Bai Giang Cong Nghe Enzyme (1)

5/25/2018 Bai Giang Cong Nghe Enzyme (1)

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Trng Cao ng Kinh t Cng ngh TPHCM

Khoa Cng ngh Sinh hc

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CCNNGGNNGGHHEENNZZYYMMEE(Ti liu lu hnh ni b)

CBDG: ThS. L Thanh Hi

HIAST

TPHCM, 03/2013


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CNG NGH ENZYME i

MC LC

1. M U ................................................................................................................................. 11.1. Lch s nghin cu v pht trin cng ngh enzyme ........................................................ 11.2. Khi nim v enzyme........................................................................................................ 2

1.2.1. Bn cht sinh hc ca enzyme ................................................................................... 31.2.2. Bn cht ha hc ca enzyme .................................................................................... 41.2.3. Cu trc ca enzyme .................................................................................................. 5

1.3. C ch tc dng ca enzyme............................................................................................. 71.3.1. C ch xc tc ca enzyme ....................................................................................... 71.3.2. Nng lng xc tc .................................................................................................... 81.3.3. S to thnh phc hp enzyme c cht .................................................................. 81.3.4. ng hc ca phn ng enzyme ................................................................................ 91.3.5. Cc yu t nh hng n phn ng enzyme .......................................................... 10

1.3.5.1. Nhit ............................................................................................................ 101.3.5.2. pH ..................................................................................................................... 111.3.5.3. Cht km hm ................................................................................................... 111.3.5.4. Cht hot ha .................................................................................................... 12

1.3.6. Tnh cht c hiu ca enzyme................................................................................ 131.4. Phn loi enzyme ............................................................................................................ 13

1.4.1. Danh php quc t ca enzyme ............................................................................... 131.4.2. Phn loi enzyme ..................................................................................................... 14

1.4.2.1. Oxydoreductase ................................................................................................ 141.4.2.2. Transpherase ..................................................................................................... 141.4.2.3. Hydrolase ......................................................................................................... 151.4.2.4. Lipase ............................................................................................................... 151.4.2.5. Isomerase .......................................................................................................... 151.4.2.6. Ligase ............................................................................................................... 15

1.5. Phng php tch v lm sch enzyme .......................................................................... 151.5.1. Cc phng php ph v t bo .............................................................................. 151.5.2. Phng php gy bin tnh chn lc ....................................................................... 16

1.6. Phng php xc nh kh nng xc tc ca enzyme ..................................................... 171.6.1. Cc nhm phng php xc nh kh nng xc tc ca E ...................................... 171.6.2. n v hot ......................................................................................................... 17

2. K THUT C BN TRONG CNG NGH ENZYME .................................................. 182.1. ngha ca k thut tinh sch trong cng ngh enzyme ............................................... 18


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CNG NGH ENZYME ii

2.2. K thut c bn ............................................................................................................... 192.2.1. Cc phng php c hc tch enzyme .................................................................... 192.2.2. Cc phng php ph v t bo sinh vt ................................................................. 19

2.2.2.1. Ph v t bo bng phng php c hc: ........................................................ 202.2.2.2.

Ph v t bo bng phng php khng phi c hc: ..................................... 20

2.2.2.3. Cc phng php c c .................................................................................. 212.2.2.4. Cc phng php tinh sch enzyme ................................................................. 212.2.2.5. To sn phm enzyme ...................................................................................... 21

3. ENZYME C NH ............................................................................................................. 223.1. Chuyn ha sinh hc ....................................................................................................... 22

3.1.1. Chuyn ha vt cht do enzyme t do hay enzyme ha tan .................................... 223.1.2. Chuyn ha vt cht bng enzyme khng ha tan .................................................. 223.1.3. Chuyn ha vt cht do cc qu trnh ln men ........................................................ 233.1.4. Chuyn ha vt cht do t bo c nh .................................................................... 23

3.2. c im ca enzyme c nh ........................................................................................ 233.3. Phng php to enzyme c nh ................................................................................... 23

3.3.1. Cc yu t nh hng n enzyme c nh ............................................................. 233.3.2. Phng php c nh enzyme ................................................................................. 25

3.3.2.1. Cht mang dng c nh enzyme ................................................................ 253.3.2.2.

Phng php c nh enzyme .......................................................................... 25

3.4. ng dng ca enzyme c nh ....................................................................................... 26

3.4.1. Sn xut fructose nh enzyme glucose isomerase .................................................. 273.4.2. Sn xut L-amino acid nh enzyme aminoacylase c nh .................................... 28

4. THU NHN ENZYME......................................................................................................... 284.1. Chn ngun nguyn liu ................................................................................................. 284.2. Thu nhn enzyme ............................................................................................................ 31

5. NG DNG CA ENZYME .............................................................................................. 325.1. Tnh hnh ng dng enzyme trong cng nghip trn th gii ........................................ 325.2. ng dng trong y hc ..................................................................................................... 345.3. ng dng trong ha hc ................................................................................................. 345.4. ng dng trong cng nghip .......................................................................................... 34

5.4.1. ng dng trong cng nghip thc phm ................................................................. 345.4.2. ng dng trong cng nghip dt ............................................................................. 365.4.3. ng dng trong cng nghip thuc da .................................................................... 36

5.5.

ng dng trong nng nghip .......................................................................................... 36

TI LIU THAM KHO ............................................................................................................. 36


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CNG NGH ENZYME 1

1. M U1.1.Lch s nghin cu v pht trin cng ngh enzymeEnzyme hc l mn hc nghin cu v vt cht phn t c hot tnh sinh hc. Mn hc ny pht trin rtmnh nh nhng thnh tu ca mn ha sinh hc, vi sinh vt hc, di truyn hc v c nhng tin b cangnh ha hc v vt l hc.

Bng 1.1. Nhng ct mc quan trng trong nghin cu v pht trin mn enzyme hc[2]

STT Nm Ni dung nghin cu, pht trin

1 1833 Payen v Persoz tch c diastase t malt

2 1874 Hansen l ngi u tin tch c rennet t bao t cu

3 1876 Kiihne l ngi u tin ngh gi cht xc tc sinh hc l enzyme

4 1897Hai anh em nh Buchner chng minh dch chit t nm men c th chuyn hang glucose thnh cn v CO2

5 1900 Rohm s dng enzyme protease trong cng ngh thuc da

6 1913 Rohm l ngi u tin s dng enzyme trong cht ty ra

7 1917Boidin v Effront nghin cu -amylase caB.subtilis v ng dng trong ngnhdt

8 1920 1928 Will Slitter tinh sch c enyzme

9 1926

Samner kt tinh c enyme urease

Northrop kt tinh c protease

10 1928 Fleming pht hin ra penicilline

11 Th chin IIBt u sn xut khng sinh theo m hnh cng nghip, s dng amyloglucosidaseng ha tinh bt. S dng penicillineacylase trong sn xut penicilline

12 1969Tanabec co xy dng quy trnh cng nghip sn xut amino acid. S dngglucose isomerase trong sn xut dch ng giu fructose


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CNG NGH ENZYME 2

13 1972Boyer et al. a ra k thut di truyn. K thut ny c tc ng tch cc trongcng ngh enzyme

14 1973Tanobe co sn xut aspartic acid bng ln men c nh t bo

Winter v Ferch a ra cng ngh sn xut protein

15 1984Nito xc lp qu trnh c bn to acrylamide v mt loi qu trnh sn xut c stham gia ca enzyme

16 1984 - nay pht hin hng trm loi enzyme khc nhau, a vo sn xut cng nghip vng dng rng ri enzyme trong sn xut v i sng. k thut enzyme c nh, t

bo c nh a cng ngh enzyme t c nhiu kt qu cao

Diastase:l enzyme xc tc cho phn ng thy phn tinh bt thnh maltose. Diastase l enzyme u tinc pht hin ra. N c chit tch t malt vo nm 1833 bi Anselme Payen v Jean-Francois Persoz

nh Ha hc ti nh my ng ca Php. Tn diastase xut pht t ting Hy Lp (diastasis), trongdch nha nng, cc enzyme ny thy phn tinh bt trong ht i mch thnh cc ng ha tan v v thc th tch phn b malt vi phn cn li ca ht. Ngy nay, diastase l , hoc -amylase.

Rennetl enzyme c sn xut trong d dy ca ng vt c v khi cn trong giai on b sa m vthng c s dng lm phomai. Rennet l mt hn hp enzyme, bao gm: cc enzyme thy phn

protein (protease) gy ng t sa, c th tch phn sa ng (phn cht rn) v phn huyt thanh sa(phn cht lng). Nhng enzyme hot ng trong d dy b c gi l chymosin hoc rennin. Ngoi ra,cng c nhng enzyme quan trng khc nh: pepsin v lipase.

Protease:(cn gi l peptidase hoc proteinase) l enzyme thy phn protein

-amylasel enzyme thy phn lin kt alpha ca phn t polysaccharide nh tinh bt v glycogen thnh

glucose v maltose. Alpha amylase c tm thy ch yu ngi v ng vt c v khc, ngoi ra,chng cn c mt trong cc loi ht c cha tinh bt v c tit ra bi mt s loi nm.

Urease: l enzyme xc tc phn ng thy phn ure thnh carbon dioxide (CO 2) v amoniac (NH3), hindin trong vi khun ng rut. Urease c tm thy trong vi khun, nm men v thc vt.

1.2.Khi nim v enzymeSinh vt c phn ra 2 nhm da vo cu to c th ca chng: sinh vt n bo v sinh vt a bo. Mcd chng c s khc bit rt ln v cu to c th v nhng c im sinh l khc nhau nhng chng uging nhau v trao i cht vi mi trng bn ngoi v mt s c im v bin d di truyn.

Sinh vt c xem nh l mt h thng m c lin quan cht ch n qu trnh trao i cht ca c th trong t bo v gia t bo vi mi trng ngoi. Qu trnh trao i cht bn trong t bo v gia t bo

vi mi trng bn ngoi l biu hin sinh ng nht ca s sng. S khc nhau gia t bo sng v vtcht khng phi s sng chnh l kh nng trao i cht ny. Khi c th khng cn kh nng trao i chtth c th s cht. Do mi quan h gi c th vi bn ngoi l mi quan h hu c.

Qu trnh trao i cht bao gm qu trnh d ha v qu trnh ng ha. Qu trnh d ha l qu trnh phngii vt cht cung cp cho t bo nng lng v vt liu xy dng t bo, qu trnh ny c th xy ratrong t bo hoc ngoi t bo.

Qu trnh di ha xy ra trong t bol qu trnh cung cp nng lng, vt cht cho qu trnh tng hp vtcht to ra sinh khi nhm lm i mi vt cht t bo.

Qu trnh d ha ngoi t bophn ln ch p ng nh cu v vt cht, gip cho t bo tng hp cccht trong t bo. Nng lng to ra t qu trnh d ha ngoi t bo thng c gii phng dng nhit

nng.


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CNG NGH ENZYME

Qu trnh tng hp vt cht cholng t cc phn ng do qu trca sinh vt i vi mi trng x

H

Sn phm ca qu trnh trao i

Sn phm bc 1 Sn phm bc 2Sn phm bc 1: l cc sn phCc sn phm ny s c tham

Sn phm bc 2: l cc sn phbo. Trong qu trnh tng hp,theo m s thot ra khi t bo.

sinh tng hp tha c lin quantruyn, nhiu khi iu chnh hcht c to ra do qu trnh ph(qu trnh d ha ngoi t bo).d ha trong t bo)

Qu trnh trao i cht lin tc cht trong thin nhin. Qu trnhnhiu chu trnh chuyn ha cckhng ch trong t bo sinh vny c xc tc bi mt loi pr

bo c gi l enzyme ngoi bni bo v enzyne ngoi bo u

1.2.1. Bn cht sinh hc ca eNhiu nh khoa hc nghin csinh vt sng tng hp nn v th

Nh vy, bn cht sinh hc ca esinh ha trong v ngoi t bo si

Enzyme c to ra trong ttp v c iu khin, kim

Enzyme tham gia phn ng c Enzyme tham gia phn ng

enzyme c tng hp v h ca c th v ca t bong trong khong 30 - 40oC

t boch xy ra trong t bo. Qu trnh ny th nh d ha. Nh vy, qu trnh trao i cht

ung quanh.

nh 1.1. H thng m ca t bo sinh vt

ht to ra 2 dng sn phm:

c to ra c trong qu trnh phn gii v c tia trc tip nn vt cht t bo v tham gia cc c

cng c to ra t qu trnh tng hp v qt s vt cht c to ra khng tham gia vo qHin tng ny c coi nh qu trnh sinh t

cht ch n h di truyn c trong t bo. Do en s thu c lng ln cc sn phm sinh tn gii s khng tham gia vo qu trnh trao it s vt cht khc thot ra khi t bo vo m

c xy ra gia trong v ngoi t bo, to nn schuyn ha theo con ng sinh vt ng vai trcht c trong thin nhin. Cc phn ng sinh hm c ngoi mi trng, bao quanh t bo .tein c bit gi l enzyme. Cc enzyme tham go. Cc enzyme thc hin trong t bo gi l enzc tng hp trong t bo.

nzyme

u v enzyme v i n thng nht: enzyme lm gia vo cc phn ng sinh hc

nzyme l sn phm ca cc qu trnh sinh hc vh vt. Cc loi enzyme u c nhng c tnh c

bo sinh vt: qu trnh tng hp enzyme l msot rt cht ch trong t bo sng v c khi enzyme c tchtrong iu kin nhit n ha. V trong qut ng trong iu kin nhit ca t bo v n

sinh vt thng l nhit thp. Phn ln nhi

3

ng phi thu nhn nngc xem nh h thng m

ong qu trnh tng hp.c qu trnh t bo.

trnh phn gii ca t trnh trao i cht tipg hp tha. Hin tng

, vic iu chnh h dig hp tha. Mi s vtcht nm ngoi t boi trng (qu trnh trnh

bin i lin tc ca vtrt quan trng trong rtc xy ra thng xuyn

. Cc phn ng sinh hcia cc phn ng ngoi tme ni bo. C enzyme

mt loi protein c

thc hin cc phn ngung nh sau:

t qu trnh ht sc phc

hi t bo sngtrnh sng ca t bo,it ca c th. Nhit c th sinh vt dao


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CNG NGH ENZYME 4

Enzyme c th tham gia xc tc cc phn ng trong v ngoi c th t giai on u n giai ongii phng hon ton nng lng d tr trong cc hp cht ha hc. Qu trnh chuyn ha ny cthc hin theo chui phn ng, mi phn ng c xc tc bi mt loi enzyme. Cc enzyme ny lnlt thay th nhau xc tc cc phn ng ln lt xy ra, cui cng to thnh CO2, H2O, mt scht khc v gii phng nng lng. Cng c th chui phn ng s to thnh chu k chuyn hakhp kn. Trong chui chuyn ha h hay chui chuyn ha khp kn, sn phm ca phn ng trcs l c cht cho phn ng sau

Enzyme c th thc hin mt phn ng: Cc phn ng thng xy ra ngoi t bo (khi ta thc hinchng trong ng nghim). Trong t bo thng khng xy ra phn ng enzyme n (mt phn ng)m thng xy ra cc phn ng theo chui phn ng.

