Trng Cao ng Kinh t Cng ngh TPHCM

Khoa Cng ngh Sinh hc

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CCNNGG NNGGHH EENNZZYYMMEE (Ti liu lu hnh ni b)

CBDG: ThS. L Thanh Hi

HIAST

TPHCM, 03/2013

CNG NGH ENZYME i

MC LC

1. M U ................................................................................................................................. 1

1.1. Lch s nghin cu v pht trin cng ngh enzyme ........................................................ 1

1.2. Khi nim v enzyme........................................................................................................ 2

1.2.1. Bn cht sinh hc ca enzyme ................................................................................... 3

1.2.2. Bn cht ha hc ca enzyme .................................................................................... 4

1.2.3. Cu trc ca enzyme .................................................................................................. 5

1.3. C ch tc dng ca enzyme............................................................................................. 7

1.3.1. C ch xc tc ca enzyme ....................................................................................... 7

1.3.2. Nng lng xc tc .................................................................................................... 8

1.3.3. S to thnh phc hp enzyme c cht .................................................................. 8

1.3.4. ng hc ca phn ng enzyme ................................................................................ 9

1.3.5. Cc yu t nh hng n phn ng enzyme .......................................................... 10

1.3.5.1. Nhit ............................................................................................................ 10

1.3.5.2. pH ..................................................................................................................... 11

1.3.5.3. Cht km hm ................................................................................................... 11

1.3.5.4. Cht hot ha .................................................................................................... 12

1.3.6. Tnh cht c hiu ca enzyme................................................................................ 13

1.4. Phn loi enzyme ............................................................................................................ 13

1.4.1. Danh php quc t ca enzyme ............................................................................... 13

1.4.2. Phn loi enzyme ..................................................................................................... 14

1.4.2.1. Oxydoreductase ................................................................................................ 14

1.4.2.2. Transpherase ..................................................................................................... 14

1.4.2.3. Hydrolase ......................................................................................................... 15

1.4.2.4. Lipase ............................................................................................................... 15

1.4.2.5. Isomerase .......................................................................................................... 15

1.4.2.6. Ligase ............................................................................................................... 15

1.5. Phng php tch v lm sch enzyme .......................................................................... 15

1.5.1. Cc phng php ph v t bo .............................................................................. 15

1.5.2. Phng php gy bin tnh chn lc ....................................................................... 16

1.6. Phng php xc nh kh nng xc tc ca enzyme ..................................................... 17

1.6.1. Cc nhm phng php xc nh kh nng xc tc ca E ...................................... 17

1.6.2. n v hot ......................................................................................................... 17

2. K THUT C BN TRONG CNG NGH ENZYME .................................................. 18

2.1. ngha ca k thut tinh sch trong cng ngh enzyme ............................................... 18

CNG NGH ENZYME ii

2.2. K thut c bn ............................................................................................................... 19

2.2.1. Cc phng php c hc tch enzyme .................................................................... 19

2.2.2. Cc phng php ph v t bo sinh vt ................................................................. 19

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

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

2.2.2.3. Cc phng php c c .................................................................................. 21

2.2.2.4. Cc phng php tinh sch enzyme ................................................................. 21

2.2.2.5. To sn phm enzyme ...................................................................................... 21

3. ENZYME C NH ............................................................................................................. 22

3.1. Chuyn ha sinh hc ....................................................................................................... 22

3.1.1. Chuyn ha vt cht do enzyme t do hay enzyme ha tan .................................... 22

3.1.2. Chuyn ha vt cht bng enzyme khng ha tan .................................................. 22

3.1.3. Chuyn ha vt cht do cc qu trnh ln men ........................................................ 23

3.1.4. Chuyn ha vt cht do t bo c nh .................................................................... 23

3.2. c im ca enzyme c nh ........................................................................................ 23

3.3. Phng php to enzyme c nh ................................................................................... 23

3.3.1. Cc yu t nh hng n enzyme c nh ............................................................. 23

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

3.3.2.1. Cht mang dng c nh enzyme ................................................................ 25

3.3.2.2. 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 .................................................. 27

3.4.2. Sn xut L-amino acid nh enzyme aminoacylase c nh .................................... 28

4. THU NHN ENZYME......................................................................................................... 28

4.1. Chn ngun nguyn liu ................................................................................................. 28

4.2. Thu nhn enzyme ............................................................................................................ 31

5. NG DNG CA ENZYME .............................................................................................. 32

5.1. Tnh hnh ng dng enzyme trong cng nghip trn th gii ........................................ 32

5.2. ng dng trong y hc ..................................................................................................... 34

5.3. ng dng trong ha hc ................................................................................................. 34

5.4. ng dng trong cng nghip .......................................................................................... 34

5.4.1. ng dng trong cng nghip thc phm ................................................................. 34

5.4.2. ng dng trong cng nghip dt ............................................................................. 36

5.4.3. ng dng trong cng nghip thuc da .................................................................... 36

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

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

CNG NGH ENZYME 1

1. M U

1.1. Lch s nghin cu v pht trin cng ngh enzyme

Enzyme hc l mn hc nghin cu v vt cht phn t c hot tnh sinh hc. Mn hc ny pht trin rt mnh nh nhng thnh tu ca mn ha sinh hc, vi sinh vt hc, di truyn hc v c nhng tin b ca ngnh 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 1897 Hai anh em nh Buchner chng minh dch chit t nm men c th chuyn ha ng 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 1917 Boidin v Effront nghin cu -amylase ca B.subtilis v ng dng trong ngnh dt

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 II Bt u sn xut khng sinh theo m hnh cng nghip, s dng amyloglucosidase ng ha tinh bt. S dng penicillineacylase trong sn xut penicilline

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

CNG NGH ENZYME 2

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

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

Winter v Ferch a ra cng ngh sn xut protein

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

16 1984 - nay pht hin hng trm loi enzyme khc nhau, a vo sn xut cng nghip v ng 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 tin c 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), trong dch nha nng, cc enzyme ny thy phn tinh bt trong ht i mch thnh cc ng ha tan v v th c th tch phn b malt vi phn cn li ca ht. Ngy nay, diastase l , hoc -amylase.

