripxin, kimotripxin, subtilizin [11]. Cc Protease ca vi sinh vt mi c ch nghin cu nhiu t nm 1950, mc d t nm 1918- 1919 Waksman pht hin c kh nng phn gii Protein ca X khun. Trong hn 10 nm nay s cng trnh nghin cu Protease vi sinh vt tng ln ng k nhiu hn cc Protease ca ng v thc vt. Nhng kt qu t c trong lnh vc ny gp phn m rng quy m sn xut chees pha enzyme v ng dng enzyme trong thc t [11].

Trong CNTP, ngi ta s dng Protease sn xut phomat t sa (Mohanty et al.,1999), xn xut bnh t bt m (Hozova et al., 2003) hay ch bin cc sn phm giu protein t u tng (Ghazi et al., 2003; Ma et al., 2004); trong cng nghip thuc da, Protease c dng thy phn mt s thnh phn phi collagen ca da v loi b cc protein phi fibrin nh albumin, globulin (Gupta, Rammani, 2006); trong cht ty ra, Protease l mt trong nhng thnh phn quan trng ca tt c cc loi cht ty ra, t cc cht ty ra dng trong gia nh n nhng cht lm sch knh hoc rng gi, kem nh rng (Rao et al., 1998). 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%) (Rao et al., 1998) [13]. T nm 1950 tr li y trn th gii c hng lot Protease ng vt, thc vt v vi sinh vt c tch chit nghin cu. Thi gian gn y cc nh khoa hc trn th gii tp trung nghin cu v Protease vi sinh vt v t nhiu thnh tu to ln v lnh vc ny (Protease t vi sinh vt chim ti 40% tng doanh thu ca enzyme ton th gii (Godfrey west, 1996) [13]). Hin nay, s lng cc enzyme c sn xut hng nm trn th gii, cc nc pht trin nht l chu u, M v Nht Bn vo khong 300.000 tn vi doanh thu t sn xut enzyme c tnh vo khong 500 triu USD. Trong khong 600 tn Protease tinh khit c sn xut t vi sinh vt bao gm khong 500 tn t vi khun v 100 tn t nm mc. Nhng nc c cng ngh sn xut v ng dng Protease tin tin trn th gii l: an Mch, Nht Bn, M, Anh, Php, H Lan, Trung Quc, c, o. Cc nc ny u t thch ng cho cng tc nghin cu, sn xut v ng dng Protease t VSV. Chnh v th nhp sn xut Protease quy m cng nghip ti cc nc pht trin hng nm tng vo khong 5%- 10%. Ngy nay ngi ta c th sn xut cc enzyme c nh trn cc cht mang khng tan cho php c th ti s dng enzyme nhiu ln. V vy m vic ng dng Protease ngy cng gia tng[4],[1].

1.2.3. Ngun thu nhn enzyme protease TRNG I HC BCH KHOA - I HC QUC GIA TP HCMKHOA
K THUT HA HcBI BO CO MN HA SINH THC PHMNGUN THU NHN V NG DNG PROTEASE

Enzyme c trong tt c cc c quan, m ca sinh vt; nhng thun li v kinh t, ngi ta ch dng nhng vt liu cho php thu mt lng ln enzyme vi hiu sut cao. Hin nay chng ta s dng 3 ngun nguyn liu sinh hc c bn thu nhn protease : - ng vt : Thng thng protease ng vt c tuyn tiu ha (nim mc d dy, nim mc rut non, tuyn ty). Vd: Pepsin t nim mc d dy v dch v ca ng vt bc cao. Chymosin rennin c trong ngn th t d c b non di 5 thng tui Tuy nhin, s dng rng ri trong cng nghip, protease ng vt t thun li do sn xut chng b hn ch v ngun nguyn liu thu nhn enzyme khng ln lm - Thc vt : T cc thc vt bc cao ngi ta cng thu c mt s ch phm enzyme quan trng Vd : Papain thu t m u xanh, Bromelin t than cy da Lng enzyme thu c t cc nguyn liu thc vt khng ln lm so vi lng nhin liu tiu hao - Vi sinh vt :Hai ngun nguyn liu trn khng th dng dng nguyn liu trong sn xut cng nghip qui m ln do nhng hn ch v nguyn liu v cng ngh. V vy, dng enzyme t vi sinh vt s khc phc c cc hn ch trn. + Ngun nguyn liu v hn + H enzyme phong ph + Hot tnh mnh + C kh nng tng cng sinh tng hp enzyme nh chn ging