Phn ng enzyme l nhng phn ng tiu hao nng lng rt t. Trong khi , cc phn ng ha hcc xc tc vi cc cht xc tc ha hc i hi nng lng rt ln. Nh c hot ng xc tc caenzyme, cc phn ng sinh ha xy ra lin tc trong iu kin nng lng n ha.

Enzyme chu s iu khin bi gen v cc iu kin phn ng. Gen quyt nh tng hp ra mt loienzyme. Mi mt enzyme quyt nh mt phn ng sinh ha. Cc nh khoa hc a ra c ch sau:

Mt gen => mt enzyme => mt phn ngNh vy, gen quyt nh bn cht sinh hc v bn cht ha hc ca enzyme. C ch ny c mt ngha rt ln trong vic iu khin tng hp enzyme trong t bo sinh vt.

1.2.2. Bn cht ha hc ca enzymeNu tch enzyme ra khi t bo v tin hnh phn tch thnh phn ha hc ca chng, ta s thy chngthuc 2 nhm:

1. Nhm enzyme n cu t:

Thuc nhm ny bao gm nhng enzyme ch c cu to mt thnh phn ha hc duy nht l protein.Nhng enzyme c to thnh ch t protein duy nht c gi l enzyme n cu t

2. Nhm enzyme a cu t

Thuc nhm ny bao gm nhng enzyme c 2 thnh phn:

Apoprotein hay apoenzyme: Phn protein thun Agon: Phn th 2 l thnh phn khng phi protein. Phn ny thng l nhng cht hu c c hiu

c vai tr thc y qu trnh xc tc.

nhng enzyme a cu t, phn apoenzyme ng vai tr xc tc nhng nu thiu thnh phn th hai(cc cht hu c c hiu) th enzyme khng th hot ng c. Chnh v th, cht hu c c hiu nycn c gi l cht cng tc (cofactor).

Cc cht hu c c hiu ny c th gn rt cht vi phn protein, cng c th gn rt lng vi phnprotein. Ta c th d dng tch chng ra khi tin hnh thm tch qua mng. y xy ra 2 hin tng:

Nhng cht hu c c hiu gn cht vi protein bng lin kt ng ha tr c gi l nhm ph(prosthetic)

Nhng cht hu c c hiu gn khng cht vi protein v d dng tch chng ra khi protein cgi l coenzyme

Tuy nhin, s phn bit ny ch mang tnh cht tng i. Ngoi ra cc nh khoa hc cng cho thy,trong thnh phn ca nhng enzyme c s hin din ca mt s kim loi. Cc kim loi ny thng l mttrong nhng thnh phn ca cht hu c c hiu

V d: trong h enzyme cytochrome, catalase, peroxydase, st (Fe) gn cht vi nhn porphyrin.

Cc kim loi c trong thnh phn ca enzyme thng rt d tch ra khi enzyme. Trong trng hpenzyme mt kim loi, chng s mt hot tnh. Khi ta a cc kim loi tng ng vo cc enzyme, hottnh enzyme li c khi phc. Tnh cht ny mang tnh cht thun nghch. Vai tr ca kim loi tronghot ng ca enzyme vn cha c lm sng t. Tuy nhin cc nh khoa hc cng cho rng, c th kimloi ng vai tr lin kt gia enzyme v c cht, lin kt gia apoenzyme v coenzyme, tham gia trc

tip vo qu trnh vn chuyn in t nh vai tr ca st trong cytochrome v peroxydase.


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CNG NGH ENZYME 5

Bng 1.2. Mt s enzyme c cha kim loi [2]

STT Enzyme Kim loi

1 H cytochrome, catalase, peroxydase St (Fe)

2 Polyphenoloxydase ng (Cu)

3 Carbonic anhydrase Km (Zn)

4 PeptidaseMangan (Mn), St (Fe),Magie (Mg)

5 Phosphatase Magie (Mg)

6 Arginase Mangan (Mn)

1.2.3. Cu trc ca enzymeEnzyme l protein c bit. Ngoi cu trc ging nh cu trc bnh thng ca mt protein, enzyme cnc cu trc rt c bit lin quan n hot ng ca enzyme. Khng phi ton b cc phn ca enzymeu tham gia vo hot ng xc tc, m ch c nhng b phn rt c bit mang tnh c hiu trong phnt protein mi tham gia xc tc phn ng. B phn c hiu ny c gi l trung tm hot ng caenzyme

Trung tm hot ng ca enzyme bao gm:

Nhng nhm ha hc, nhng lin kt peptide tip xc trc tip vi c cht. Nhng nhm ha hc, nhng lin kt peptide khng tip xc trc tip vi c cht nhng c chcnng trc tip trong qu trnh xc tc

Phn cn li ng vai tr nh mt ci khung, gi cho cu trc khng gian thch hp vi kh nng xc tc.Nu b tc ng bi cc iu kin bn ngoi nh nhit , pH, nng cc cht, b khung ny s bin icu trc khng gian. T lm thay i su sc hot tnh enzyme.

Phn ca phn t enzyme khng c lin quan n hot tnh enzyme, nu b tc ng, b mt i hoc bbin i s hon ton khng nh hng n hot tnh ca enzyme

Trung tm hot ng ca enzyme thng cha cc amino acid c nhm ha hc hot ng mnh nhamino acid serine, histidine, cysteine, lysine, arginine, glutamic acid, aspartic acid, tryptophan. Cc nhmha hc ny hot ng c kh nng gn vi c cht to thnh phc cht enzyme - c cht. Nhng

nhm ha hc hot ng mnh bao gm:

Nhm NH2ca lysine Nhm -SH ca cysteine Nhm -carboxyl ca glutamic acidCc gc amino acid to ra trung tm hot ng ca enzyme thng khng nm cnh nhau trong chui

polypeptide thng (cu trc bc 1). Trong thc t, chui popypeptide ca enzyme tn ti trng thikhng gian (cu trc bc 3, bc 4) nn cc amino acid thng tn ti gn nhau theo cu trc khng gian.Do cu trc khng gian nh vy, trung tm hot ng c to thnh.

Trong trung tm hot ng ca enzyme ngi ta cn thy c ion kim loi. Cc ion kim loi c mt trongtrung tm hot ng ca enyzme c vai tr xc tc rt ln.

Ngoi cc gc amino acid, ion kim loi, cc nh khoa hc cn cho thy cc nhm chc ca coenzymecng c coi nh mt phn cu to ca trung tm hot ng ca enzyme.


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CNG NGH ENZYME 6

Mi mt enzyme thng c 1 trung tm hot ng. Tuy nhin cng c enzyme c 2 trung tm hot ng(alcohol hydrogenase ca gan), thm ch c enzyme c ti 4 trung tm hot ng (alcohol dehydrogenaseca nm men)

Hot ng xc tc ca trung tm hot ng c lin quan ti c cht. Cc nh khoa hc khi nghin cu vn ny a ra 3 quan im quan trng:

1. Ch nhng c cht c cu trc phn t thch hp vi trung tm hot ng ca enzyme mi c th

kt hp c vi trung tm hot ng ca enzyme to thnh phc hp enzyme-c cht.Theo quan im ny, c cht c cu trc phn t trng vi trung tm hot ng l hin tng kt hp rtcht ch v mang tnh c hiu cao. Chnh v th, mi loi enzyme ch c th tham gia xc tc phn ngcho mt loi c cht nht nh.

2. Cc loi enzyme thng to ra trung tm hot ng c cu trc khng gian nht nh.

Cc trung tm hot ng thng c hnh thnh sn cc enzyme. Chnh nhng trung tm hot ngca enzyme quyt nh cho php nhng c cht cu trc tng ng vi trung tm hot ng mi c kthp vo. Quan im ny do Fisher xng. Thuyt ca Fisher tuy gii thch c cc hin tng gnkt gia c cht v trung tm hot ng ca enzyme nhng nhiu kt qu thc nghim, thuyt ny chagii thch c trn vn

3. Thuyt trung tm hot ng linh hot ca Koshland c nhiu nh khoa hc chp nhn hn.

Theo thuyt ny, cc nhm chc ca trung tm hot ng ca enzyme t do cha c th tham gia xc tcngay. Chnh c cht l tc nhn tc ng lm thay i cu trc khng gian ca trung tm hot ng caenzyme. Chnh tc ng cm ng ny ca c cht lm cho cc gc amino acid, cc nhm chc ca trungtm hot ng di chuyn, nh hng mt cch thch hp, chnh xc gn c cht vo trung tm hotng v thc hin qu trnh xc tc.

Nh vy, Koshland cho rng trung tm hot ng ca enzyme ch c to thnh khi c s tc ng cmng ca c cht. Chnh c ch mm do ny ca trung tm hot ng gii thch c s cnh tranhgia c cht v cc cht khng phi c cht nhng li c cu trc khng gian ging c cht. Khi , cccht ging c cht chim ch trong trung tm hot ng ca enzyme v phn ng c cht khng xy ra.

Hnh 1.2. M hnh ca Fisher gii thch c ch tc ng ca enzyme vi c cht


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CNG NGH ENZYME 7

Hnh 1.3. M hnh ca Kosland gii thch c ch tc ng ca enzyme vi c cht

1.3.C ch tc dng ca enzyme1.3.1. C ch xc tc ca enzymeMi vi sinh vt u c cu to t t bo. Cc t bo lun lun tin hnh qu trnh trao i cht vi mitrng bn ngoi bng nhng phn ng sinh ha. Cc phn ng xy ra lun lun c xc tc bi ccenzyme ca t bo. Nh vy, enzyme ng vai tr rt quan trng trong sinh l ca t bo. Tc ng caenzyme vo cc phn ng sinh ha mang 2 ngha i vi t bo:

1. Lm gim nng lng hot ha phn ng

Tt c cc phn ng sinh ha trong t bo sinh vt u c thc hin trong iu kin n ha, trng vinhit ca c th. Cc phn ng enzyme thng khng i hi nhit cao, do n m bo cho mihot ng sinh l bnh thng ca t bo. Trong khi , nng lng chi ph cho nhng phn ng ha hcthng rt cao.

c im lm gim nng lng hot ha ca phn ng enzyme c ngha rt ln trong sinh l ca sinhvt. c im ny gn lin vi qu trnh tin ha ca sinh vt, nu nhit c th tng s lm ri lonton b qu trnh sinh l ca t bo. Khi c th s chuyn t trng thi sinh l bnh thng sang trngthi bnh l. C th trng thi bnh l l kt qu ca s ri lon cc phn ng enzyme. Chnh v th,vic duy tr trng thi sinh l bnh thng ca t bo hay ca c th ng ngha vi vic duy tr hot ng

hi ha ca cc phn ng enzyme2. Enzyme tham gia vo cc phn ng sinh ha thng lm tng tc phn ng

Tc phn ng tng s lm tng mc chuyn ha c cht. Nh vy qu trnh trao i cht ca t bos tng. Kt qu l t bo s tng nhanh v s lng v khi lng. Nh vy, enzyme khng ch ng vaitr duy tr trng thi sinh l ca t bo m cn ng vai tr quan trng trong s pht trin v sinh sn cat bo, duy tr s chuyn ha vt cht trong cc chu trnh chuyn ha trong thin nhin. Nh c hot ngca enzyme m khi lng c cht c chuyn ha trong mt n v thi gian trong t bo ln gp hngngn ln khi lng t bo.

Bn cht ca cc phn ng enzyme l khi c s tham gia xc tc ca cc enzyme, cc c cht s chot ha mnh, t lm thay i tnh cht ha hc ca c cht, kt qu sau phn ng s to ra nhng sn

phm ca phn ng. Di tc dng ca enzyme, c cht c th c nhng thay i khng ch v cu trcha hc, m cn thay i tnh cht ha hc. Qu trnh xc tc ca enzyme xy ra qua 3 giai on:

Giai on th nht: Enzyme s kt hp vi c cht bng nhng lin kt yu, nh s to ra phc henzyme c cht. Phc h ny thng khng bn. Phn ng to ra phc h enzyme c cht thng xyra rt nhanh v i hi t nng lng.

Giai on th hai: khi c cht to phc vi enzyme s b thay i c cu hnh khng gian, c v mc bn vng ca cc lin kt. Kt qu l cc lin kt b ph v v to ra sn phm

Giai on th ba: y l giai on cui cng, sn phm qu trnh phn ng c to thnh v tch rakhi enzyme

C ch xc tc tng qut ca enzyme:

Trong : E enzyme (enzyme) S C cht (substrate) P Sn phm (products)

E + S ES E + P


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CNG NGH ENZYME 8

Hnh 1.4. C ch xc tc ca enzyme

1.3.2. Nng lng xc tcEnzyme tham gia hu ht cc phn ng sinh ha trong c th sng. Nh hot ng xc tc ca enzyme,cc t bo c th chuyn ha lng c cht rt ln. Enzyme s dng ngun nng lng no v s dng

nng lng bng cch no tin hnh qu trnh xc tc m kh nng xc tc li ln nh vy?Cc nh khoa hc a ra 2 cch s dng nng lng lin kt tin hnh qu trnh xc tc:

Th nht: enzyme s dng nng lng lin kt lm gim nng lng hot ha. Nng lng ny cgii phng trong qu trnh hnh thnh lin kt yu trong tng tc qua li gia enzyme v c cht.