Rennet l enzyme c sn xut trong d dy ca ng vt c v khi cn trong giai on b sa m v thng 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

-amylase l 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 (CO2) v amoniac (NH3), hin din trong vi khun ng rut. Urease c tm thy trong vi khun, nm men v thc vt.

1.2. Khi nim v enzyme

Sinh vt c phn ra 2 nhm da vo cu to c th ca chng: sinh vt n bo v sinh vt a bo. Mc d chng c s khc bit rt ln v cu to c th v nhng c im sinh l khc nhau nhng chng u ging 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 vt cht khng phi s sng chnh l kh nng trao i cht ny. Khi c th khng cn kh nng trao i cht th 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 phn gii vt cht cung cp cho t bo nng lng v vt liu xy dng t bo, qu trnh ny c th xy ra trong t bo hoc ngoi t bo.

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

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

CNG NGH ENZYME

Qu trnh tng hp vt cht cho tlng t cc phn ng do qu trnh dca sinh vt i vi mi trng xung quanh.

Hnh

Sn phm ca qu trnh trao i ch

Sn phm bc 1 Sn phm bc 2

Sn phm bc 1: l cc sn phm Cc sn phm ny s c tham gia tr

Sn phm bc 2: l cc sn phm cbo. Trong qu trnh tng hp, mtheo m s thot ra khi t bo. Hisinh tng hp tha c lin quan chtruyn, nhiu khi iu chnh h gen scht c to ra do qu trnh phn gi(qu trnh d ha ngoi t bo). Md ha trong t bo)

Qu trnh trao i cht lin tc cht trong thin nhin. Qu trnh chuynhiu chu trnh chuyn ha cc chkhng ch trong t bo sinh vt m cny c xc tc bi mt loi protein bo c gi l enzyme ngoi bo. Cc enzyme thni bo v enzyne ngoi bo u

1.2.1. Bn cht sinh hc ca enzyme

Nhiu nh khoa hc nghin csinh vt sng tng hp nn v tham gia vo c

Nh vy, bn cht sinh hc ca enzyme l ssinh ha trong v ngoi t bo sinh v

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

Enzyme tham gia phn ng c Enzyme tham gia phn ng trong i

enzyme c tng hp v ho ca c th v ca t bo sinh vng trong khong 30 - 40oC

t cho t bo ch xy ra trong t bo. Qu trnh ny thng do qu trnh d ha. Nh vy, qu trnh trao i cht c xem nh h

ng xung quanh.

Hnh 1.1. H thng m ca t bo sinh vt

i cht to ra 2 dng sn phm:

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

m cng c to ra t qu trnh tng hp v qu trnh phn gip, mt s vt cht c to ra khng tham gia vo qu trnh trao

bo. Hin tng ny c coi nh qu trnh sinh tng hc lin quan cht ch n h di truyn c trong t bo. Do , vi

gen s thu c lng ln cc sn phm sinh tng ho ra do qu trnh phn gii s khng tham gia vo qu trnh trao i ch

bo). Mt s vt cht khc thot ra khi t bo vo mi tr

c c xy ra gia trong v ngoi t bo, to nn s t trong thin nhin. Qu trnh chuyn ha theo con ng sinh vt ng vai tr r

n ha cc cht c trong thin nhin. Cc phn ng sinh ht m c ngoi mi trng, bao quanh t bo . Cc ph

i protein c bit gi l enzyme. Cc enzyme tham gia cc phi bo. Cc enzyme thc hin trong t bo gi l enzym

u c tng hp trong t bo.

a enzyme

u v enzyme v i n thng nht: enzyme l mp nn v tham gia vo cc phn ng sinh hc

a enzyme l sn phm ca cc qu trnh sinh hc v thbo sinh vt. Cc loi enzyme u c nhng c tnh chung nh sau:

bo sinh vt: qu trnh tng hp enzyme l mt qu trnh hm sot rt cht ch

ng c trong t bo sng v c khi enzyme c tch khng trong iu kin nhit n ha. V trong qu trnh s

p v hot ng trong iu kin nhit ca t bo v nhibo sinh vt thng l nhit thp. Phn ln nhit

C

3

ng phi thu nhn nng c xem nh h thng m

trong qu trnh tng hp. bo v tham gia cc cc qu trnh t bo.

qu trnh phn gii ca t o ra khng tham gia vo qu trnh trao i cht tip

ng hp tha. Hin tng bo. Do , vic iu chnh h di

ng hp tha. Mi s vt i cht nm ngoi t bo

bo vo mi trng (qu trnh trnh

bin i lin tc ca vt rt quan trng trong rt c xy ra thng xuyn

bo . Cc phn ng sinh hc i l enzyme. Cc enzyme tham gia cc phn ng ngoi t

i l enzyme ni bo. C enzyme

enzyme l mt loi protein c

c v thc hin cc phn ng c tnh chung nh sau:

t qu trnh ht sc phc

c tch khi t bo sng trong qu trnh sng ca t bo,

bo v nhit ca c th. Nhit t c th sinh vt dao

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 on gii phng hon ton nng lng d tr trong cc hp cht ha hc. Qu trnh chuyn ha ny c thc hin theo chui phn ng, mi phn ng c xc tc bi mt loi enzyme. Cc enzyme ny ln lt thay th nhau xc tc cc phn ng ln lt xy ra, cui cng to thnh CO2, H2O, mt s cht khc v gii phng nng lng. Cng c th chui phn ng s to thnh chu k chuyn ha khp kn. Trong chui chuyn ha h hay chui chuyn ha khp kn, sn phm ca phn ng trc s 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 hin chng 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 hc c xc tc vi cc cht xc tc ha hc i hi nng lng rt ln. Nh c hot ng xc tc ca enzyme, 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 loi enzyme. 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 ng Nh 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 enzyme

Nu tch enzyme ra khi t bo v tin hnh phn tch thnh phn ha hc ca chng, ta s thy chng thuc 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 ny cn 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 phn protein. 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 c gi 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 mt trong 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 hp enzyme mt kim loi, chng s mt hot tnh. Khi ta a cc kim loi tng ng vo cc enzyme, hot tnh enzyme li c khi phc. Tnh cht ny mang tnh cht thun nghch. Vai tr ca kim loi trong hot ng ca enzyme vn cha c lm sng t. Tuy nhin cc nh khoa hc cng cho rng, c th kim loi 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.