+ Vi sinh vt sinh sn vi tc cc nhanh + Thc n nui d kim, r rin. 1.2.4. ng dng ca enzyme protease http://tailieu.vn/xem-tai-lieu/thu-nhan-va-ung-dung-cuaenzyme-protease.206830.html

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: 1.2.4.1. 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). 1.2.4.2. Trong ch bin thu sn - Khi sn xut nc mm th 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 Protease rt ngn thi gian lm v ci thin hng v ca nc mm. Phng php s dng Protease trong xn 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 polypeptid

Protease peptid amino acid

Trong 24 gi u tin, di tc ng ca cc h Protease ni ti ca c mt phn protein tht c b thy phn thnh peptid, polypeptid ha tan trong nc bi. Sau protein tht c, polypeptid v cc peptid c to thnh ny tip tc b thy phn mnh m bi cc Protease kim loi to thnh cc aa t do, cc peptid ngn v c s hnh thnh hng v ca nc mm. 1.2.4.3. 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 mt [1] Trn Xun Ngch (2007), Cng ngh enzym, Trng i Hc Bch Khoa Nng. . 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. 1.2.4.4. 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 [6]. L Xun Phng (2001), Vi sinh vt cng nghip, NXB Xy dng, H Ni.

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 [7]. Nguyn c Lng ( ch bin),(2004), Cng Ngh EnZym, Nh Xut Bn i Hc Quc Gia thnh ph H Ch Minh. 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, - Sn xut keo ng vt, cht git tng hp git ty cc cht bn protein, sn xut m phm, 1.3. Tng quan v vi sinh vt 1.3.1. Gii thiu chung v vi sinh vt - Trong nui cy vi sinh vt thu nhn enzyme c mt s k thut chung v mt s k thut ring. Nhng k thut chung bao gm: [7] + K thut to ging + La chn phng php nui cy + Thit k thit b nui cy + K thut ln men

+ Tch, tinh ch thu nhn E. - Nhiu vi sinh vt c kh nng tng hp mnh Protease, cc enzyme ny c th trong t bo hoc c tit vo mi trng nui cy. Mt s Protease ngoi bo sn xut quy m cng nghip v c s dng rng ri trong nhiu ngnh cng nghip, trong nng nghip v trong y hc. - Ngun vi sinh vt thu nhn enzyme Protease ch yu gm: vi khun, nm mc, x khun. 3.1 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]. 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 S5 c kh nng tng hp Protease mnh nht. Cc vi khun thng tng hp cc Protease hot ng thch hp vng pH trung tnh v kim yu.[2] 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. Chng cn c kh nng i lc cao i vi cc acid amin a bo v thm v c sinh ra nhiu bi B.subtilis, B.mesentericus, B. thermorpoteoliticus v mt s ging thuc chi Clostridium [2].

Protease ca Bacillus a kim c im ng in bng 11, khi lng phn t t 20.000-30.000dalton. n nh trong khong pH 6-12 v hot ng trong khong pH rng 7-12. [2] 3.2 Nm mc Nhiu loi nm mc c kh nng tng hp mt lng ln Protease c ng dng trong cng nghip thc phm nh cc chng: Aspergillus oryzae, A. terricola, A. fumigatus, A. saitoi, Penicilliumchysogenum)cc loi nm mc ny c kh nng tng hp c ba loi P: 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 [2]. 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 phomt. 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 c mt s chng c kh nng tng hp cao nh: Streptomyces grieus, S. fradiae, S. trerimosus[2]. Cc ch phm 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 phn ti 90% lin kt peptide ca nhiu protein thnh acid amin. T S. fradiae cng c th tch chit c keratinase thy phn karetin. M, CP c sn xut c tn l M-Zim dng trong sn xut da [2]. Protease t S. fradiae cng c hot tnh elastase cao, do chng c dng trong cng nghip ch bin tht. 1.3.2. Phng php thu nhn enzyme protease t vi sinh vt 1.3.2.1. 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. C th thng qua cc phng php sau: - Phng php gy t bin - Phng php bin np - Phng php tip hp gene - Phng php ti np 1.3.2.2. Mi trng nui cy vi sinh vt tng hp enzyme Protease 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. a) Ngun cacbon [1],[11]