Th hai: enzyme s dng nng lng lin kt to phn ng xc tc c hiu thng qua c ch sau:

Gim entropy: enzyme v c cht lun trng thi chuyn ng trong dung dch lng. Nu khng cnng lng lin kt, enzyme s rt kh nh hng tc ng ln c cht, gi c cht v nh hng

phn ng. Lm mt v nc bao quanh: nh tng tc yu gia enzyme v c cht c kh nng lm gim ton

b lin kt hydrogen tn ti gia c cht v nc bao quanh Nng lng lin kt do cc tng tc yu to ra trng thi chuyn tip c s dng lm cng hoc

un khc c cht, to iu kin cho enzyme xc tc phn ng d dng Nng lng lin kt to ra cu hnh khng gian ca enzyme sao cho ph hp vi cu hnh khng gian

ca c cht

1.3.3. S to thnh phc hp enzyme c chtBrown v Henri l nhng nh khoa hc u tin a ra thuyt phc hp trung gian. Sau , Michaelisv Menten pht trin thm.

Theo thuyt ny, enzyme s kt hp vi c cht, to ra mt phc hp khng gian gia enzyme v c cht.Do tc dng ca enzyme, c cht b bin i mnh v cui cng to ra sn phm. S to thnh phc hpenzyme - c cht thng xy ra rt nhanh v v bn cht th phc hp ny hon ton khng bn vng.

Cc tnh cht ny ca phc hp enzyme v c cht ph thuc rt nhiu bn cht ha hc ca c cht to thnh phc hp enzyme - c cht c n nm loi lin kt tham gia. Cc lin kt ny c c tnhring v nng lng lin kt khc nhau. Cc lin kt ny bao gm: lin kt phi tr, lin kt hydrogen, linkt ion, lin kt k nc lc Van der Waals v lin kt do chuyn dch in tch

Enzyme l nhng phn t protein c phn t lng ln. Trn b mt ca phn t enzyme tn ti rt nhiunhm ha hc c hiu. Cc nhm chc ny ng vai tr quan trng trong nh hng v gip c chtenzyme tip cn vi nhau. Khi enzyme v c cht tng tc vi nhau thng to ra lin kt yu, to ranng lng kt hp . Nng lng c to tng lin kt yu ny thng nh, nhng c nhiu nnglng lin kt nn tng nng lng trong phn ng enzyme l rt ln.

Enzyme s s dng ngun nng lng ny lm gim nng lng trong phn ng enzyme. Nng lng

gim trong phn ng enzyme c chuyn qua h thng cc cht tham gia phn ng. Lng nng lnggim trong qu trnh hot ha phi bng lng nng lng cc cht tham gia phn ng nhn c.


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CNG NGH ENZYME 9

Theo kin ca cc nh khoa hc, u tin c cht phi c hot ha chuyn sang trng thi chuyntip tm thi tng tc vi c cht to nn phc enzyme c cht (ES). Sau phc hp ny li chot ha chuyn thnh trng thi chuyn tip tm thi sang phc hp EP (P l sn phm). Sau phchp EP li chuyn sang trng thi tm thi khc to thnh P v gii phng E t do. Nng lng ca sn

phm s thp hn mc nng lng ca c cht ban u. Ton b qu trnh phn ng c din gii chnhthc nh sau

E + S => ES => EP => E + PNng lng cn thit lm gim nng lng hot ha qu trnh xc tc, ng thi cng to ra tnh chiu cho phn t enzyme. Tnh c hiu ca enzyme l kh nng nhn bit, chn lc c cht tin hnhcc phn ng ring theo bn cht tng loi enzyme.

1.3.4. ng hc ca phn ng enzymeNm 1913 hai nh khoa hc Michaelis v Menten a ra m hnh ng hc gii thch phn ng cxc tc bi enzyme v lp phng trnh phn nh quan h gia vn tc phn ng vi nng c cht venzyme. Theo m hnh ny, enzyme v c cht s kt hp vi nhau, to nn phc hp enzyme c cht(ES). Phc hp ES s li c chuyn ha tip tc to thnh sn phm (P) v gii phng enzyme (E).Enzyme c gii phng li thc hin nhng phn ng mi.

Hai nh khoa hc trn a ra m hnh chuyn ha trong phn ng enzyme vi mt c cht duy nhtnh sau:

Trong : k1, k2, k3, k4: l hng s vn tc ca cc phn ng tng ng.

Khi nghin cu ng hc phn ng enzyme, ngi ta thng xc nh vn tc ban u ca phn ng khi

Lng sn phm P to thnh cha ng k nn K4 l hng s vn tc phn ng to ES t P v E nhnht v xp x bng 0.

ng thi nng c cht rt ln so vi nng enzyme tng [E o]. Ngoi ra, phn ng chuyn hat ES thnh E + P l phn ng quan trng nht, quyt nh ton b qu trnh chuyn ha S thnh P.

Vn tc phn ng ES thnh P + E t l vi nng ES, khi nng ES cng cao th vn tc phn ngcng cao nn:

Vi= k3[ES] (1)

Gi s nng enzyme ban u c k hiu l [Eo], nng phc enzyme c cht l [ES], nng enzyme t do khi phn ng t c im cn bng l [E]. Ta s c:

[E] = [Eo] [ES] (2)

giai on u ca phn ng, nu nng c cht thp th tc phn ng enzyme s ph thuc tuyntnh vi nng c cht. Ta c th tnh c vn tc phn ng trong trng hp sau:

Vn tc phn ng thun: V1= k1[E][S] (3)Vn tc phn ng nghch: V2= k2[ES] (4)

Vn tc phn ng to ra sn phm: V3= k3[ES] (5)

Trong giai on cn bng, s phn ly ES s cn bng vi s to ra ES. Khi vn tc phn ng s l:V1 = V2 + V3 (6)

Thay (2, 3, 4, 5) vo (6) ta c:

k1([Eo] [ES])[S] = (k2+ k3)[ES] (7)

T ta c: [ES] = k1[Eo][S]/(k1[S] + [k2+ k3]) (8)

Nu chia 2 v cho k1, thay (k2 + k3)/k1 = km (hng s michaelis menten) ta s c:[ES] = [Eo][S]/(km+ [S]) (9)

k4k2

k1 k3E + S ES E + P


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CNG NGH ENZYME 10

Khi [S] >> [Eo] tt c enzyme u tham gia to phc ES v vn tc phn ng Vi s t cc i (Vmax),ta c [ES] = [Eo]

Vn tc cc i: Vmax= k3[Eo] (10)

C th vit:

Vi/Vmax= [ES]/[Eo] = 1/(1+km/[S]) (11)

Phng trnh Michaelis MentenVi= Vmax[S]/(km+ [S]) (12)

Hnh 1.5. Phng trnh Michaelis Menten

Nu [S]km: vn tc phn ng t cc i, khng ph thuc vo [S]. Nh vy [S] ln n

mc no , nu tip tc tng [S], V cng khng tng theo Nu [S] = km; vn tc phn ng bng vn tc cc i

Ngi ta d dng xc nh c km v Vmax bng cch nghch o c 2 v ca phng trnh michaelis menten:

1/Vi= (km+ [S])/(Vmax[S]) (13)Phng trnh ny l phng trnh tuyn tnh c dng y = ax + b

Nu v th, ng thng s ct trc tung 1/Vmax v ct trc honh -1/km v nghing bngkm/Vmax

Hnh 1.6. Phng trnh nghch o ca Michaelis Menten

T phng trnh trn, ta d dng xc nh c v tr Vmax, km trong th nghim xc nh tc Vi caphn ng enzyme vi nng c cht ban u khc nhau.

1.3.5. Cc yu t nh hng n phn ng enzyme1.3.5.1. Nhit

Nhit c nh hng rt ln n phn ng enzyme. Tc phn ng enzyme khng phi lc no cngt l thun vi nhit phn ng. Tc phn ng ch tng n mt gii hn nhit nht nh. Vt

qu nhit , tc phn ng enzyme s gim n mc trit tiu.Ngi ta thng s dng h s nhit Q10 biu th nh hng ca nhit n tc phn ng. Nhit tng ng vi tc phn ng enzyme cao nht c gi l nhit ti u. Phn ln enzyme hot


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CNG NGH ENZYME 11

ng mnh nht nhit 40 50oC. Nhit ti u ca nhng enzyme khc nhau l hon ton khcnhau. Mt s enzyme khc c nhit ti u 60oC, mt s khc li c nhit ti u 70oC, thm chc mt s enzyme ca vi khunBacillus subtilisli hot ng mnh 90oC

Nu a nhit cao hn mc nhit ti u, hot tnh enzyme s b gim. Khi enzyme khng c khnng phc hi li hot tnh.

Ngc li, nhit 0oC enzyme b hn ch hot ng rt mnh, nhng khi a nhit ln t t hot

tnh enzyme s tng dn u n mc ti u.Nhit ti u ca mt enzyme ph thuc rt nhiu vo s c mt ca c cht, kim loi, pH, cc cht bov. Ngi ta thng s dng nhit iu khin hot ng ca enzyme v tc phn ng trong ch

bin v bo qun thc phm.

Hnh 1.7. nh hng ca nhit n phn ng enzyme

1.3.5.2. pHpH mi trng thng nh hng n mc ion ha c cht, enzyme v c bit nh hng n bnca enzyme. Chnh v th pH c nh hng rt mnh n phn ng ca enzyme

Nhiu enzyme hot ng rt mnh pH trung tnh. Tuy nhin cng c nhiu enzyme hot ng pH acidyu. Mt s khc li hot ng mnh pH kim v c pH acid.

VD: mt s protease hot ng pH kim (protease kim), mt s li hot ng pH acid (protease acid)v mt s protease li hot ng pH trung tnh (protease trung tnh)

Ngi ta thng s dng nh hng ca pH iu ha phn ng trong bo qun, ch bin lng thc,thc phm, trong tuyn chn ging vi sinh vt

Hnh 1.8. nh hng ca pH n phn ng enzyme

1.3.5.3. Cht km hmCc cht km hm hot ng ca enzyme thng l cc cht c mt trong cc phn ng enzyme, lm gimhot tnh ca enzyme nhng li khng b enzyme lm thay i tnh cht ha hc, cu to ha hc v tnhcht vt l ca chng.

Cc cht gy km hm hot ng ca cc enzyme bao gm cc ion, cc phn t v c, cc cht hu c vc protein. Cc cht km hm c ngha rt ln trong iu khin cc qu trnh trao i t bo sinh vt.

C ch km hm ca cc cht km hm c th l thun nghch hoc khng thun nghch. Trong trng

hp cc cht km hm thun nghch, phn ng gia enzyme v cht km hm s nhanh chng t ctrng thi cn bng.


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CNG NGH ENZYME 12

Cc cht km hm kt hp vi enzyme bng lin kt ng ha tr. Theo , cc cht km hm gn rt chtvo enzyme, to thnh phc enzyme cht km hm (EI). Phc hp ny b phn r rt chm. Ty thucvo bn cht ca cht km hm m ngi ta chia ra nhng cht km hm sau:

Cht km hm cnh tranh: cc cht km hm cnh l nhng cht c cu trc tng t nh cu trc cac cht. Chng thng l nhng cht km hm thun nghch. Chng c kh nng kt hp vi trung tmhot ng ca enzyme. Khi , chng s chim v tr ca c cht trong trung tm hot ng. Nu cht km

hm chin c v tr trong trung tm hot ng th c cht s khng cn c hi tip cn vi trung tmny. C ch loi tr ln nhau ca cht km hm v trung tm lm gim s lng cc enzyme kt hp vic cht. Kt qu l hot ng ca enzyme s gim.

V c cu trc khng gian ging nhau, nn cc cht cnh tranh v c cht u c xu hng chim v trtrong trung tm hot ng. Vn tc phn ng lc ny ph thuc vo 2 yu t:

Ph thuc vo nng c cht v nng cht canh tranh. Nu nng c cht ln s loi trc hin tng cnh tranh.

Ph thuc vo i lc gia c cht v cht cnh tranh vi enzymeCht km hm khng cnh tranh:

Nu nh trong c ch km hm cnh tranh, cc cht km hm chim trung tm hot ng ca enzyme th

c ch km hm khng cnh tranh, cht km hm khng chim trung tm hot ng ca enzyme m l mt v tr ngoi trung tm hot ng ca enzyme. Kt qu s kt hp ny, cht km hm lm thay i cutrc khng gian ca phn t enzyme theo chiu hng bt li cho hot ng xc tc. V th cc cht kmhm lm gim hot ng ca enzyme.

Khi cht km hm khng cnh tranh kt hp vi enzyme s lm vn tc phn ng enzyme b gim mc dqu trnh ny khng lm thay i i lc gia enzyme v c cht. Mc km hm ca cht km hmkhng cnh tranh khng ph thuc vo tng quan nng c cht v cht km hm. C cht d clng ln bao nhiu cng khng loi tr c tc dng km hm ca cht km hm.

Cc cht km hm khng cnh tranh c th l cc cht sau:

1. Km hm bi sn phm ca phn ng: cc sn phm ca phn ng c th ng vai tr nh cht km

hm khng cnh tranh. Nu nh phn ng xy ra do cht A v cht B, c s tham gia ca enzyme tothnh sn phm P1 v P2 th enzyme c i lc vi c P1, P2 v c cht A v B. Khi sn phm P1, P2c xem l cht km hm khng cnh tranh

2. Km hm do tha c cht: trong phn ng enzyme thng thng th: E + S ES => E + P

Khi ES c to thnh rt c th c mt c cht gn vi ES to thnh ESS lm chng khng th chuynha to tip tc c to sn phm v gii phng enzyme t do.