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 Peptidase Mangan (Mn), St (Fe), Magie (Mg)

5 Phosphatase Magie (Mg)

6 Arginase Mangan (Mn)

1.2.3. Cu trc ca enzyme

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

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 chc

nng 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 i cu 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 b bin 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 nh amino acid serine, histidine, cysteine, lysine, arginine, glutamic acid, aspartic acid, tryptophan. Cc nhm ha 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 NH2 ca lysine Nhm -SH ca cysteine Nhm -carboxyl ca glutamic acid

Cc 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 thi khng 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 trong trung 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 coenzyme cng c coi nh mt phn cu to ca trung tm hot ng ca enzyme.

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 dehydrogenase ca 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 rt cht ch v mang tnh c hiu cao. Chnh v th, mi loi enzyme ch c th tham gia xc tc phn ng cho 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 ng ca enzyme quyt nh cho php nhng c cht cu trc tng ng vi trung tm hot ng mi c kt hp vo. Quan im ny do Fisher xng. Thuyt ca Fisher tuy gii thch c cc hin tng gn kt gia c cht v trung tm hot ng ca enzyme nhng nhiu kt qu thc nghim, thuyt ny cha gii 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 tc ngay. Chnh c cht l tc nhn tc ng lm thay i cu trc khng gian ca trung tm hot ng ca enzyme. Chnh tc ng cm ng ny ca c cht lm cho cc gc amino acid, cc nhm chc ca trung tm hot ng di chuyn, nh hng mt cch thch hp, chnh xc gn c cht vo trung tm hot ng 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 cm ng ca c cht. Chnh c ch mm do ny ca trung tm hot ng gii thch c s cnh tranh gia c cht v cc cht khng phi c cht nhng li c cu trc khng gian ging c cht. Khi , cc cht 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

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 enzyme

1.3.1. C ch xc tc ca enzyme

Mi vi sinh vt u c cu to t t bo. Cc t bo lun lun tin hnh qu trnh trao i cht vi mi trng bn ngoi bng nhng phn ng sinh ha. Cc phn ng xy ra lun lun c xc tc bi cc enzyme ca t bo. Nh vy, enzyme ng vai tr rt quan trng trong sinh l ca t bo. Tc ng ca enzyme 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 vi nhit ca c th. Cc phn ng enzyme thng khng i hi nhit cao, do n m bo cho mi hot ng sinh l bnh thng ca t bo. Trong khi , nng lng chi ph cho nhng phn ng ha hc thng rt cao.

c im lm gim nng lng hot ha ca phn ng enzyme c ngha rt ln trong sinh l ca sinh vt. c im ny gn lin vi qu trnh tin ha ca sinh vt, nu nhit c th tng s lm ri lon ton b qu trnh sinh l ca t bo. Khi c th s chuyn t trng thi sinh l bnh thng sang trng thi 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 enzyme

2. 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 bo s tng. Kt qu l t bo s tng nhanh v s lng v khi lng. Nh vy, enzyme khng ch ng vai tr duy tr trng thi sinh l ca t bo m cn ng vai tr quan trng trong s pht trin v sinh sn ca t bo, duy tr s chuyn ha vt cht trong cc chu trnh chuyn ha trong thin nhin. Nh c hot ng ca enzyme m khi lng c cht c chuyn ha trong mt n v thi gian trong t bo ln gp hng ngn 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 c hot 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 trc ha 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 h enzyme c cht. Phc h ny thng khng bn. Phn ng to ra phc h enzyme c cht thng xy ra 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 ra khi 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

CNG NGH ENZYME 8

Hnh 1.4. C ch xc tc ca enzyme

1.3.2. Nng lng xc tc

Enzyme 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 c gii 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 c nng 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 cht

Brown v Henri l nhng nh khoa hc u tin a ra thuyt phc hp trung gian. Sau , Michaelis v 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 hp enzyme - 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 tnh ring v nng lng lin kt khc nhau. Cc lin kt ny bao gm: lin kt phi tr, lin kt hydrogen, lin kt 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 nhiu nhm ha hc c hiu. Cc nhm chc ny ng vai tr quan trng trong nh hng v gip c cht enzyme tip cn vi nhau. Khi enzyme v c cht tng tc vi nhau thng to ra lin kt yu, to ra nng lng kt hp . Nng lng c to tng lin kt yu ny thng nh, nhng c nhiu nng lng 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 lng gim trong qu trnh hot ha phi bng lng nng lng cc cht tham gia phn ng nhn c.

CNG NGH ENZYME 9

Theo kin ca cc nh khoa hc, u tin c cht phi c hot ha chuyn sang trng thi chuyn tip tm thi tng tc vi c cht to nn phc enzyme c cht (ES). Sau phc hp ny li c hot ha chuyn thnh trng thi chuyn tip tm thi sang phc hp EP (P l sn phm). Sau phc hp 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 chnh thc nh sau

E + S => ES => EP => E + P

Nng lng cn thit lm gim nng lng hot ha qu trnh xc tc, ng thi cng to ra tnh c hiu cho phn t enzyme. Tnh c hiu ca enzyme l kh nng nhn bit, chn lc c cht tin hnh cc phn ng ring theo bn cht tng loi enzyme.