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 hp 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 Protease ca Asp. oryzae 79c th theo th t: fructoza sacaroza maltoza glucoza manit arabinoza galactoza. - Tinh bt l ngun cacbon ca nhiu chng vi khun sinh tng hp enzyme Protease. b) Ngun nit [1] Ngun nit 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. 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 2274%. Cn trng hp dng cc ngun nit v c duy nht trong mi trng s dn n ngng sinh tng hp Protease ni chung. Cc acid 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%. 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 VSV. c) Ngun cc nguyn t khong v cc yu t kch thch sinh trng [1]

- 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 E, nhng khi cng cn tnh n kh nng bin i nhanh chng ch s bi VSV. + 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 cho qu trnh sinh tng hp enzyme mong mun l cao nht 1.3.2.3. Nui cy thu nhn ch phm enzyme Protease bng phng php b mt [3] L phng php to mi trng cho vi sinh vt pht trin trn b mt mi trng. C th l mi trng lng hoc c. 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. - 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. 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 d thc hin v d dng x l khi b nhim vi sinh vt l, c th thc hin qui m gia nh, trang tri cng nh qui m ln n 20Tn/ngy. + 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. Ngoi ra phng php nui cy b mt c mt nhc im rt ln l tn nhiu din tch. Do vy m phng php ny dn c thay th bng nui cy chm nui cy vi khun. 1.3.3. Thu nhn enzyme (E) 1.3.3.1. Tch v lm sch ch phm enzyme [3] Enzyme thng cha cc t bo sinh vt gi l cc enzyme ni bo (intracellular enzyme), nhng n cng c th c cc sinh vt tit ra mi trng sng. l cc enzyme ngoi bo (extracellular enzyme). Enzyme vi sinh vt thng chit l enzyme ngoi bo. - Cc phn t enzyme ni bo 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 ny, 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. 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. - trch ly enzyme ra khi t bo trc ht cn 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. i vi trng hp enzyme cn nm trong t bo (E ni bo nui bng phng php b mt) th cn phi gii phng enzyme bng cch ph v t bo thu nhiu cch nh: + Nghin nh, nghin vi ct, nghin vi vn thy tinh, nghin bi. + t bo t phn hy + Dng tc dng ca siu m hoc to p sut thm thu cao, trch ly bng mui, dung dch mui trung tnh, dung mi hu c. + Kt ta enzyme bng cc cht in ly thch hp. - Thm tch [9] Trong qu trnh tinh sch cc enzyme loi b cc phn t ha tan nh khng mong mun khi dch trch ly enzyme nh amoni sulfat sau khi kt ta, dng mt mui kh hp th enzyme trong sc k trao i ion, hoc mt phi t cnh tranh dng trong sc k i lcngi ta thng dng phng php thm tch. Ti thm tch c cu to bng mt mng bn thm, cha dch chit E, c t vo trong mt dung dch m khng c cha cht ha tan cn c loi b. Cht ha tan khuch tn ra khi mng cho n khi nng cht ha tan trong ti v bn ngoi dch m s bng nhau. - Lm c [9]