Hnh 1.9. Cht km hm cnh tranh Hnh 1.10. Cht km hm khng cnh tranh

1.3.5.4. Cht hot haCc cht c tc dng lm tng hot tnh ca enzyme gi l cc cht hot ha enzyme. Cc cht hot haenzyme c bn cht ha hc rt khc nhau. Chng c th l nhng anion, cc ion kim loi t th 11 n th 55 trong bng h thng tun hon, cc cht hu c c cu trc phc tp. Tuy nhin, cc cht hot

ha ch c tc dng mt nng nht nh. Vt qu nng ny, chng s gy c ch hot ng caenzyme


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CNG NGH ENZYME

nng hot ha, cc cht hokh nng ph v mt s lin ktchc nng trong trung tm hot

1.3.6. Tnh cht c hiu caTnh cht c hiu l biu hincht c hiu rt cao. Tnh chtcht xc tc khc.

Mi mt loi enzyme ch c khmt kiu phn ng nht nh. T

1. c hiu kiu phn ng

Tnh cht c hiu kiu phn nchuyn ha nht nh trong ccthy phn

2. c hiu c cht

Khi tham gia vo cc phn ngnhin khng phi c cht no cenzyme. Ngi ta phn tch c

A c hiu tuyt i

L kh nng xc tc ca enzymec kh ngng xc tc i vi m

B c hiu tng i

L kh nng xc tc ca enzyme

C c hiu quang hc (c hi

L kh nng xc tc ca enzymev trans)

1.4.Phn loi enzyme1.4.1. Danh php quc t caKhi s lng enzyme mi cngi tm ra n. Dn dn, theo thy c tnh cht gn ging nhaunhng quy c quc t v tn genzyme thuc nhm no v b

Theo quy c quc t, tn gi ckiu phn ng m chng tham gi

t ha thng lm nhim v chuyn nhm hydrtrong phn t tin enzyme hoc cc cht c tcng ca enzyme

Hnh 1.11. Cht hot ha enzyme

enzyme

h nng xc tc ca enzyme i vi c cht nhc hiu ca enzyme cho thy s khc bit rt

nng xc tc cho s chuyn ha mt hay nhih cht c hiu ca enzyme biu hiu mt s k

biu hin ch, mi enzyme ch c th xc tiu phn ng nh phn ng oxy ha kh, phn

, cc loi c cht phi gn vi trung tm hotng c kh nng tip cn v gn c vo vi tiu c cht theo mc sau:

i vi mt c cht nht nh. Ngoi c cht nc cht no khc na.

tc dng ln mt kiu lin kt ha hc nht nh

lp th)

n mt trong 2 dng ng phn quang hc c

enzyme

pht hin cn t, ngi ta thng gi tn enzyi gian, s lng enzyme tm ra ngy mt nhiuoc ging nhau, ngi ta phn loi chng th

i enzyme khi nghin cu v ng dng chngn cht ha hc ca chng ra sao.

a enzyme thng c gi theo c cht c hiu. Theo , tn gi ca 1 enzyme thng c 2 ph

13

gen hot nhng cht cng phc hi cc nhm

t nh. Enzyme c tnhln gia enzyme v cc

c cht nht nh theoiu c hiu sau:

cho mt kiu phn ngng chuyn v, phn ng

ng ca enzyme. Tuyung tm hot ng ca

y ra, enzyme ny khng

c cht (ng phn cis

e theo thch ca tngv nhng enzyme tm rah tng nhm v a rai cng c th hiu c

ca chng cng vi tnn:


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CNG NGH ENZYME 14

Phn u l tn c cht: trong trng hp phn ng l phn ng lng phn th phn th nht l tngi ca 2 c cht vit cch nhau hai chm.

Phn sau ch khi qu bn thn ca phn ng:

V d: enzyme urease c tn gi h thng l carbanite-amideohydrolase

Cc enzyme thng cui tn c ui ase

Tuy nhin, c nhiu loi enzyme v thi quen nn vn c gi theo tn c, khng theo h thng phnloi. V d nh trypsin, chimotrypsin, pepsin

1.4.2. Phn loi enzymeNm 1960, hip hi ha sinh quc t (IUB) thng nht phn loi enzyme ra lm 6 lp v c nh sth t t 1 n 6. Cc s th t ny l c nh cho mi lp. Mi lp li chia ra lm nhiu t, mi t lichia ra lm nhiu nhm, chnh v th, theo h thng phn loi, mi enzyme thng c 4 cha s: s thnht ch lp, s th 2 ch t, s th 3 ch nhm, s th 4 ch enzyme

1. Oxydoreductase: cc enzyme xc tc cho phn ng oxy ha kh

2. Transpherase: cc enzyme xc tc cho phn ng chuyn v

3. Hydrolase: cc enzyme xc tc cho phn ng thy phn4. Liase: cc enzyme xc tc cho phn ng phn ct khng cn nc, loi nc to thnh lin kt ihoc kt hp phn t nc vo lin kt i

5. Isomerase: cc enzyme xc tc cho phn ng ng phn ha

6. Ligase: cc enzyme xc tc cho phn ng tng hp c s dng lin kt giu nng lng ca ATP

1.4.2.1. Oxydoreductasey l enzyme xc tc cho phn ng oxy ha kh. Cc enzyme ny l nhng enzyme phc tp. Chng

bao gm 2 thnh phn. Phn coenzyme ca chng l NAD+, NADP+, FMN, FAD, heme. Lp enzymeny c phn ha thnh nhng nhm sau:

A- Dehydrogenase: nhm ny tham gia vo cc phn ng tch H trc tip t c cht v chuyn chng nNAD+, NADP+, FMN, FAD.

Cc dehyrogenase xc tc cho giai on u ca chui h hp, vn chuyn ng thi c proton velectron. Ngoi ra, chng cn tham gia xc tc cho chiu ngc li, chuyn H t NADH+H+ hoc

NADPH+H+, FMNH2, FAD-H2 n c cht v kh c cht. y l nhng phn ng ng vai tr rtquan trng trong qu trnh tng hp

B Oxydase: y l phn ng xc tc cho qu trnh chuyn electron n oxy. Chng hot ha oxy lmcho chng c kh nng kt hp vi proton c trong mi trng. Lp enzyme ny tc dng trc tip vioxy

C Oxygenase: y l enzyme xc tc cho phn ng kt hp trc tip oxy vo phn t ca hp cht hu

c c vng thmNhm ny c 2 loi, oxygenase v hydroxylase, oxygenase xc tc cho phn ng kt hp ton b phn toxy. Hydrogenase xc tc cho phn ng kt hp mt na phn t oxy (thng dng OH) vo hp chthu c

D Peroxydase: y l enzyme c coenzyme l heme, xc tc cho phn ng oxy ha cc hp cht hu ckhi c H2O2

1.4.2.2. TranspheraseTranspherase cng thuc lp enzyme phc tp. Coenzyme ca transpherase c bn cht ha hc rt khcnhau ty theo vo bn cht ca nhm c chuyn v. Lp transpherase bao gm nhng enzyme tiu biusau:

A acyltranspherase: enzyme ny tham gia chuyn ha nhm acyl thng qua coenzyme A, to thnhphc CoAS-acyl. Khi , nhm carboxyl ca acid kt hp vi nhm SH ca coenzyme A to thnh lin


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CNG NGH ENZYME 15

kt thioester giu nng lng. Cc enzyme ny c vai tr quan trng trong chuyn ha lipid v phn giiglucose

Glucosyltransferase: enzyme ny tham gia xc tc cho phn ng vn chuyn ng hexose, pentose tcht cho n cht nhn khc nhau, thng gp nht l nhm OH ca mt gc saccharide khc hoc cagc phosphate, nguyn t nitrogen ca nhn vng.

Ngoi ra, enzyme ny cng tham gia xc tc cho qu trnh to cu trc phn nhnh trong phn t

glycogen, amylopectinPhosphotransferase: enzyme ny tham gia xc tc chuyn ha gc phosphoryl. Trong phn ng nythng c s tham gia ca ATP vi ngha nh mt cht cho. Gc phosphate c chuyn t ATP nnhm hydroxyl ca alcohol hay saccharide

1.4.2.3. HydrolaseHydrolase tham gia xc tc cho cc qu trnh thy phn. Chnh v th, nc l thnh phn khng th thiuc ca nhng phn ng do enzyme ny tham gia. C rt nhiu enzyme thuc lp enzyme ny nh:

A peptide hydrolase: enzyme ny tham gia xc tc phn ng thy phn peptide, to thnh nhng phnt thp v cc amino acid.

Cc enzyme peptide hydrolase thy phn cc lin kt peptide gia chui peptide gi l endopeptidehydrolase hay proteinase. i din cho endopeptide hydrolase l pepsin, trypsin, chymotrypsin. Ccenzyme peptide hydrolase thy phn cc lin kt peptide u chui peptide gi l exo peptide hydrolasehay peptidase

Nm 1960, Hartley chia proteinase ra lm 4 nhm chnh:

Proteinase serine: trong enzyme ny, gc serine ng vai tr xc tc trung tm hot ng. Nhmny bao gm trypsin, chymotrypsin, elastase, cc proteinase lm ng mu, acrosin

Proteinase thiol: trong trung tm hot ng c nhm SH trc tip tham gia phn ng, bao gmpapain, bromelin, ficin

Proteinase kim loi: trong trung tm hot ng, kim loi c ng vai tr quan trng trong xc tc Proteinase acid (aspartic proteinase): trong trung tm hot ng c nhm gammar carboxyl

B lipase: enzyme ny tham gia qu trnh xc tc cho phn ng thy phn triglycerol (du thc vt vm ng vt) to thnh acid bo t do v glycerol. Chng xc tc phn ng thy phn ln lt tng linkt ch khng phi ct t c 3 lin kt ester cng mt lc.

1.4.2.4. LipasePyruvate decarboxylase: enzyme ny tham gia xc tc cho phn ng loi CO2 ra khi phn t pyruvicacid, to thnh aldehyde tng ng l acetaldehyde. y l phn ng quan trng trong ln men ru.Enzyme ny l enzyme a cu t, coenzyme ca chng ta l thiamipiro phosphate

Fumarate hydrolase: enzyme ny l xc tc cho phn ng tch thun nghch phn t nc khi malicacid to thnh fumaric acid (acid c mt ni i)

1.4.2.5. IsomeraseEnzyme ny xc tc cho phn ng chuyn ha tng h phc tp gia galactose v glucose. NAD+ trongenzyme ny tham gia trc tip trong phn ng.

1.4.2.6. LigaseEnzyme ny tham gia xc tc cho phn ng carboxyl ha pyruvic acid, to thnh oxaloacetic acid. Chngcn acetyl-coA v Mg2+cho phn ng xc tc. Biotin l cht mang CO2 hot ha v chuyn thnh acid

pyruvic

1.5.Phng php tch v lm sch enzyme1.5.1. Cc phng php ph v t boEnzyme l loi protein c to thnh trong t bo, phn ln chng tn ti trong t bo. Cc enzyme nythng khng c kh nng di chuyn qua mng sinh hc. Do , vic tch chng ra khi t bo l diu rtkh khn.


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CNG NGH ENZYME 16

thu nhn enzyme ni bo, ngi ta phi ph v t bo bng nhiu phng php. Cc phng php phv t bo thng s dng nh:

1. Phng php c hc: nghin bi, nghin c cht tr nghin nh bt thy tinh, ct thch anh, ng habng thit b ng ha

2. Phng php vt l: dng sng siu m

3. Phng php ha hc: dng cc loi dung mi, butylic, acetone, glycerol, ethylacetate.C 3 kh khn trong khi tch enzyme khi t bo cn phi ht sc lu :

Enzyme c trong t bo sinh vt vi lng khng ln so vi cc thnh phn khc. Do , vic tch thu nhn thnh phn nh ny l iu rt kh.

Enzyme l cht hu c khng bn, chng rt d b bin tnh khi b tc ng ca cc yu t bn ngoi. Enzyme l protein. Protein enzyme lun lun i cng nhng loi protein khng phi enzyme nhng

li c tnh cht ha l rt ging protein enzyme. Do , vic tch protein enzyme ra khi cc loiprotein khng phi lc no cng t c kt qu tt v khng phi khng gp nhng kh khn nhtnh

1.5.2. Phng php gy bin tnh chn lcSau khi ph v t bo, ngi ta thng s dng nc, dung dch m, dung dch mui trung tnh tchenzyme ra khi sinh khi x l theo nhng phng php trn. Dch chit thu c ngi ta gi l ch

phm enzyme th. s di gi l ch phm enzyme th v trong ch phm ny ngoi enzyme cn c nc,protein khng phi enzyme cc thnh phn ca t bo.

Nh vy vic lm sch enzyme chnh l vic loi cc protein khng phi l enzyme (i khi loi b c ccprotein enzyme khng mong mun), nc, cc thnh phn khc ca t bo khi enzyme. Cng vic nyhon ton khng d dng. Do , vic lm sch enzyme i hi ngi lm nghin cu v enzyme, ngois hiu bit v enzyme ra cn phi nm vng rt nhiu thao tc k thut khc nhau trnh gy mt tnhhot ng ca enzyme.

loi b nhng thnh phn khng phi l enzyme ta ang cn, ngi ta thc hin mt s phng phpsau:

1. Phng php gy bin tnh chn lc: phng php ny da trn s tc ng ca nhit , pH lmbin tnh chn lc cc loi protein tp. Phng php ny ch thch hp vi enzyme chu aicd v bn nhit.Ngi ta a nhit ca dung dch enzyme ln 50 70oC v pH < 5. iu kin ny nhng enzymekhng bn nhit v khng chu c pH thp s b bin tnh. Sau bng phng php ly tm hoc

phng php lc loi b cc thnh phn khng phi enzyme m ta mong mun.