1.3.4. ng hc ca phn ng enzyme

Nm 1913 hai nh khoa hc Michaelis v Menten a ra m hnh ng hc gii thch phn ng c xc tc bi enzyme v lp phng trnh phn nh quan h gia vn tc phn ng vi nng c cht v enzyme. 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 nht nh 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 nh nht v xp x bng 0.

ng thi nng c cht rt ln so vi nng enzyme tng [Eo]. Ngoi ra, phn ng chuyn ha t 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 ng cng 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 tuyn tnh 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)

k4 k2

k1 k3 E + S ES E + P

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 Menten

Vi = 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 bng km/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 ca phn ng enzyme vi nng c cht ban u khc nhau.

1.3.5. Cc yu t nh hng n phn ng enzyme

1.3.5.1. Nhit

Nhit c nh hng rt ln n phn ng enzyme. Tc phn ng enzyme khng phi lc no cng t 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

CNG NGH ENZYME 11

ng mnh nht nhit 40 50oC. Nhit ti u ca nhng enzyme khc nhau l hon ton khc nhau. Mt s enzyme khc c nhit ti u 60oC, mt s khc li c nhit ti u 70oC, thm ch c mt s enzyme ca vi khun Bacillus subtilis li hot ng mnh 90oC

Nu a nhit cao hn mc nhit ti u, hot tnh enzyme s b gim. Khi enzyme khng c kh nng 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 bo v. 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. pH

pH mi trng thng nh hng n mc ion ha c cht, enzyme v c bit nh hng n bn ca 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 acid yu. 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 hm

Cc cht km hm hot ng ca enzyme thng l cc cht c mt trong cc phn ng enzyme, lm gim hot tnh ca enzyme nhng li khng b enzyme lm thay i tnh cht ha hc, cu to ha hc v tnh cht 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 v c 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 c trng thi cn bng.

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 cht vo enzyme, to thnh phc enzyme cht km hm (EI). Phc hp ny b phn r rt chm. Ty thuc vo 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 ca c cht. Chng thng l nhng cht km hm thun nghch. Chng c kh nng kt hp vi trung tm hot 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 tm ny. C ch loi tr ln nhau ca cht km hm v trung tm lm gim s lng cc enzyme kt hp vi c 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 tr trong 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 tr c hin tng cnh tranh.

Ph thuc vo i lc gia c cht v cht cnh tranh vi enzyme

Cht 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 cu trc khng gian ca phn t enzyme theo chiu hng bt li cho hot ng xc tc. V th cc cht km hm 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 d qu trnh ny khng lm thay i i lc gia enzyme v c cht. Mc km hm ca cht km hm khng cnh tranh khng ph thuc vo tng quan nng c cht v cht km hm. C cht d c lng 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 to thnh sn phm P1 v P2 th enzyme c i lc vi c P1, P2 v c cht A v B. Khi sn phm P1, P2 c 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 chuyn ha 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 ha

Cc cht c tc dng lm tng hot tnh ca enzyme gi l cc cht hot ha enzyme. Cc cht hot ha enzyme 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 ca enzyme

CNG NGH ENZYME

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

1.3.6. Tnh cht c hiu ca enzyme

Tnh cht c hiu l biu hin khcht c hiu rt cao. Tnh cht cht xc tc khc.

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

1. c hiu kiu phn ng

Tnh cht c hiu kiu phn ng bichuyn ha nht nh trong cc kithy phn

2. c hiu c cht

Khi tham gia vo cc phn ng , cc lonhin khng phi c cht no cng c khenzyme. Ngi ta phn tch c hi

A c hiu tuyt i

L kh nng xc tc ca enzyme c kh ngng xc tc i vi mt c ch

B c hiu tng i

L kh nng xc tc ca enzyme tc d

C c hiu quang hc (c hiu l

L kh nng xc tc ca enzyme v trans)

1.4. Phn loi enzyme

1.4.1. Danh php quc t ca enzyme

Khi s lng enzyme mi c pht hingi tm ra n. Dn dn, theo thy c tnh cht gn ging nhau honhng 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 gia. Theo , tn g

t ha thng lm nhim v chuyn nhm hydrogen hot trong phn t tin enzyme hoc cc cht c tc d

t ng ca enzyme

Hnh 1.11. Cht hot ha enzyme

a enzyme

n kh nng xc tc ca enzyme i vi c cht nht c hiu ca enzyme cho thy s khc bit rt l

nng xc tc cho s chuyn ha mt hay nhiu c chnh. Tnh cht c hiu ca enzyme biu hiu mt s ki

ng biu hin ch, mi enzyme ch c th xc tc cho mnh trong cc kiu phn ng nh phn ng oxy ha kh, phn

ng , cc loi c cht phi gn vi trung tm hot ng c kh nng tip cn v gn c vo vi trung tm ho

c hiu c cht theo mc sau:

a enzyme i vi mt c cht nht nh. Ngoi c cht ny ra, enzyme t c cht no khc na.

a enzyme tc dng ln mt kiu lin kt ha hc nht nh

u lp th)

a enzyme n mt trong 2 dng ng phn quang hc ca c ch

a enzyme

c pht hin cn t, ngi ta thng gi tn enzyme theo thch cn, theo thi gian, s lng enzyme tm ra ngy mt nhiu v nhng nhau hoc ging nhau, ngi ta phn loi chng thnh t

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

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

13

n nhm hydrogen hot nhng cht c t c tc dng phc hi cc nhm

t nh. Enzyme c tnh t ln gia enzyme v cc

u c cht nht nh theo kiu c hiu sau:

xc tc cho mt kiu phn ng ng chuyn v, phn ng

t ng ca enzyme. Tuy i trung tm hot ng ca

t ny ra, enzyme ny khng

a c cht (ng phn cis

i tn enzyme theo thch ca tng u v nhng enzyme tm ra

i chng thnh tng nhm v a ra ng chng ai cng c th hiu c

u ca chng cng vi tn n:

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 tn gi 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 phn loi. V d nh trypsin, chimotrypsin, pepsin

1.4.2. Phn loi enzyme

Nm 1960, hip hi ha sinh quc t (IUB) thng nht phn loi enzyme ra lm 6 lp v c nh s th 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 li chia ra lm nhiu nhm, chnh v th, theo h thng phn loi, mi enzyme thng c 4 cha s: s th nht 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 phn

4. Liase: cc enzyme xc tc cho phn ng phn ct khng cn nc, loi nc to thnh lin kt i hoc 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. Oxydoreductase

y 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 enzyme ny 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 n NAD+, NADP+, FMN, FAD.