Trong qu trnh tinh sch enzyme, c th phi lm c dch E, nht l sau khi qua giai on sc k dch cha enzyme b pha long ra nhiu. Lm c dch cha enzyme bng cch cho bc hi trong chn khng, bng cch thm thu ngc ( to p sut thm thu lm cho dung dch i qua mt mng bn thm v gi li cc protein E) hoc bng siu lc (dung dch enzyme c dn qua mt mng c l nh bng p sut hoc bng ly tm). 1.3.3.2. Cc dng ch phm enzyme thu c a) Thu ch phm k thut Ch phm enzyme k thut l ch phm enzyme cha c tinh ch, n c th cha mt hoc vi enzyme ch yu ty theo yu cu s dng, ngoi ra trong thnh phn cn c th c cc protein khng hot ng, cc cht n nh v cc tp cht khc. thu c ch phm enzyme k thut bc u ngi ta c c cc dch enzyme c nng cht kh thp 4-6 g/l ln ti 15 20 g/l nhit 35oC trong thit b c chn khng cao. Sau : + Hoc l ta s c c tip nhit 40- 45oC t nng cht kh 30-35g/l, b sung thm cc bo qun NaCl, Glixerol, Socbitol, Benzoat v thu c ch phm dng lng, c th bo qun nhit thng t 1-2 nm. + Hoc l b sung thm cc cht n nh t nng cht kh 30-40 g/l ri sy phun vi nhit sy c nhit u vo 120oC v u ra 60oC. Kt qu thu c CP dng bt. + Hoc ngi ta c th kt ta enzyme bng cc loi mui trung tnh nh (NH4)2SO4 hoc cn, trn thm cc cht n nh ri sy kh, nghin mn. Kt qu ta thu c ch phm dng bt. b) Thu ch phm enzyme tinh khit

Vic tinh ch enzyme c th tin hnh theo nhiu phng php v qua nhiu giai on khc nhau. Cc protein khng hot ng c th c loi khi dch enzyme bng phng php lm bin tnh chn lc nh tc dng ca pH, nhit. Cc protein b bin tnh c th tch khi dung dch khi ly tm hoc loi b kt ta. C th dng phng php tch phn on khc nhau nh kt ta bng dung mi hu c, kt ta bng mui, hp ph chn lc, trao i ion thu nhng phn c hot lc enzyme cao nht.

Chng

II:

QUY

TRNH

SN

XUT

ENZYME

PROTEASE

http://www.scribd.com/doc/66914567/27/Quy-trinh-thu-nh%E1%BA%ADn-enzyme-proteaset%E1%BB%AB-n%E1%BA%A5m-m%E1%BB%91c-Asp-oryzae

Nc

Nguyn liu dinh dng (bt bp, cao nm men, pepton)

Khong hn hp

Hp thanh trng Lm ngui n 300C ln khay Ging Asp.oryzae 0,5-2% Nui cy nhit phng Thu nhn sinh khi (enzyme th) Ch phm enzyme th em tinh ch Sy Nghin mn Trch ly Lc B Dng trong chn nui Ch phm ezyme th em s dng

Kt ta enzyme Thu nhn kt ta Sy Tinh ch Ch phm ezyme K thut Thu nhn ch phm enzyme tinh khit

Chng III: THUYT MINH QUY TRNH http://tailieu.vn/xem-tai-lieu/thu-nhan-va-ungdung-enzyme-protease-vi-sinh-vat.138826.html

3.1. i tng nghin cu Asp. oryzae l mt loi nm vi th thuc b Plectascales, lp Ascomycetes. C th sinh trng ca n l mt h si bao gm nhng si rt mnh, chiu ngang 5-7 m, phn nhnh rt nhiu v c vch ngang , chia si thnh nhiu bao t bo (nm a bo). c im ca ging Asp.oryzae giu cc enzyme thy phn ni bo v ngoi bo (amylase, protease, pectinasa,), ta rt hay gp chng cc kho nguyn liu, trong cc thng cha ng 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 mc mi. 3.2. Thuyt minh quy trnh 3.2.1. K thuyt nui cy Sau khi trn ging, mi trng c tri u ra cc khay vi chiu di 2-3cm, ri c a vo phng 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 28320C. Nhit thp qu hoc cao qu 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 36-60 gi. iu ny cn ph thuc vo chng nm mc Asp.oryzae v iu kin mi trng cng nh ph thuc vo iu kin nui cy.