2. Phng php kt ta phn on: da trn c s v kh nng kt ta ca cc enzyme nng muikhc nhau, ngi ta thng s dng (NH4)2SO4 kt ta phn on enzyme. Bng cch ny, ngi tac mt s protein tp giai on u ca qu trnh lm sch enzyme. Ngoi mui (NH4)2SO4 ra,ngi ta cn s dng mt s dung mi hu c kt ta enzyme. Phng php kt ta ny thng lm

bin tnh rt nhanh. V th trc khi tin hnh kt ta, ngi ta tin hnh lm lnh cc cht kt ta v cdung dch enzyme

3. Phng php hp th chn lc: ngi ta c th hp th trc tip vo dung dch enzyme, hoc chodung dch enzyme chy qua mt ct, trong ct c cha cht hp th. Cht hp th c s dng rng ril hydrocyapatite. Ngi ta c th thc hin mt trong hai cch sau: hoc l hp th enzyme hoc l hpth protein tp. trnh lm bin tnh enzyme, ngi ta thng thc hin qu trnh hp th nhit 0oC. Sau khi hp th ngi ta li tin hnh qu trnh chit enzyme (qu trnh phn hp th) bng dungmi thch hp.

4. Phng php sc k: da trn c s ca phn ng trao i gia protein tan trong nc v tc nhn traoi ion, ngi ta p dng phng php ngi ta s dng phng php sc k trao i ion lm schenzyme. Ngi ta thng s dng tc nhn trao i ion sau:

Nha trao i ion Dn xut ester ca cellulose nh carboxymethyl-cellulose, diethylamino-ethyl-cellulose (DEAE-cellulose), triethylamino-ethyl-cellulose


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CNG NGH ENZYME 17

Ngi ta thng s dng DEAE cellulose hn c. u im ca DEAE-cellulose l ngoi tp chtprotein, cc loi nucleic acid u c loi khi enzyme.

Sephadex l dn xut ca polysaccharide dextran. Trong cht ny cc phn t ca chng thng linkt vi nhau bi cc lin kt ngang nh tc dng ca epichohydrin, to thnh sng phn t. Slng lin kt ngang cng nhiu, kch thc sng phn t cng nh.

Khi tin hnh lc, cc cht phn t c knh thc nh s khuch tn vo bn trong cc ht sephadex

c ngm trong dung dch m. Cc phn t c kch thc ln hn khng c kh nng i vo ht sc chit nhanh ra khi ct. phng php ny c ng dng rng ri trong k thut enzyme.

Hin nay, cha c mt phng php chun no tht s hiu qu trong vic lm sch enzyme. Do , viclm sch enzyme ch tht s c hiu qu khi ta bit la chn cc phng php ring, kt hp vi nhau, tora mt h thng ni tip nhau mt cch hi ha nht.

1.6.Phng php xc nh kh nng xc tc ca enzyme1.6.1. Cc nhm phng php xc nh kh nng xc tc ca E

Ngi ta xc nh kh nng xc tc ca enzyme thng qua vic xc nh hot hot ng ca enzyme.Ngi ta cng khng th nh lng enzyme trc tip m phi xc nh gin tip thng qua hot hotng ca chng hoc thng qua kh nng lm gim c cht sau mt thi gian phn ng.

Hin nay, nhiu phng th nghim s dng mt trong 3 nhm phng php sau xc nh kh nng xctc ca enzyme:

Tin hnh o lng c cht b mt i hay lng sn phm c to thnh sau mt thi gian nht nhv lng enzyme xc nh trc. phng php ny c p dng rng ri v c xc nh ltng i chnh xc.

Tin hnh xc nh thi gian cn thit thu nhn c mt lng bin i nht nh ca lng ccht hay lng sn phm tng ng vi mt lng enzyme nht nh

Tin hnh chn nng enzyme cn thit trong mt thi gian nht nh s thu c s bin inht nh v c cht hay v sn phm.

Bng 1.3. Cc nhm phng php xc nh kh nng xc tc ca enzyme [2]

Phng php Cc thng s c nh Cc thng s thay i

1Thi gian

Nng enzymeLng c cht v sn phm

2Lng c cht hay sn phm

Nng enzymeThi gian

3Thi gianLng c cht hay sn phm Nng enzyme

1.6.2. n v hot Kh nng xc tc ca enzyme c xc nh thng qua hot hot ng ca enzyme. Hot hot ngca enzyme c xc nh thng qua n v hot . Ngi ta biu din n v hot qua nhng n vsau:

a - n v hot quc t (UI): n v hot quc t l lng enzyme c kh nng xc tc lm chuynha 1 micromol c cht trong 1 pht iu kin tiu chun

b - katal (kat): n v kat l lng enzyme c kh nng xc tc lm chuyn ha c 1mol c cht sau 1giy iu kin tiu chun

1UI = 16,67nano kat (nkat)


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CNG NGH ENZYME 18

c - hot ring: hot ring ca mt ch phm enzyme l s n v UI (hoc mt n v katal) ngvi 1 ml dung dch (nu l dung dch) hoc 1mg protein (nu l bt kh) ca ch phm enzyme. Khi bitc khi lng phn t ca enzyme ta hon ton c th tnh c hot ring ca phn t.

d - hot ring ca phn t: hot ring ca phn t enzyme l s phn t c cht c chuyn habi mt phn t enzyme trong mt n v thi gian.

Vic xc nh hot ca enzyme thng c cc nh khoa hc a ra nhng phng php rt c th

cho tng loi enzyme, ng vi tng loi c cht. Tuy nhin, khi tin hnh xc nh hot ca enzymecn lu nhng im c bn sau:

Khi tin hnh xc nh hot ca enzyme cn phi cho lng c cht trong mt gii hn thch hp tha bo ha enzyme. Lng c cht cho vo khng c qu nhiu. Nu lng c cht qunhiu s gy c ch hot ng ca enzyme

i vi nhng enzyme d mt hot tnh cn phi c cht hot ha hoc lm bn enzyme, th cn phicho nhng cht ny vo trc khi cho c cht vo thc hin phn ng.

Khi xc nh hot enzyme cn ch phi thc hin trong iu kin pH c nh v thch hp vihot ng ti u ca enzyme nghin cu. pH thng thay i ty thuc vo c cht v dung dch m

Khi xc nh hot enzyme cn lu n nhit . Cc nh khoa hc cho rng khng nn xc nhhot enzyme nhit ti u ca enzyme m nhit thp hn nhit ti u mt t

trnh kh nng lm bin tnh enzyme khi c s bin ng v nhit Thi gian xc nh hot enzyme ch nn ko di khong 5 - 10 pht, khng nn ko di qu, d

gy gim hot ca enzyme. Tuy nhin, cng c nhng trng hp ko di n 24 gi i vinhng enzyme c kh nng hot ng yu. Trong trng hp phi ko di thi gian, cn thit phicho cht c ch hot ng hoc tiu dit vi sinh vt

Ngoi ra, hin nay trn th gii c rt nhiu cch biu th n v hot ca enzyme nh:

MWU n v biu th lng enzyme cn thit chuyn ha mt mg tinh bt ha tan n dextrin trong 30pht iu kin th nghim;

SKB l lng enzyme cn thit dextrin ha 1g beta-dextrin gii hn kch thc xc nh sau 1 gi iu kin th nghim

2. K THUT C BN TRONG CNG NGH ENZYME2.1. ngha ca k thut tinh sch trong cng ngh enzymeCng ngh enzyme c bt u t giai on sn xut ch phm enzyme th v kt thc giai on tothnh ch phm tinh khit.

Bt u t ch phm enzyme th, ngi ta tin hnh tinh sch enzyme bng nhiu k thut khc nhau.Cc k thut tinh sch thng a n nhng kt qu quan trng nh sau:

Khi lng ch phm enzyme s c gim dn qua tng bc tinh sch. Ch phm enzyme th thngcha nhng thnh phn c bn sau:

Nc chim khi lng ln nht Protein khng c hot tnh sinh hc Cc tp cht t t bo (nu l ngun ng vt v thc vt), mi trng nui cy (nu l ngun t vsv) Enzyme (hay protein c hot tnh sinh hc)

Nh vy, qu trnh tinh sch l qu trnh loi b ba phn trn, ch nhn sn phm cui l protein c hottnh sinh hc. Vic loi b thnh phn u khng th thc hin trong mt giai on m phi thc hin quanhiu giai on khc nhau, mi giai on ta s loi b c mt phn khng phi l enzyme ra khi hnhp. Khi cc thnh phn khng phi l enzyme c loi dn ra, khi lng ch phm s gim theo.

Nh vy, nu enzyme c tinh sch theo nhng k thut khc nhau s thu nhn nhng gi tr quan trngsau:

Gim khi lng. Tr s ny c ngha cho vic ng bao b, vn chuyn, s dng v bo qun. Tng c hot tnh enzyme c ngha rt ln trong qu trnh s dng sau ny. Tng gi tr kinh t thng qua gi thnh sn phm gii quyt c trong nhng mc tiu trn, ta thc hin nhng k thut c bn sau:


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CNG NGH ENZYME 19

2.2.K thut c bn2.2.1. Cc phng php c hc tch enzymeCc phng php c hc c ng dng nhiu tch enzyme khi t bo v cc thnh phn khc gm 2

phng php:

Phng php ly tm: ly tm l tch vt cht rn ra khi dung dch. Trong cng ngh enzyme, phngphp ly tm thng c ng dng kh rng ri thu nhn dung dch enzyme, dung dch ny chacc enzyme ngoi bo. Enzyme ni bo nm trong t bo sinh vt mun thu nhn enzyme ni bo tacn phi tin hnh ph v t bo.

Phn ln enzyme ngoi bo l enzyme ha tan. Nh vy khi tin hnh ly tm, dung dch c tch khicc thnh phn rn l dung idjch enzyme th. Dung dch enzyme th ny cn cha thnh phn sau:

Protein c hot tnh sinh hc Cc cht ha tan khc Nc

Phng php ly tm ch tch c thnh phn rn c t trng ln hn dung dch. Dch thu c cha phil ch phm enzyme tinh khit m l ch phm enzyme th, v cn cha protein khng hot ng, ncv cc cht ha tan khc.

trnh hin tng bin tnh ca enzyme, trong nhng mu nghin cu, ngi ta thng tin hnh lytm lnh, cn trong sn xut theo quy m cng nghip, ngi ta thng tin hnh trong iu kin nhit thp.

Trong quy m cng nghip, ngi ta thng tin hnh ly tm thu nhn dung dch enzyme bng my lytm lin tc. Phng php ly tm lin tc c nhng u im l thi gian thu nhn sn phm nhanh, lintc v khng gy nh hng ln n hot tnh enzyme.

Phng php lc: trong cng ngh enzyme, sau khi ph v thnh t bo sinh vt hay sau khi ln men,ngi ta thng s dng qu trnh lc thu nhn dung dch enzyme ni bo, enzyme ngoi bo hatan.

Lc cng l phng php tch thnh phn rn ra khi dung dch. Lc l mt phng php khi thc hinthng gp nhiu kh khn nh kch thc vt cht di t bo thng rt nh v nht ca dung dchthng rt cao. C hai yu t ny u lm cn tr qu trnh lc. Tc lc ph thuc rt ln vo: dintch b mt vt liu lc, p sut khi lc, nht dch lc

Trong cng nghip sn xut enzyme, ngi ta thng s dng cc kiu sau:

Lc p: thng c s dng trong trng hp dung dch cn lc c khi lng nh. Phngphp ny s dng lc enzyme rt c hiu qu.

Lc chn khng: l phng php c ng dng nhiu nht trong nghin cu v trong sn xutcc sn phm sinh hc c hot tnh. Trong lc chn khng quay c s dng nhiu hn c.

Lc theo dng chy ct ngang: trong nhng nm gn y, nhiu nh my sn xut enzyme sdng phng php lc theo dng chy ct ngang. Theo , dng dung dch lc s chy song song

b mt nguyn liu lc. Phn lc s c thot qua vt liu lc v i xung pha di. Phngphp ny c u im l lm gim sc khng qu trnh lc ca vt liu rn. Lc thng thng: phng php lc thng thng c t rt lu, hin nay, mt s nh my

vn cn s dng. Phng php ny dn c thay bng phng php khc nhanh hn, c hiuqu hn.

2.2.2. Cc phng php ph v t bo sinh vtMc ch ca vic ph v t bo l gii phng ton b cc cht c trong t bo. Cng vi cc cht ctrong t bo c gii phng ra ngoi l enzyme ni bo.

i vi t bo ng vt v t bo thc vt ta phi tin hnh nghin nt trc khi thc hin cc qu trnhtrch ly, lc, c c, lm sch v sy

i vi t bo vsv phi tin hnh tch bng phng php ly tm hoc bng phng php lc. Phn dchlc thu c ca mt trong hai phng php ny l dch ch phm enzyme ngoi bo. Phn sinh khi vsvsau khi ly tm hoc lc cha enzyme ni bo. Vic ph v t bo ng vt v thc vt khng my kh


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CNG NGH ENZYME 20

khn nhng ph v t bo vsv rt phc tp v t bo vsv va c kch thc nh, va c thnh t bo ccu trc kh c trng. Do , ph v t bo vsv i hi phi c nhng phng php c bit.

2.2.2.1. Ph v t bo bng phng php c hc:Mc ch ca ph v t bo l gii phng cc cht c trong t bo v m bo c hot tnh ca enzymeni bo.

i vi t bo ng vt (thng s dng ton b c quan hay m bo) c cha enzyme v cn phic tr nhit thp trnh mt hot tnh enzyme. Trc khi tr lnh cn loi b m hot ccthnh phn khc bm theo m bo , m cn c x l nhanh v phi c thu nhn enzyme trongthi gian khng qu 4 gi k t khi git m.

i vi t bo v m thc vt cng cn phi c lm sch v c tr lnh nu cha tin hnh thunhn enzyme ngay.