Cc dehyrogenase xc tc cho giai on u ca chui h hp, vn chuyn ng thi c proton v electron. 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 rt quan 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 lm cho chng c kh nng kt hp vi proton c trong mi trng. Lp enzyme ny tc dng trc tip vi oxy

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 thm

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

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

1.4.2.2. Transpherase

Transpherase cng thuc lp enzyme phc tp. Coenzyme ca transpherase c bn cht ha hc rt khc nhau ty theo vo bn cht ca nhm c chuyn v. Lp transpherase bao gm nhng enzyme tiu biu sau:

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

CNG NGH ENZYME 15

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

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

Phosphotransferase: enzyme ny tham gia xc tc chuyn ha gc phosphoryl. Trong phn ng ny thng c s tham gia ca ATP vi ngha nh mt cht cho. Gc phosphate c chuyn t ATP n nhm hydroxyl ca alcohol hay saccharide

1.4.2.3. Hydrolase

Hydrolase tham gia xc tc cho cc qu trnh thy phn. Chnh v th, nc l thnh phn khng th thiu c 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 phn t thp v cc amino acid.

Cc enzyme peptide hydrolase thy phn cc lin kt peptide gia chui peptide gi l endopeptide hydrolase hay proteinase. i din cho endopeptide hydrolase l pepsin, trypsin, chymotrypsin. Cc enzyme peptide hydrolase thy phn cc lin kt peptide u chui peptide gi l exo peptide hydrolase hay 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. Nhm ny 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 gm papain, 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 v m ng vt) to thnh acid bo t do v glycerol. Chng xc tc phn ng thy phn ln lt tng lin kt ch khng phi ct t c 3 lin kt ester cng mt lc.

1.4.2.4. Lipase

Pyruvate decarboxylase: enzyme ny tham gia xc tc cho phn ng loi CO2 ra khi phn t pyruvic acid, 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 malic acid to thnh fumaric acid (acid c mt ni i)

1.4.2.5. Isomerase

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

1.4.2.6. Ligase

Enzyme ny tham gia xc tc cho phn ng carboxyl ha pyruvic acid, to thnh oxaloacetic acid. Chng cn 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 enzyme

1.5.1. Cc phng php ph v t bo

Enzyme l loi protein c to thnh trong t bo, phn ln chng tn ti trong t bo. Cc enzyme ny thng khng c kh nng di chuyn qua mng sinh hc. Do , vic tch chng ra khi t bo l diu rt kh khn.

CNG NGH ENZYME 16

thu nhn enzyme ni bo, ngi ta phi ph v t bo bng nhiu phng php. Cc phng php ph v 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 ha bng 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 loi protein khng phi lc no cng t c kt qu tt v khng phi khng gp nhng kh khn nht nh

1.5.2. Phng php gy bin tnh chn lc

Sau khi ph v t bo, ngi ta thng s dng nc, dung dch m, dung dch mui trung tnh tch enzyme 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 cc protein enzyme khng mong mun), nc, cc thnh phn khc ca t bo khi enzyme. Cng vic ny hon ton khng d dng. Do , vic lm sch enzyme i hi ngi lm nghin cu v enzyme, ngoi s hiu bit v enzyme ra cn phi nm vng rt nhiu thao tc k thut khc nhau trnh gy mt tnh hot ng ca enzyme.

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

1. Phng php gy bin tnh chn lc: phng php ny da trn s tc ng ca nhit , pH lm bin 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 enzyme khng 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 mui khc nhau, ngi ta thng s dng (NH4)2SO4 kt ta phn on enzyme. Bng cch ny, ngi ta c 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 c dung dch enzyme

3. Phng php hp th chn lc: ngi ta c th hp th trc tip vo dung dch enzyme, hoc cho dung dch enzyme chy qua mt ct, trong ct c cha cht hp th. Cht hp th c s dng rng ri l hydrocyapatite. Ngi ta c th thc hin mt trong hai cch sau: hoc l hp th enzyme hoc l hp th 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 dung mi thch hp.

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

CNG NGH ENZYME 17

Ngi ta thng s dng DEAE cellulose hn c. u im ca DEAE-cellulose l ngoi tp cht protein, 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 lin kt vi nhau bi cc lin kt ngang nh tc dng ca epichohydrin, to thnh sng phn t. S lng 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 s c 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 , vic lm sch enzyme ch tht s c hiu qu khi ta bit la chn cc phng php ring, kt hp vi nhau, to ra mt h thng ni tip nhau mt cch hi ha nht.

1.6. Phng php xc nh kh nng xc tc ca enzyme

1.6.1. 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 hot ng 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 xc tc ca enzyme:

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

Tin hnh xc nh thi gian cn thit thu nhn c mt lng bin i nht nh ca lng c cht 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 i nht 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

1 Thi gian Nng enzyme

Lng c cht v sn phm

2 Lng c cht hay sn phm Nng enzyme

Thi gian

3 Thi gian Lng 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 ng ca enzyme c xc nh thng qua n v hot . Ngi ta biu din n v hot qua nhng n v sau:

a - n v hot quc t (UI): n v hot quc t l lng enzyme c kh nng xc tc lm chuyn ha 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 1 giy iu kin tiu chun

1UI = 16,67nano kat (nkat)