Qu trnh pht trin ca nm mc trong mi trng bn rn khi nui bng phng php b mt ny tri qua cc giai on sau: . Giai on 1: Giai on ny ko di 10-14 gi k t thi gian 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 c 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. Enzym protease c tng hp mnh. Lng O2 trong khng kh gim v CO2 s tng dn, do trong giai on ny cn phi c thng kh mnh v nhit c gng duy tr trong khong 29-300C 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 phng nui cy/ 1gi. Nhit d nui duy tr 300C, trong giai on ny, bo t c hnh thnh nhiu do lng Enzym protease to ra s gim xung. Chnh v th vic xc nh thi im cn thit thu nhn enzym rt cn thit. 3.2.2. Thu nhn sn phm: Kt thc qu trnh nui cy ta thu nhn c ch phm enzym protease, ch phm ny c gi l ch phm enzym th. Ty theo mc ch s dng ta c th dng ch phm th ny ngay khng cn phi qu trnh tinh sch. Trong nhng trng hp cn thit khc, ta phi tin hnh lm sch enzym. sn xut enzym tinh khit ngi ta ph tin hnh nh sau: . Nghin mn Ton b khi lng enzym th protease c em i nghin nh. Mc ch ca qa trnh ny 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 enzym ni bo cha thot ra khi t bo s d dng

thot khi t bo. Phn ln enzym protease ngoi bo khi c tng hp v thot khi t bo ngay lp tc thm vo thnh phn mi trng. Khi ta nghin nh, enzym thot ra khi cc thnh phn ny d dng hn. Trong khi nghin ngi ta thng s dng nhng cht tr nghin nh 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 thy tinh phi c ra sch, sy kh nhit ln hn 1000C 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 cho 4-5 phn nc, khuy nh v sau lc ly dch, phn b thu ring dng lm thc phm gia sc (ch cn loi b ct 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 c cha nc, cc cht ha tan khc t khi mi trng nui cy. Vic tip theo l lm sao tch enzym ra khi vt cht ny. . Kt ta enzym protease lm vic trn ngi ta tin hnh kt ta enzyme nh nhng tc nhn gy ta. Trong cng ngh tinh ch enzyme, 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 ta enzym vo dung dch enzym th phi ht sc t t trnh hin tng bin tnh. Cc enzym s c to kt ta v lng xung y, 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 ta sy kt ta enzyn protease 40OC cho n khi m cui cng t 5-8% W (thit b sy thng dng l my sy phun sng. Trong nhiu trng hp ch phm enzyme protease dng kt ta vn hon ton cha sch v mt ha hc v trong cn cha mt s enzym ngoi enzyme ta quan tm.

KT LUN T cc kt qu tham kho nhng ti, ti liu mang tnh thit thc, qua ti Nghin cu quy trnh sn xut protease t vi sinh vt em rt ra c mt s kt lun sau: Bit c quy trnh sn xut enzyme protease t vi sinh vt, c bit l t nm mc Aps. Oryzae, c tnh ca nm mc Aps. Oryzae dng sn xut protease, tnh hnh sn xut enzyme protase trong nc v trn th gii, cu to v phn loi ca enzyme protease, ng dung ca enzyme protease

TI LIU THAM KHO

Ti liu tham kho ting Vit [9]. H Th Tuyt Mai(2006), bi ging Cng ngh E, Trng Cao ng Lng Thc Thc Phm. [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 TP H Ch Minh. [3]. Lng c Phm (2004), Cng ngh vi sinh vt, Nh xut bn Nng nghip, H Ni. [6]. L Xun Phng (2001), Vi sinh vt cng nghip, NXB Xy dng, H Ni. [7]. Nguyn c Lng ( ch bin),(2004), Cng Ngh EnZym, Nh Xut Bn i Hc Quc Gia thnh ph H Ch Minh.

[11]. enzyme Vi Sinh Vt, tp 1, L Ngc T, La Vn Ch, Phm Trn Chu, NXB Jhoa hc k thut H Ni 1982. Ti liu tham kho mng-internet. ht tp://www.scribd.com/ http://www.e-socrates.org