T bo ng vt v thc vt thng rt d ph v bng cc phng php c hc. Ring i vi t bovsv, vic ph v t bo gp phi kh khn nht nh: Th nht: t bo vsv c kch thc qu nh, vic ph v t bo bng phng php nghin nu

khng c cht tr nghin s khng c hiu qu. Th hai: vsv l c th n bo, khi pht trin trong mi trng (b l mt lng) c nhiu c cht

thy phn, chng s to ra nhiu enzyme ngoi bo tng ng. Cc enzyme ngoi bo ny ha

han trong mt v d dng tch chng ra khi dung dch nui cy. Do trong cng nghip, trtrng hp phi thu nhn enzyme ni bo ngi ta mi tin hnh nghin t bo vsv

Phng php ng ha p lc cao: l phng php c ng dng rng ri nht ph v t bo vsv.Huyn ph tb vsv s c nn vi mt p sut cao, chng va chm rt mnh vo vnh ng. Tb b ph v

bi lc ct v sc nn.

Phng php nghin m: l phng php c s dng rng ri trong cng nghip sn xut enzyme.Trong qu trnh nghin, ngi ta s dng nhng vin bi thy tinh c kch thc 0,2 1mm tng qutrnh ph v t bo. Trong nhiu trng hp, ngi ta khng dng vin bi thy tinh m ngi ta dng htthy tinh v ht thy tinh c ma st cao hn

2.2.2.2. Ph v t bo bng phng php khng phi c hc:Ngoi 2 phng php c hc trn, ngi ta cn ph v t bo bng phng php sau:Phng php ha hc: da vo kh nng to ra p sut thm thu mnh, hoc kh nng oxy ha mnhca cc cht ha hc ph v thnh t bo. Phng php ny khng i hi p sut cao, nn t chi ph vd thc hin. Tuy nhin, v trong qu trnh thc hin, ngi ta s dng ha cht nn cc ha cht nythng ln vo trong hn hp, i hi qu trnh tch rt phc tp. Mt trong nhng ha cht s dngnhiu l acetone

Phng php vt l: l s dng siu m, thng c s dng trong quy m ptn cha s dng trong cngnghip. Phng php vt l l to shock nhit hoc shock thm thu. Nguyn tc ca phng php ny lkhi a nhit ca huyn ph tb xung nhit rt thp, ngay lp tc nng nhit ln 40oC khi thnht bo s b ph v v ngi ta thu c huyn ph t bo v. Phng php ny thng d thc hin, m

bo c hot tnh ca enzyme nhng hiu sut ph v t bo khng cao.

Phng php sinh hc: c 2 phng php hin ang c s dng rng ri l phng php t phn vphng php thy phn bng enzyme a t bn ngoi vo.

Phng php t phn l to iu kin ti u cho mt s enzyme c kh nng phn gii mt s thnh phnca thnh t bo, cc enzyme ny phi l nhng enzyme c trong tb v l ca tb . Bnh thng ccenzyme ny khng hot hot ng mnh, nhng nu iu kin nhit , pH, nc bn ngoi tb m trngvi mc hot ti u ca chng, th chng s hot ng mnh v thy phn thnh t bo, lm cht t

bo.

T phn l qu trnh c p dng nhiu trong ph v thnh t bo cc loi nm men. Ngi ta thngtin hnh t phn huyn ph t bo nm men nhit 48 52 oC trong khong thi gian 6 24 gi.Phng php ny c nhiu nhc im v trong qu trnh thy phn, cc enzyme c trong t bo khng

ch thy phn cc cht thnh t bo m c nhng cht c trong t bo, thm ch enzyme cng b phhy. Phng php ny hin khng c s dng nhiu trong cng nghip.


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CNG NGH ENZYME 21

2.2.2.3. Cc phng php c cDung dch enzyme th thng cha lng enzyme khng nhiu. x l mt khi ln dung dch enzyme

phi tn t nhiu cng sc chi ph cho x l ny rt cao. Mt khc lng enzyme c trong dch enzymeth qu t, hot tnh enzyme khng cao. Chnh v th phi c c dung dch enzyme nhm mc ch:

Lm tng hot tnh ca enzyme Lm gim chi ph v cng sc cho vic x l sau ny Tng kh nng bo qun enzyme Gim chi ph vn chuyn, bo qun Tng hiu sut tc ng ca enzyme i vi c cht t c mc ch , ngi ta tin hnh c c enzyme bng phng php sau:

Phng php nhit: s dng nhit c c enzyme thng kh khn v enzyme l protein rt nhy cmvi nhit . Qu trnh c c nhit l qu trnh lm bc hi nc, lm cho lng nc trong dung dchgim i, hm lng enzyme trong dung dch s tng ln, hot tnh enzyme cng tng cao. Tuy nhin phich n iu kin nhit khng ch n trong mt khong cho php th hot tnh enzyme mi c

bo ton.

Phng php kt ta: enzyme l mt phc hp protein c kh nng kt ta vi mt s dung mi

Kt ta bng mui: mui nng cao c th tc dng vi phn t nc bao quanh protein enzyme vlm chuyn ha in tch, lm thay i tnh ha tan ca enzyme. Trong cng nghip thng s dngmui ammonium sulfate. Nhit tin hnh kt ta: 35 40oC, nng mun: 20 80%

Kt ta bng dung mi hu c: dung mi hu c c nh hng rt ln n kh nng ha tan caenzyme, nh hng solvat ha ca phn t nc xung quanh phn t enzyme b thay i, tng tcgia cc phn t enzyme s tng ln. Trong cng nghip sn xut enzyme ngi ta thng s dngethanol v acetone kt ta enzyme. Nhit kt ta: 3 10oC, t l v nng cc dung mi huc dng kt ta enzyme c xc nh bng thc nghim cho tng loi enzyme v tng nng enzyme c trong dung dch enzyme.

Kt ta enzyme im ng in: protein l cht lng cc. Mc ha tan ca enzyme ph thucvo pH. Kt ta enzyme im ng in thng thc hin quy m nh. Thi gian to im ng

in v thi gian lu to kt ta bn vng thng lm thay i hot tnh enzyme.Phng php siu lc: l phng php s dng mng lc c l lc siu nh. c s dng rng ri thu nhn cc cht c khi lng phn t trong khong 1000 300.000Da. Ngi ta thng s dngcellulose acetate v cc polymer hu c nh: poly sulfone, polyvinylidene v polypropylene lm nguynliu mng lc.

2.2.2.4. Cc phng php tinh sch enzymeC 3 phng php tinh sch enzyme chnh:

Phng php kt tinh Phng php in di Phng php sc k Sc k lc gel Sc k trao i ion Sc k i lc

2.2.2.5. To sn phm enzymeSau khi c lm sch, ngi ta tin hnh thc hin giai on cui cng l to sn phm, ng bao. Ch

phm enzyme c th c to thnh cc loi sn phm sau: enzyme dng dung dch, enzyme dng huynph, enzyme dng bt kh, enzyme dng vin nh

Enzyme dng dung dch hay dng huyn ph: c nhc im l rt kh bo qun. do ngi tathng a chng vo dung dch cc cht bo qun. mt khc khi s dng ngi ta cng phi gi ntrong iu kin nhit nht nh, khng c bo qun nhit cao

Enzyme dng bt kh hoc dng vin nh: thng d dng bo qun v kh nng bo qun rt lu.ngoi ra, cc ch phm enzyme ny thng d vn chuyn.


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CNG NGH ENZYME 22

3. ENZYME C NH3.1.Chuyn ha sinh hcQu trnh chuyn ha sinh hc l qu trnh vt cht trong t nhin c sinh vt chuyn t dng ny sangdng khc thng qua hot ng sng ca t bo. Hot ng sng ca t bo l qu trnh trao i cht gia

bn ngoi v bn trong t bo thng qua hot ng ca enzyme v ca t bo.

Nh hot ng xc tc ca enzyme to cho t bo sinh vt v c th sinh vt nh mt h thng m. nng lng cha trong vt cht lun lun c bin i, lun lun c trao i gia bn trong v bnngoi c th.

Ton b chuyn ha sinh hc c thc hin bi 4 qu trnh sau:

Qu trnh hot ng ca enzyme ngoi bo (enzyme t do) Qu trnh hot ng ca cc vi sinh vt trong qu trnh ln men. Qu trnh hot ng ca t bo c nh Qu trnh chuyn ha ca enzyme c nh3.1.1. Chuyn ha vt cht do enzyme t do hay enzyme ha tanEnzyme t do hay enzyme c tch khi t bo, chng c kh nng ha tan trong mi trng nc v

thc hin cc phn ng ngoi t bo sinh vt. Nhng enzyme ny cn c gi l enzyme ngoi bo. Ccenzyme ny ng vai tr rt quan trng trong s chuyn ha vt cht ngoi thin nhin v c trong snxut v i sng. Tuy nhin, khi s dng cc enzyme ny, cc nh khoa hc cng cho thy chng cnhng nhc im sau:

Trong qu trnh tham gia phn ng, cc loi enzyme ny thng ln vo sn phm. Vic tch snphm cui ra khi enzyme ha tan l mt cng vic rt kh khn v i hi chi ph khng nh. Nhiusn phm ca qu trnh thy phn i hi khng c cha tp cht v cc thnh phn khc nhenzyme. V d nh trong sn xut glucose s dng trong y hc phi tuyt i sch c v phng dinha hc v v sinh hc.

Sau phn ng, nu tch c enzyme ra khi phn ng thc hin cc phn ng tip theo, enzymes khng gi c hot tnh nh hot tnh ban u, nh vy vic ti s dng enzyme s khng c hiu

qu, trong nhiu trng hp enzyme ny khng s dng c na.Cc enzyme ha tan thng km bn nhit, acid, kim, dung mi hu c hay mt s ion kim loi nng.Do , vic s dng cc bin php bo v chng trong phn ng l iu rt cn thit

3.1.2. Chuyn ha vt cht bng enzyme khng ha tanEnzyme khng ha tan hay enzyme c nh c hiu theo c ngha hp v ngha rng

Theo ngha hp: enzyme khng ha tan l nhng enzyme c a vo nhng pha ring r, pha nyc tch ring vi pha dung dch t do. Pha enzyme khng ha tan trong nc v c gn vi nhng

polimer a nc c trng lng phn t ln

Theo ngha rng: cc cht xc tc c nh l cc enzyme, t bo trng thi sng trng thi cho phps dng li. Nh vy, theo ngha rng enzyme khng ha tan bao gm c enzyme c c nh vo mtcht mang, bao gm c enzyme c trong c th sng c c nh trong cc bnh phn ng sinh hc cgn kt mt cht mang cho php ta s dng nhiu ln.

Enzyme khng ha tan hay cn gi l enzyme c nh thng l nhng enzyme ha tan c gn vo mtcht mang bng nhiu k thut khc nhau. Nh qu trnh ny m enzyme t trng thi ha tan chuynsang dng khng ha tan. khi chuyn t trng thi ha tan sang trng thi khng ha tan, enzyme khngha tan c nhng u im sau:

Enzyme khng ha tan c th c s dng nhiu ln, hot tnh ca enzyme khng ha tan t b thayi trong nhng ln ti s dng. c im ny ca enzyme khng ha tan c ngha rt ln trong kthut, nh ta c th ti s dng nhiu ln v s lm gim chi ph cho vic sn xut enzyme. y lu im ln nht ca vic thu nhn v ng dng enzyme khng ha tan.

Enzyme khng ha tan khng ln vo sn phm cui ca phn ng enzyme, do chng ta khngphi chi ph cho vic tch enzyme ra khi sn phm. Sn phm cui thu c s coi nh sn phmtng i sch.


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CNG NGH ENZYME 23

T 2 c im trn cho thy s dng enzyme khng ha tan c ngha kinh t hn s dng enzyme hatan nhiu ln.

3.1.3. Chuyn ha vt cht do cc qu trnh ln menQu trnh ln men l qu trnh chuyn ha sinh hc c thc hin bi cc t bo vi sinh vt sng t do.Ln men l qu trnh chuyn ha vt cht c trng cho ring vi sinh vt. Cc qu trnh ln men l tphp rt nhiu cc qu trnh enzyme. , ton b cc phn ng xy ra trong t bo theo mt trt t c

quyt nh bi h thng gen c trong nucleic acid.Khc vi enzyme ngoi t bo, qu trnh ln men thng xy ra phc tp hn, cc c cht ngoi t bo

phi thm thu c vo trong t bo. Ti y s to ra cc sn phm bc mt (sinh khi), sn phm bchai (sn phm trao i cht). Qu trnh to ra cc sn phm trn ch c thc hin trong t bo sng. Nut bo cht th cc qu trnh chuyn ha li mang ngha khc. l qu trnh thy phn bi enzymengoi bo.

Qu trnh chuyn ha sinh hc ny xy ra thng xuyn, lin tc trong thin nhin v c loi nging dng vo sn xut hng ngn nm nay nh sn xut ru, bia, nc gii kht, phomai, tng, chao,nc mm v pht trin mnh trong cng ngh ln men hin i nh amino acid, khng sinh, cc chtkch thch tng trng.

3.1.4. Chuyn ha vt cht do t bo c nhT bo c nh l t bo vi sinh vt c gn vo mt cht mang. Cc cht mang v vi sinh vt gn vo c a vo bnh phn ng sinh hc (bioreactor). C cht c a vo t nh ca bnh phn ngsinh hc v sn phm c ly ra y bnh phn ng sinh hc. Qu trnh ny c thc hin lin tc.

T bo c nh c ng dng rng ri, em li hiu qu kinh t rt r rt

3.2.c im ca enzyme c nhKhi gn enzyme ha tan vo mt s cht mang, enzyme ny c nhng c im khc hn enzyme ha tan.