CNG NGH ENZYME 18

c - hot ring: hot ring ca mt ch phm enzyme l s n v UI (hoc mt n v katal) ng vi 1 ml dung dch (nu l dung dch) hoc 1mg protein (nu l bt kh) ca ch phm enzyme. Khi bit c 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 ha bi 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 enzyme cn 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 qu nhiu 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 phi cho 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 vi hot 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 nh hot 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 vi nhng enzyme c kh nng hot ng yu. Trong trng hp phi ko di thi gian, cn thit phi cho 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 30 pht 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 ENZYME

2.1. ngha ca k thut tinh sch trong cng ngh enzyme

Cng ngh enzyme c bt u t giai on sn xut ch phm enzyme th v kt thc giai on to thnh 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 thng cha 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 hot tnh sinh hc. Vic loi b thnh phn u khng th thc hin trong mt giai on m phi thc hin qua nhiu giai on khc nhau, mi giai on ta s loi b c mt phn khng phi l enzyme ra khi hn hp. 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 trng sau:

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:

CNG NGH ENZYME 19

2.2. K thut c bn

2.2.1. Cc phng php c hc tch enzyme

Cc 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, phng php ly tm thng c ng dng kh rng ri thu nhn dung dch enzyme, dung dch ny cha cc enzyme ngoi bo. Enzyme ni bo nm trong t bo sinh vt mun thu nhn enzyme ni bo ta cn 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 khi cc 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 phi l ch phm enzyme tinh khit m l ch phm enzyme th, v cn cha protein khng hot ng, nc v cc cht ha tan khc.

trnh hin tng bin tnh ca enzyme, trong nhng mu nghin cu, ngi ta thng tin hnh ly tm 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 ly tm lin tc. Phng php ly tm lin tc c nhng u im l thi gian thu nhn sn phm nhanh, lin tc 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 ha tan.

Lc cng l phng php tch thnh phn rn ra khi dung dch. Lc l mt phng php khi thc hin thng gp nhiu kh khn nh kch thc vt cht di t bo thng rt nh v nht ca dung dch thng rt cao. C hai yu t ny u lm cn tr qu trnh lc. Tc lc ph thuc rt ln vo: din tch 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. Phng php 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 xut cc 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 s dng 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. Phng php 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 hiu qu hn.

2.2.2. Cc phng php ph v t bo sinh vt

Mc ch ca vic ph v t bo l gii phng ton b cc cht c trong t bo. Cng vi cc cht c trong 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 trnh trch 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 dch lc thu c ca mt trong hai phng php ny l dch ch phm enzyme ngoi bo. Phn sinh khi vsv sau khi ly tm hoc lc cha enzyme ni bo. Vic ph v t bo ng vt v thc vt khng my kh

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 c cu 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 enzyme ni bo.

i vi t bo ng vt (thng s dng ton b c quan hay m bo) c cha enzyme v cn phi c tr nhit thp trnh mt hot tnh enzyme. Trc khi tr lnh cn loi b m hot cc thnh phn khc bm theo m bo , m cn c x l nhanh v phi c thu nhn enzyme trong thi 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 thu nhn enzyme ngay.

T bo ng vt v thc vt thng rt d ph v bng cc phng php c hc. Ring i vi t bo vsv, 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, tr trng 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 qu trnh ph v t bo. Trong nhiu trng hp, ngi ta khng dng vin bi thy tinh m ngi ta dng ht thy 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 mnh ca cc cht ha hc ph v thnh t bo. Phng php ny khng i hi p sut cao, nn t chi ph v d thc hin. Tuy nhin, v trong qu trnh thc hin, ngi ta s dng ha cht nn cc ha cht ny thng ln vo trong hn hp, i hi qu trnh tch rt phc tp. Mt trong nhng ha cht s dng nhiu l acetone

Phng php vt l: l s dng siu m, thng c s dng trong quy m ptn cha s dng trong cng nghip. Phng php vt l l to shock nhit hoc shock thm thu. Nguyn tc ca phng php ny l khi a nhit ca huyn ph tb xung nhit rt thp, ngay lp tc nng nhit ln 40oC khi thnh t 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 v phng 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 phn ca thnh t bo, cc enzyme ny phi l nhng enzyme c trong tb v l ca tb . Bnh thng cc enzyme ny khng hot hot ng mnh, nhng nu iu kin nhit , pH, nc bn ngoi tb m trng vi 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 thng tin hnh t phn huyn ph t bo nm men nhit 48 52oC 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 ph hy. Phng php ny hin khng c s dng nhiu trong cng nghip.

CNG NGH ENZYME 21

2.2.2.3. Cc phng php c c

Dung 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 enzyme th 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 cm vi nhit . Qu trnh c c nhit l qu trnh lm bc hi nc, lm cho lng nc trong dung dch gim i, hm lng enzyme trong dung dch s tng ln, hot tnh enzyme cng tng cao. Tuy nhin phi ch 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 v lm chuyn ha in tch, lm thay i tnh ha tan ca enzyme. Trong cng nghip thng s dng mui 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 ca enzyme, nh hng solvat ha ca phn t nc xung quanh phn t enzyme b thay i, tng tc gia cc phn t enzyme s tng ln. Trong cng nghip sn xut enzyme ngi ta thng s dng ethanol v acetone kt ta enzyme. Nhit kt ta: 3 10oC, t l v nng cc dung mi hu c 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 thuc vo 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 dng cellulose acetate v cc polymer hu c nh: poly sulfone, polyvinylidene v polypropylene lm nguyn liu mng lc.

2.2.2.4. Cc phng php tinh sch enzyme

C 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 enzyme

Sau 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 huyn ph, 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 ta thng a chng vo dung dch cc cht bo qun. mt khc khi s dng ngi ta cng phi gi n trong 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.

CNG NGH ENZYME 22

3. ENZYME C NH

3.1. Chuyn ha sinh hc

Qu trnh chuyn ha sinh hc l qu trnh vt cht trong t nhin c sinh vt chuyn t dng ny sang dng 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 bn ngoi 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 nh

3.1.1. Chuyn ha vt cht do enzyme t do hay enzyme ha tan

Enzyme 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. Cc enzyme ny ng vai tr rt quan trng trong s chuyn ha vt cht ngoi thin nhin v c trong sn xut v i sng. Tuy nhin, khi s dng cc enzyme ny, cc nh khoa hc cng cho thy chng c nhng nhc im sau:

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

Sau phn ng, nu tch c enzyme ra khi phn ng thc hin cc phn ng tip theo, enzyme s 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 tan

Enzyme 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 ny c 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 php s dng li. Nh vy, theo ngha rng enzyme khng ha tan bao gm c enzyme c c nh vo mt cht mang, bao gm c enzyme c trong c th sng c c nh trong cc bnh phn ng sinh hc c gn 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 mt cht mang bng nhiu k thut khc nhau. Nh qu trnh ny m enzyme t trng thi ha tan chuyn sang dng khng ha tan. khi chuyn t trng thi ha tan sang trng thi khng ha tan, enzyme khng ha 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 thay i trong nhng ln ti s dng. c im ny ca enzyme khng ha tan c ngha rt ln trong k thut, nh ta c th ti s dng nhiu ln v s lm gim chi ph cho vic sn xut enzyme. y l u 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 khng phi chi ph cho vic tch enzyme ra khi sn phm. Sn phm cui thu c s coi nh sn phm tng i sch.

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 ha tan nhiu ln.

3.1.3. Chuyn ha vt cht do cc qu trnh ln men

Qu 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 tp hp 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 bc hai (sn phm trao i cht). Qu trnh to ra cc sn phm trn ch c thc hin trong t bo sng. Nu t bo cht th cc qu trnh chuyn ha li mang ngha khc. l qu trnh thy phn bi enzyme ngoi bo.

Qu trnh chuyn ha sinh hc ny xy ra thng xuyn, lin tc trong thin nhin v c loi ngi ng 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 cht kch thch tng trng.

3.1.4. Chuyn ha vt cht do t bo c nh

T 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 ng sinh 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 nh

Khi 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 in

tch ca enzyme, cu trc khng gian ca enzyme c s thay i mc nht nh. V s thay i cu trc khng gian ca enzyme nn s tng tc ca enzyme v c cht chm li, phn ng s xy ra yu hn. Mc gim hot tnh ca enzyme ph thuc vo mc thay i cu trc khng 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 phn t enzyme to thnh mt vt cn i vi c cht ca enzyme. Do , s tip xc gia trung tm hot ng ca enzyme vi c cht gp kh khn hn so vi s tip xc gia c cht vi enzyme t do

Enzyme khng ha tan hon ton tun theo nh lut Michaelis menten. Tuy nhin, trong phn ng gia 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 cng nghip

3.3. Phng php to enzyme c nh

3.3.1. Cc yu t nh hng n enzyme c nh

Khi gn ln cht mang, enzyme b gii hn trong mt phm vi mi trng xc nh, cu to khng gian ca phn t c th b thay i, do lm bin i mt s tnh cht ca enzyme ban u (enzyme ha tan).

CNG NGH ENZYME 24

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

Mt trong nhng nguyn nhn lm gim hot tnh ca enzyme c nh l tng tc protein protein gia cc 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 cht mang 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 mang

Cc tnh cht l hc ca cht mang nh tnh ha tan, tnh bn vng c hc, trng, in tch, tnh ho nc v k nc u c nh hng nht nh n lng enzyme c lin kt, tnh bn v hot tnh sinh hc ca dn xut enzyme c nh. Bn cht ha hc ca cht mang cng c nh hng ng k n kh nng to thnh dn xut enzyme, v ti kh nng cht mang hp ph khng c trng cc cht t mi trng 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 kh nng 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 nhau ca 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 (hoc y 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 ti enzyme 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 c nh. 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 gia vng mi trng xung quanh enzyme v dung dch t do. Hot tnh xc tc ca enzyme dn ti s thay i nng ca c cht v sn phm phn ng vng mi trng vi m xung enzyme. iu ny lm xut 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 trong gel 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 xung quanh ht polymer. Cc cht khuch tn vo lp ny nh c s kt hp ca khuch tn phn t th ng v s i lu. dy ca lp ny ph thuc vo tc khuy trn ca dung mi xung quanh cc ht cha 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 cht mang 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.

CNG NGH ENZYME 25

c) nh hng ca pH ln enzyme khng ha tan

in tch ca cht mang c nh hng ng k ln pH ti u ca enzyme c nh. pH ti u ca enzyme c nh c th dch chuyn v pha pH kim hay pH acid so vi pH ti u ca enzyme ha tan. S dch chuyn pH ti u l do nh hng ca trng tnh in ca cht mang to nn. S c mt ca trng tnh in 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 sang pha pH kim so vi enzyme ha tan, ngc li nu enzyme lin kt ng ha tr vi cht mang mang in tch dng th pH ti u s chuyn dch sang pha pH acid. Cn khi lc ion ln in tch ca cht mang c lm trung ha bi cc ion tri du v pH ti u ca enzyme c nh gn ging nh ca enzyme ha tan.

3.3.2. Phng php c nh enzyme

3.3.2.1. Cht mang dng c nh enzyme

Mc ch cui cng ca vic c nh enzyme l gn c enzyme vo mt cht mang no ta c th thc 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 mang

Yu 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 lin quan 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 iu kin 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 khng c 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 lm tng 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 mng

Tt 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) v polymer 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 oxide

3.3.2.2. Phng php c nh enzyme

Trong t bo sinh vt tn ti sn c enzyme dng ha tan v c enzyme khng ha tan. Mun thc hin c 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 bc hay mt polymer. Cc cht tham gia phn ng v sn phm ca phn ng c th thm thu vo trong hoc 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 vi s c mt ca mt hay nhiu tc nhn khu mch, to ra phc chelate. Khi enzyme s c bao bc trong 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 tt hn phng php nht enzyme trong gel. Cc si s dng nht enzyme thng l nhng si nhn

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. Cellulose acetate 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 thc 1 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 s dng 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 c bit 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 cht