Hot tnh ca enzyme khng ha tan thng nh hn hot tnh ring ca enzyme ha tan cng loi.S d c s thay i v hot tnh ring ca enzyme khng ha tan l do nhng nguyn nhn sau:

a)

Do nh hng in tch ca cht mang. Khi gn enzyme vo cht mang c in tch khc vi intch ca enzyme, cu trc khng gian ca enzyme c s thay i mc nht nh. V s thayi cu trc khng gian ca enzyme nn s tng tc ca enzyme v c cht chm li, phn ngs xy ra yu hn. Mc gim hot tnh ca enzyme ph thuc vo mc thay i cu trckhng gian ca enzyme khi ta gn chng vo cht mang.

b) Do enzyme b nht vo mt gel. Khi ta nht mt enzyme vo mt gel no , gel s bao ly phnt enzyme to thnh mt vt cn i vi c cht ca enzyme. Do , s tip xc gia trung tmhot ng ca enzyme vi c cht gp kh khn hn so vi s tip xc gia c cht vi enzymet do

Enzyme khng ha tan hon ton tun theo nh lut Michaelis menten. Tuy nhin, trong phn nggia c cht vi enzyme khong ha tan c nhng sai khc nht nh:a) C th xy ra hin tng cnh tranh c cht vi enzyme v cht mang

b) Hin tng cn tr s khuch tn c cht v cc sn phm ca phn ng lm gim gim tc phn ng Enzyme khng ha tan c tnh bn nhit hn enzyme ha tan cng loi v chng c bo v bi cht

mang Enzyme khng ha tan c xu hng chuyn dch pH ti u sang kim hoc acid so vi pH ti u ca

enzyme ha tan ch khng trng vi pH ti u ca enzyme ha tan cng loi Enzyme khng ha tan c kh nng bo qun tt hn enzyme ha tan cng loi Ta c th ti s dng enzyme khng ha tan nhiu ln. Trong khi enzyme ha tan ch c th s

dng mt ln. c im ny rt c ngha v kinh t khi ta tin hnh cc phn ng theo quy m cngnghip

3.3.Phng php to enzyme c nh3.3.1. Cc yu t nh hng n enzyme c nhKhi gn ln cht mang, enzyme b gii hn trong mt phm vi mi trng xc nh, cu to khng gianca phn t c th b thay i, do lm bin i mt s tnh cht ca enzyme ban u (enzyme ha tan).


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CNG NGH ENZYME 24

Enzyme c th thay i v pH, nhit hot ng, cng nh cc gi tr ca hng s Michaelis mentenv tnh cht c hiu, tuy nhin nhng thay i ny cn ph thuc nhiu vo bn cht ha hc ca chtmang. Ni chung enzyme c nh thng bn vi cc tc nhn bin tnh hn, nhng hot ring thngthp hn enzyme ha tan.

Mt trong nhng nguyn nhn lm gim hot tnh ca enzyme c nh l tng tc protein protein giacc phn t enzyme c lin kt. Trong mi trng hp u thy hot ring ca ch phm b gim

i cng vi s tng lng enzyme c kt hp vo trn mt n v trng lng ca cht mang.Nhn chung, cc enzyme c nh vn gi c tnh c hiu ca mnh. Thm vo , nh lin kt vi chtmang m chng c bn vng ln i vi tc ng khc nhau.

a) Hot tnh ca enzyme khng tan ph thuc vo bn cht v tnh cht ha hc ca cht mangCc tnh cht l hc ca cht mang nh tnh ha tan, tnh bn vng c hc, trng, in tch, tnh honc v k nc u c nh hng nht nh n lng enzyme c lin kt, tnh bn v hot tnhsinh hc ca dn xut enzyme c nh. Bn cht ha hc ca cht mang cng c nh hng ng k nkh nng to thnh dn xut enzyme, v ti kh nng cht mang hp ph khng c trng cc cht t mitrng phn ng.

Dn xut enzyme khng tan thng c tnh bn nhit cao so vi enzyme tan do kt qu ca s nh v

enzyme to nn. Cht mang cng c tc dng hn ch s bin tnh ca enzyme trong cc dung mi c khnng lm t mi lin kt hydrogen v lin kt k nc.

Khi enzyme c gii hn trong phm vi mi trng xc nh, s xy ra nhiu kiu tng tc khc nhauca cht mang polymer ln mi trng vi m bao xung quanh phn t enzyme c nh.

Th nht: polymer nh vo nhng tnh cht l ha hc c trng s ko ti b mt ca enzyme (hocy khi enzyme) c cht, sn phm ca phn ng v cc phn t khc, iu ny lm tng (hay gim)nng ca nhng cht ny trong mi trng vi m st enzyme.

Th hai: bn thn cht mang polymer ngn cn s khuch tn t do ca cc phn t theo hng tienzyme cng nh i khi enzyme, t nh hng n hiu qu xc tc ca enzyme.b) Hot tnh ca enzyme khng tan ph thuc vo s khuch tn ca c cht, sn phm v cc

phn t khc

Tc khuch tn ca c cht, sn phm v cc cht khc ph thuc vo cc yu t:

Kch thc l gel ca cht mang polymer Trng lng phn t ca c cht S chnh lch nng gia vng mi trng vi m xung quanh enzyme v dung dch t do

Nhng hn ch v mt khuch tn cng c ngha ng k trong qu trnh s dng h thng enzyme cnh. ng knh l ca cht mang polymer v trng lng phn t ca c cht ng vai tr hng u.Song nhng hn ch khuch tn khng ch n gin nh vy bi v cn c s khc bit nng giavng mi trng xung quanh enzyme v dung dch t do. Hot tnh xc tc ca enzyme dn ti s thayi nng ca c cht v sn phm phn ng vng mi trng vi m xung enzyme. iu ny lmxut hin gradient nng ca c cht v sn phm ca dung dch v cht mang cha enzyme c nh.

S xut hin ca gradient nng ni trn c gii thch bi tc khuch tn thp cc phn t tronggel v hot tnh xc tc cao ca enzyme

Nhng gii hn khuch tn c th c th hin hai dng: hng ro khuch tn bn ngoi v bn trong.Ro khuch tn bn ngoi xut hin l do c s tn ti ca lp mng dung mi khng pha trn bao xungquanh ht polymer. Cc cht khuch tn vo lp ny nh c s kt hp ca khuch tn phn t th ngv s i lu. dy ca lp ny ph thuc vo tc khuy trn ca dung mi xung quanh cc htcha enzyme c nh. Vic gia tng tc pha trn s lm gim ro khuch tn bn ngoi, v vy trong

bt k quy trnh cng ngh no, tc khuy o ng vai tr ht sc quan trng.

Ro khuch tn bn trong l s gii hn khuch tn t do bn trong cht mang polymer do chnh chtmang gy ra. Trong gii hn ca ht polymer ch c s khuch tn phn t th ng khng b nh hng

bi tc pha trn. Ro khuch tn bn trong s biu hin r rng hn nu nh enzyme c c nh

bng cch a n vo trong cht mang polymer ch khng phi gn n vo b mt ca cht mang.


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c) nh hng ca pH ln enzyme khng ha tanin tch ca cht mang c nh hng ng k ln pH ti u ca enzyme c nh. pH ti u ca enzymec nh c th dch chuyn v pha pH kim hay pH acid so vi pH ti u ca enzyme ha tan. S dchchuyn pH ti u l do nh hng ca trng tnh in ca cht mang to nn. S c mt ca trng tnhin mnh dn ti nng H+ gn cht mang v trong dung dch khc nhau. Khi lc ion thp th nng H+ gn cht mang in tch m cao hn, cn gn cht mang in tch dng thp hn trong dung dch.

Kt qu l pH ti u ca enzyme lin kt ng ha tr vi cht mang c in tch m s chuyn dch sangpha pH kim so vi enzyme ha tan, ngc li nu enzyme lin kt ng ha tr vi cht mang mangin tch dng th pH ti u s chuyn dch sang pha pH acid. Cn khi lc ion ln in tch ca chtmang c lm trung ha bi cc ion tri du v pH ti u ca enzyme c nh gn ging nh caenzyme ha tan.

3.3.2. Phng php c nh enzyme3.3.2.1. Cht mang dng c nh enzymeMc ch cui cng ca vic c nh enzyme l gn c enzyme vo mt cht mang no ta c ththc hin phn ng enzyme nhiu ln. Cng vic ny cn thc hin nhng bc sau:

Chn cht mang ph hp vi enzyme cn gn vo n Hot ha cht mang cho kh nng gn enzyme tt hn Tin hnh cc k thut ph hp gn enzyme vo cht mangYu cu ca mt cht mang l tng:

mt cht mang l tng s dng trong c nh enzyme iu trc ht l cn phi r. iu ny c linquan n hiu qu kinh t ca quy trnh cng ngh, c bit c ngha khi quy trnh ng dng quy m cng nghip

Cht mang phi c tnh cht c l bn vng, n nh. Nh m cht mang mi chu c cc iukin ca mi trng nh khuy trn, p lc trong cc quy trnh sn xut

V mt ha hc, cht mang phi bn vng, khng tan trong mi trng phn ng. Cht mang khngc lm mt hot tnh enzyme. Cht mang khng gy ra nhng phn ng hp ph khng c hiu.

Cht mang phi c tnh khng khun cao, bn vng vi s tn cng ca vi sinh vt. Cht mang phi c trng tt, c din tch b mt tip xc ln. Tnh cht ny ca cht mang va

lm tng kh nng c nh enzyme va tng kh nng tip xc ca c cht vi enzyme, nh lmtng hot tnh ca enzyme c nh v s ln ti s dng

Cht mang c th c cu trc l xp, siu l, c th dng ht, dng mng, dng phim mngTt c cc cht mang dng trong c nh enzyme c chia lm hai nhm: cht mang polymer hu c,cht mang v c

Cht mang l polymer hu c: polymer tng hp (polyacrylamide, polyester, polyvinylalcohol) vpolymer t nhin (polysaccharide: cellulose, agarose, dextran, sephadex v cc dn xut ca chng,alginate, carrageenan, chitin, chitosan; protein: gelatin, keratin, albumin)

Cht mang v c: si bng thy tinh, silicum oxide, alluminium oxide, mangesium oxide3.3.2.2. Phng php c nh enzymeTrong t bo sinh vt tn ti sn c enzyme dng ha tan v c enzyme khng ha tan. Mun thc hinc cc phng php to enzyme khng ha tan ta phi tch v thu nhn c enzyme t t bo vsv.

Phng php nht enzyme: c 2 phng php:

Nht enzyme trong gel: nht enzyme trong gel l phng php da trn c s to ra mt mng bchay mt polymer. Cc cht tham gia phn ng v sn phm ca phn ng c th thm thu vo tronghoc ra ngoi thng qua mang bao bc ny v enzyem s c gi nguyn trong khun gel .

Cch thc hin: enzyme c trn vo trong mt polymer. Sau tin hnh cc qu trnh trng hp vis c mt ca mt hay nhiu tc nhn khu mch, to ra phc chelate. Khi enzyme s c bao bctrong khong khng ca gel mi to thnh. Polymer c s dng y l acrylamide

Nht enzyme trong h si: Phng php nht enzyme trong h si c kh nng xc tc phn ng tthn phng php nht enzyme trong gel. Cc si s dng nht enzyme thng l nhng si nhn


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CNG NGH ENZYME 26

to. Cc loi si ny c bn vi acid, kim, cc loi ion v cc dung mi hu c ha tan. Celluloseacetate thng c s dng nhiu hn c

Phng php to vi nang nht enzyme: cc vi nang c to ra nht enzyme thng c kch thc1 100m. Cc vi nang c tnh cht l cho cc c cht v sn phm phn ng qua li t do.

Phng php ny c u im l enzyme c nht trong kh bn vi mi tc ng bn ngoi v sdng c nhiu ln.

Phng php to lin kt enzyme vi cht mang:

y l nhng phng php khng phi nht enzyme vo mt cht mang m gn enzyme vo cht mang.Nh vy, khng phi tt c cc vt liu u c th s dng lm cht mang m chng phi c tnh cht cbit mi gn enzyme c vo cht mang. Tnh cht c bit ny:

Cht mang phi bn vi acid, kim Cht mang pahri gn c vi enzyme theo nhng c ch nht nh Cht mang khng c tham gia phn ng vi c chtTy theo tnh cht ca cht mang, ngi ta thc hin gn enzyme vo cht mang theo nhng c ch sau:

Phng php hp ph: nh kh nng hp ph ca nguyn liu lm cht mang, enzyme s c gnvo b mt ca cht mang. Kh nng hp ph ca cht mang cng cao, enzyme cng c gn nhiuvo cht mang. Do , kh nng ti s dng enzyme c nh cng cao. Trong thc t ngi ta thngs dng cc cht c kh nng hp ph cao nh: cellulose, agarose, chitin, polyacrylamide, nylon,nha trao i ion, silicagel, thy tinh.

Phng php to lin kt ion: da vo kh nng to lin kt ion gia cht mang v enzyme ngi tagn enzyme vo cht mang. Lin kt ion thng khng bn bng s hp ph ca enzyme i vi chtmang. Cht mang thng s dng: DEAE-sephadex

Phng php to lin kt kim loi: ngi ta lm tng hot tnh b mt ca cht mang bng nhng hpcht kim loi. T cc enzyme s gn cht hn vo cc cht mang. Nhng cht mang thng chot ha b mt bng kim loi: cellulose, gelatin, bng thy tinh, nylon, nhm, silicagel

Phng php to lin kt ng ha tr: da trn nguyn tc ca s tng tc ng ha tr gia phnt enzyme v cht mang. Cc cht mang ny phi l nhng cht khng c tan trong nc. Phng

php ny c ng dng rt rng trong quy m cng nghip.

3.4.ng dng ca enzyme c nhNgy nay, c mt vi quy trnh cng nghip quy m ln ang hot ng m c s dng enzyme cnh. V d in hnh l qu trnh sn xut fructose siro t tinh bt bp v qu trnh sn xut L-amino acidt hn hp L v D-amino acid. Enzyme c nh penicillin acylase cng c s dng rng ri trong qutrnh sn xut penicillin bn tng hp.