Ty 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 gn vo b mt ca cht mang. Kh nng hp ph ca cht mang cng cao, enzyme cng c gn nhiu vo cht mang. Do , kh nng ti s dng enzyme c nh cng cao. Trong thc t ngi ta thng s 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 ta gn enzyme vo cht mang. Lin kt ion thng khng bn bng s hp ph ca enzyme i vi cht mang. 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 hp cht kim loi. T cc enzyme s gn cht hn vo cc cht mang. Nhng cht mang thng c hot 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 phn t 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 nh

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

CNG NGH ENZYME 27

3.4.1. Sn xut fructose nh enzyme glucose isomerase

D-glucose khng th c thay th trc tip t ng sucrose bi v glucose t ngt hn. Hn na, tinh th glucose trong dung dch m c c th lm cho qu trnh kh thc hin hn. loi b nhng tr ngi ny ngi ta chuyn i t dng ng phn glucose sang fructose, s dng enzyme glucose isomerase.

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

Enzyme ngoi bo glucose isomerase c to ra bi nhiu vi sinh vt, c th l Arthobacter v Streptomyces sp. Nhu cu ph v t bo m khng ph hy enzyme lm cho glucose isomerase co gi thnh cao hn so vi cc enzyme thy gii ngoi bo khc. Hn na, glucose isomerase rt nht cm vi mt 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 isomerase di 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 iu kin c bn ca enzyme hc: ti u ha n nh nhit ca -amylase c s dng trong giai on ha lng (nhit ca giai on ny vo khong 105oC) th cn s dng ion canxi. Mt khc, nhng ion ny c ch glucose isomerase, v chng c loi b nh qu trnh trao i ion trc khi dung dch ng dextrose c cho vo thit b thc hin phn ng isomer ha.

CNG NGH ENZYME 28

3.4.2. Sn xut L-amino acid nh enzyme aminoacylase c nh

Nhu cu s dng L-amino acid trong thc phm v y hc gia tng nhanh chng. L-amino acid c tin hnh nghin cu sn xut t cng ngh vi sinh vt hay bng phng php tng hp ha hc. Cc amino 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 xut ngi 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 bi enzyme aminoacylase:

Phn ng xy ra trong mt thit b dng ct c cha enzyme aminoacylase c nh. L-amino acid mong mun c tch ra khi dng acyl D-amino acid da trn s khc bit v ha tan. Sau D-acylamino acid c racemic ha thnh dng DL-amino acid tip tc quay tr li l phn ng c cha enzyme aminoacylase 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 c hot tnh cao, d dng chun b, c kh nng ti sinh v n nh.

4. THU NHN ENZYME

4.1. Chn ngun nguyn liu

Enzyme 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 iu khng tng, nn ngi ta thng thu nhn chng t cc ngun sinh hc. Mc d enzyme c trong tt c cc c quan, m ca ng vt thc vt cng nh trong t bo vi sinh vt, song vic tch enzyme p ng yu 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 enzyme khc. Lng enzyme li thay i ty theo giai on sinh trng pht trin ca sinh vt v ty theo loi nn chng ta phi chn ngun nguyn liu thch hp cho vic chit rt v tinh ch enzyme. C ba ngun nguyn liu sinh hc c bn:

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 tch enzyme 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 xut fomat. Ngi ta cng sn xut pepsin t d dy ng vt. Nhng khc vi pepsin, renin c kh nng ng t sa cao m khng thy phn su sc casein. Renin l ch phm enzyme c gi tr ln trong cng nghip.

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 giu cht g th nhiu enzyme chuyn ha cht y. V d trong ht cy thu du c nhiu lipase, trong ht u tng c nhiu enzyme urease.

Thc ny mm cha nhiu - amylase, c khoai lang li c nhiu - amylase. Ngi ta thu c mt 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, cn fixin 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 ta thy 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 c tnh cht c o vt xa so vi ngun nguyn liu t ng vt, thc vt, cng nh s khc phc c mi 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 l ngun 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 nh enzyme c th chuyn ha mt lng ln c cht. S liu tnh ton cho bit, trong vng 24 gi, vi sinh vt 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 khc nhau, trong c nhng enzyme ng, thc vt khng tng hp c. V d cellulase, raxemase...Phn ln cc thc n nui vi sinh vt li d kim v gi r. Nhiu vi sinh vt cho enzyme thng c kh nng 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 vi sinh vt. V vy dng vi sinh vt lm ngun thu enzyme s mang li gi thnh r, thi gian nhanh v hiu qu kinh t cao.

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

CNG NGH ENZYME 30

Vi sinh vt rt nhy cm i vi tc ng ca mi trng, thnh phn dinh dng nui chng cng nh mt s tc nhn l ha, c hc khc. Do c th thay i nhng iu kin nui cy chn ging to nhng chng t bin cho ta hm lng enzyme ng k vi hot tnh xc tc cao. C th ni rng, nh ngun enzyme vi sinh vt, ngi ta c th iu khin s tng hp enzyme d dng hn cc ngun nguyn liu 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 ngun nguyn 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 tng hm lng mt s cht hoc thm mt s cht mi vo mi trng nui cy, c bit l c cht ca enzyme, th s tng hp enzyme tng ng tng ln mt cch ng k, khc thng c khi cn tng hp enzyme mi: hin tng trn gi l s cm ng sinh tng hp enzyme. Cht gy nn s cm ng sinh tng 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 nui cy 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 hot cht 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 vt tng hp enzyme mi trng c sy nh, nghin nh. Ch phm thu c dng rn - th. Mun c ch 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 pht trin 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 hp enzyme 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 vt c hot tnh enzyme cao, tng hp c enzyme cn thit v vi s lng nhiu. Cc chng c phn lp theo phng php thng thng ch tng hp mt lng nh enzyme (enzyme bn th), do cn tin hnh gy t bin bng cc phng php sinh hc, l, ha hc... to chng c kh nng siu tng hp enzyme. 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 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