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CNG NGH ENZYME 27

3.4.1. Sn xut fructose nh enzyme glucose isomeraseD-glucose khng th c thay th trc tip t ng sucrose bi v glucose t ngt hn. Hn na, tinhth glucose trong dung dch m c c th lm cho qu trnh kh thc hin hn. loi b nhng trngi ny ngi ta chuyn i t dng ng phn glucose sang fructose, s dng enzyme glucoseisomerase.

H s cn bng ca phn ng ny 50 oC gn bng 1, gia tr ny khng thay i ln theo nhit bi vc lng nhit ca phn ng ny l 1kcal/mol. Kt qu l sn phm c cha glucose v frucose theo tl 1:1. Hn hp ny ngt hn so vi glucose n l v rt ph hp thay th ng trong nhiu ngdng nh sn xut nc gii kht, thc phm v lm bnh ko. S d sn phm cui ngt hn l do ccha nhiu fructose.

Enzyme ngoi bo glucose isomerase c to ra bi nhiu vi sinh vt, c th l Arthobacter vStreptomyces sp.Nhu cu ph v t bo m khng ph hy enzyme lm cho glucose isomerase co githnh cao hn so vi cc enzyme thy gii ngoi bo khc. Hn na, glucose isomerase rt nht cm vimt s tc nhn km hm. C hai yu t ny cho thy duy tr enzyme dng c nh di nhng iu kin

phn ng c kim sot tt l mt bin php cn c thc hin. Ngi ta c nh glucose isomerasedi dng kt hp vi collagen, bng hay mt vi loi tc nhn gn kt khc.

Hnh 3.1. S sn xut fructose siro t tinh bt bp s dng enzyme glucose isomerase c nh

C nhiu cng on tch lc v x l gia qu trnh ng ha v ng phn ha phi tha mn cc iukin c bn ca enzyme hc: ti u ha n nh nhit ca -amylase c s dng trong giai onha lng (nhit ca giai on ny vo khong 105oC) th cn s dng ion canxi. Mt khc, nhng ionny c ch glucose isomerase, v chng c loi b nh qu trnh trao i ion trc khi dung dchng dextrose c cho vo thit b thc hin phn ng isomer ha.


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CNG NGH ENZYME 28

3.4.2. Sn xut L-amino acid nh enzyme aminoacylase c nhNhu cu s dng L-amino acid trong thc phm v y hc gia tng nhanh chng. L-amino acid ctin hnh nghin cu sn xut t cng ngh vi sinh vt hay bng phng php tng hp ha hc. Ccamino acid c tng hp bng phng php ha hc thng gp mt s bt li do to ra c dng ng

phn D-amino acid, dng ng phn ny khng c gi tr dinh dng. Do , trong qu trnh sn xutngi ta mong mun ch thu c dng ng phn L trong sn phm cui.

Nguyn l chung ca phn ng chuyn ha Dl-amino acid thnh dng L-amino acid di s xc tc bienzyme aminoacylase:

Phn ng xy ra trong mt thit b dng ct c cha enzyme aminoacylase c nh. L-amino acid mongmun c tch ra khi dng acyl D-amino acid da trn s khc bit v ha tan. Sau D-acylaminoacid c racemic ha thnh dng DL-amino acid tip tc quay tr li l phn ng c cha enzymeaminoacylase c nh.

Hnh 3.2. S sn xut L-amino acid s dng amino acylase ca cng ty Taabe Seiyaku, Nht Bn

Hin nay, loi aminoacylase gn vi DEAE-Sephadex bng lin kt ion c chn s dng bi v n chot tnh cao, d dng chun b, c kh nng ti sinh v n nh.

4. THU NHN ENZYME4.1.Chn ngun nguyn liuEnzyme l nhng cht xc tc sinh hc, c nhiu trong c th sng. Vic iu ch chng bng phng

php ha hc vi s lng ln l vic lm rt kh khn v y tn km nu khng mun ni l iukhng tng, nn ngi ta thng thu nhn chng t cc ngun sinh hc. Mc d enzyme c trong tt ccc c quan, m ca ng vt thc vt cng nh trong t bo vi sinh vt, song vic tch enzyme p ngyu cu v mt kinh t ch c th tin hnh khi nguyn liu c cha mt lng ln enzyme cng nh cho

php thu c enzyme vi hiu sut cao v d dng tinh ch chng. Vic phn b ca enzyme trong t

bo cng khng ng u, trong mt loi t bo cng c th c nhiu enzyme ny song khng c enzymekhc. Lng enzyme li thay i ty theo giai on sinh trng pht trin ca sinh vt v ty theo loinn chng ta phi chn ngun nguyn liu thch hp cho vic chit rt v tinh ch enzyme. C ba ngunnguyn liu sinh hc c bn:


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CNG NGH ENZYME 29

Cc m v c quan ng vt M v c quan thc vt T bo vi sinh vt.Cc m v c quan ng vt

Trong tt c cc nguyn liu c ngun gc ng vt th tuyn tu, mng nhy d dy, tim... dng tchenzyme rt thun li. Dch tu tng c cha amylase, lipase, protease, ribonuclease v mt s enzyme

khc.T ngn t ca d dy b ngh ngi ta c th thu nhn ch phm renin lm ng sa trong sn xutfomat. Ngi ta cng sn xut pepsin t d dy ng vt. Nhng khc vi pepsin, renin c kh nngng t sa cao m khng thy phn su sc casein. Renin l ch phm enzyme c gi tr ln trong cngnghip.

M v c quan thc vt

thc vt: thng thng enzyme hay c mt cc c quan d tr nh ht, c, qu. C quan d tr giucht g th nhiu enzyme chuyn ha cht y. V d trong ht cy thu du c nhiu lipase, trong ht utng c nhiu enzyme urease.

Thc ny mm cha nhiu - amylase, c khoai lang li c nhiu - amylase. Ngi ta thu cmt s ch phm enzyme thy phn nh papain, bromelain, fixin t thc vt bc cao.

Papain thu c t mu nha u xanh, bromelain thu c t cc b phn (l, thn, qu) cy da, cnfixin c tch t dch p thn v l cy Ficus.

Qua cc ngun nguyn liu ng, thc vt chnh c th t chit xut cc ch phm enzyme, chng tathy rng hai ngun nguyn liu ny khng th dng sn xut cc ch phm enzyme vi quy m ln

bi cc nhc im sau y:

Chu k sinh trng ca chng di Ngun nguyn liu ny khng ci to c. Nhiu nguyn liu dng lm thc phm (dng n) khng th dng lm nguyn liu sn xut vi

quy m ln cc ch phm enzyme nhm tho mn cc nhu cu ca nn kinh t quc dn.

Dng vi sinh vt lm ngun nguyn liu sn xut cc ch phm enzyme c nhiu u im ni bt v ctnh cht c o vt xa so vi ngun nguyn liu t ng vt, thc vt, cng nh s khc phc cmi kh khn v hn ch trn.

T bo vi sinh vt

Trc ht vi sinh vt l ngun nguyn liu v tn sn xut enzyme vi s lng ln. y cng lngun nguyn liu m con ngi ch ng to ra c. Chu k sinh trng ca vi sinh vt ngn (t 16 -100 gi) v vy c th nui cy hng trm ln trong nm.

Enzyme vi sinh vt c hot tnh rt mnh, vt xa cc sinh vt khc. V vy ch cn mt lng nhenzyme c th chuyn ha mt lng ln c cht. S liu tnh ton cho bit, trong vng 24 gi, vi sinhvt c kh nng chuyn ha mt lng thc n gp 30 - 40 ln so vi trng lng c th chng. Trong khi, h enzyme ca con ln trn 50 kg ch c th chuyn ha c vi kg thc n trong ngy.

H enzyme vi sinh vt v cng phong ph. Vi sinh vt c kh nng tng hp nhiu loi enzyme khcnhau, trong c nhng enzyme ng, thc vt khng tng hp c. V d cellulase, raxemase...Phnln cc thc n nui vi sinh vt li d kim v gi r. Nhiu vi sinh vt cho enzyme thng c khnng pht trin trn cc mi trng n gin, gi r, d kim nh cc ph liu ca cc ngnh sn xut.Hn na, c th dng nhng nguyn liu khng phi thc phm, nhng dung dch mui v c nui visinh vt. V vy dng vi sinh vt lm ngun thu enzyme s mang li gi thnh r, thi gian nhanh v hiuqu kinh t cao.

Vi sinh vt sinh sn pht trin vi tc cc k nhanh chng, khi lng li nh, kch thc b, nhng tl enzyme trong t bo tng i ln nn quy trnh sn xut ch phm enzyme kh d dng, hiu sut thuhi cao. Lng enzyme c th c sn xut ra trong mt thi gian ngn. i vi mt s trng hp cth dng 100% sinh khi vi sinh vt lm ngun enzyme.


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Vi sinh vt rt nhy cm i vi tc ng ca mi trng, thnh phn dinh dng nui chng cng nhmt s tc nhn l ha, c hc khc. Do c th thay i nhng iu kin nui cy chn ging tonhng chng t bin cho ta hm lng enzyme ng k vi hot tnh xc tc cao. C th ni rng, nhngun enzyme vi sinh vt, ngi ta c th iu khin s tng hp enzyme d dng hn cc ngun nguynliu khc tng lng enzyme c tng hp hoc tng hp nh hng enzyme.

Tuy vy trong qu trnh chn ngun nguyn liu t vi sinh vt, cn lu mt s vi sinh vt c kh nng

sinh c t c bin php x l thch hp. Ni chung cc vi sinh vt mun c s dng lm ngunnguyn liu tch enzyme cn phi tho mn cc iu kin sau:

Kh nng tng hp enzyme mnh trong mt thi gian ngn. D tch enzyme v khng sinh c t.C mt iu l th l: trong iu kin bnh thng, vi sinh vt ch tng hp ra mt lng enzyme va cho hot ng sinh l c th ca chng (thng c gi l s tng hp enzyme "bn th"). Nu khi tnghm lng mt s cht hoc thm mt s cht mi vo mi trng nui cy, c bit l c cht caenzyme, th s tng hp enzyme tng ng tng ln mt cch ng k, khc thng c khi cn tng hpenzyme mi: hin tng trn gi l s cm ng sinh tng hp enzyme. Cht gy nn s cm ng sinhtng hp gi l cht cm ng. S tng hp mt lng ng k enzyme gi l siu tng hp enzyme.

thu c ngun enzyme di do t vi sinh vt, cn phi nui cy chng. C hai phng php nuicy vi sinh vt thu enzyme: phng php nui cy b mt v phng php nui cy b su hay l

phng php ni v phng php chm.

Trong phng php nui cy b mt, ngi ta cho vi sinh vt pht trin v bao ph trn b mt cc hotcht dinh dng rn, c lm m, dng lm mi trng (cm go, cm np, cm m, bp xay nh...). mi trng xp ngi ta trn thm mt lng nh mt ca... Sau khi nui thi gian vi sinh vttng hp enzyme mi trng c sy nh, nghin nh. Ch phm thu c dng rn - th. Mun cch phm tinh khit phi qua giai on tch v tinh ch enzyme.

Khc vi phng php nui cy b mt, trong phng php nui cy b su ngi ta cho vi sinh vt phttrin trong mi trng lng. Nguyn liu chnh v ph bin l dch ng glucose, fructose, maltose,saccharose... dch thy phn cellulose, tinh bt... Ngun nit hu c thng dng l nc chit

bp, chit malt, dch t phn nm men. Cn chn pH ph hp vi chng vi sinh vt v s tng hpenzyme theo mong mun. Sau khi nui, ta thu c canh trng lng - dng th.

lm tng lng enzyme vi sinh vt chng ta cn ch tuyn la v chn ging cc chng vi sinh vtc hot tnh enzyme cao, tng hp c enzyme cn thit v vi s lng nhiu. Cc chng c phnlp theo phng php thng thng ch tng hp mt lng nh enzyme (enzyme bn th), do cn tinhnh gy t bin bng cc phng php sinh hc, l, ha hc... to chng c kh nng siu tng hpenzyme. Vi sinh vt sau khi c tuyn chn, cn c nhn ging v nui trong iu kin ti u chng sinh trng tt, tng hp nhiu enzyme.

Ngoi ra cn phi chn mi trng v thnh phn mi trng dinh dng c nh hng trc tip n ssinh 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 nhtrong thnh phn mi trng c cc cht cm ng th cht hay sn phm phn gii ca n s km hmhoc lm yu tc dng km to ca cht km hm nhm bo m kh nng sinh tng hp enzyme chokhng b cn tr. Cht cm ng tng hp enzyme cho thm vo mi trng nui thng l c cht tngng ca enzyme cn tng hp.

V d: Mun tch - amylase nm mc (Asp.Oryzae), ngi ta cho vo mi trng nui cy tinh bt,maltose, isomaltose, oligosaccharid... c cha lin kt - 1,6 glucozid. Mun tch pectinase Asp.Niger,ngi ta cho thm vo mi trng pectin. i vi hemicellulase th cht cm ng l hemicellulose; cni vi proteinase cht cm ng c hiu lc l protein, bt u nnh, lng, sng nghin nh (Actinomyces fradiae). Cht cm ng cng c th l nhng cht ging c cht v nhng sn phm thy

phn ca chng. V d: thay cho protein th peptid v thay cho tinh bt th erithrodextrin u c tc dng

cm ng.C nhiu yu t nh hng i vi mi trng nui cy. Nhit nui cy thng thng t 25 30 oC.Tr s pH ban u ca mi trng (ch yu mi trng nc) cng c th gy nh hng no n s


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CNG NGH ENZYME

to thnh enzyme, nhng khi vt. Thng thng i vi - acho hot ng ca n (pH = 4,7

pH ti u cho s sinh tng hpthit cho vic sinh tn

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Bai Giang Cong Nghe Enzyme (1)