Enzyme

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XVII. ENZYME C NH. 1. ngha ca enzyme c nh. Trong nhng nm cui ca th k 20 bt u ph bin k thut c nh enzyme. Enzyme ha tan sau khi s dng thng ln vo cng vi sn phm khng tch ra c. Nu tch ra th enzyme b mt hot tnh, v vy vi mt lng enzyme nht nh ch c th s dng c mt ln. S dng enzyme c nh c mt s u im sau: - Mt lng enzyme c th s dng lp i lp li c nhiu ln trong mt thi gian di; - Enzyme khng ln vo trong sn phm, do trnh c nh hng khng tt n cht lng sn phm; - Dng enzyme c nh c th dng nhanh chng phn ng khi cn thit bng cch tch n ra khi c cht. 2. Cc phng php iu ch enzyme c nh. Theo k thut ch to enzyme c nh, enzyme c gn vo mt vt mang no . Nhng vt mang nh vy c th l cellulose v cc dn xut ca n, cc ht knh xp, gel polyacrylamide, sephadex, collodium, tinh bt, cc loi allumosilicate v oxide kim loi...V nguyn tc, c 3 phng php iu ch cc enzyme c nh: - Gn bng lin kt ng ha tr phn t enzyme vo cht mang khng ha tan hoc gn cc enzyme vi nhau to nn i phn t khng ha tan (hnh 16);

Hnh 16. S iu ch enzyme c nh bng cch nh mch ngang nh cc tc nhn lng chc hoc a chc.

GS.TS. Mai Xun Lng

Khoa Sinh hc

Enzyme

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- nh enzyme ln b mt cht mang hoc vo trong lng khun gel c kch thc l kh nh gi enzyme, cn cc cht khc qua li t do (Hnh 17);

Hnh 17. Hp h enzyme trn b mt cht mang (a) v trong lng cht mang (b)

- Hp ph enzyme ln trn cc cht mang khng ha tan c mang hoc khng mang in tch (Hnh 18).

Hnh 18. S hp ph enzyme trn cht mang khng c v c in tch.

a/ Phng php gn enzyme bng lin kt ng ho tr. a s enzyme c nh thu c bng phng php ny. Vi phng php ny c th thu enzyme c nh bng hai cch. Kt hp phn t protein enzyme vo cht mang khng ha tan. Cc cht mang gn enzyme phi tha mn nhng yu cu sau: C ha tan thp v bn vng i vi cc tc ng c hc v ha hc; Khng gy tc ng km hm n hot tnh enzyme; Khng hp ph phi chn lc i vi cc protein khc; Cht mang tt hn c l c tnh ho nc, v cht mang k nc c th gy tc dng c ch enzyme lin kt;

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Khoa Sinh hc

Enzyme Vic gn enzyme s c hiu qu hn khi in tch ca enzyme v ca cht mang c du ngc nhau.

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Cc cht mang loi ny thng l polypeptide, cc dn xut ca cellulose v dextran (DEAE-cellulose, CM-cellulose, DEAE- sephadex, CM, sephadex), agarose v cc polimer tng hp khc nh polyacrilamide, polystyrol, polyamide v cc cht v c nh silicagen, bentonit, hydroxide nhm v.v... Cht mang ph bin hn c l dextran c lin kt ngang (Sephadex) v agarose ht (sepharose). Cc hai loi cht ny u c cu trc l xp khin cho cc phn t ln c th xm nhp vo gel mt cch d dng. Poliacrylamide cng l cht mang rt tin li vi c bn vng c hc v ha hc cao. Cht mang v c thng rt bn vi nhit, c hc, vi dung mi hu c v vi cc vi sinh vt, nht l khng b bin i cu hnh ca khun khi thay i tnh cht ca mi trng chung quanh. Nhiu dn liu cho thy rng enzyme nh vi khun v c khi bo qun thng bn vng hn enzyme gn vi khun polymer hu c. Tham gia to thnh cc lin kt ng ha tr c th c cc nhm chc ca phn t protein enzyme nh nhm - v -carboxyl COOH ca acid asparaginic v acid glutamic, nhm -NH2 ca lysine, nhm -SH ca cysteine, vng phenol ca tyrosine, nhn imidasol ca histidine, cc nhm OH ca serine, threonine, nhm guanidine ca arginine v imidasol ca tryptophan. Qu trnh kt hp enzyme s xy ra mt cch trc tip khi cht mang c cha cc nhm chc c kh nng lin kt trc tip ni trn trong phn t protein enzyme. V d gc anhydride maleic lin kt vi nhm -NH2 ca lysine. Trong cc trng hp khc s lin kt gia cht mang v protein enzyme ch xy ra sau khi cht mang c hot ha . Vic hot ha s b cht mang c th c thc hin bng nhiu cch. V d, cc nhm hydroxyl ca dextran hoc cc polyoside khc c th c hot ha bng bromur cyan (BrCN). Sau cht mang c hot ha mi lin kt vi enzyme (hnh 19).

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Enzyme

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Hnh 19. Gn enzyme vi vt mang c hot ha bng bromur cyan (BrCN).

Cc cht mang c cha nhm amin nh aminobenzoylcellulose, polyaminostyrol c th c hot ha bng phn ng diazo. V d, polyaminostyrol trc ht c diazo ha bng natri nitrit trong mi trng acid thnh mui diazo, sau mi kt hp vi enzyme: R NH2
R: Gc alkyl, aryl

NaNO2/HCl

R N2+X

Mui diazo ca cht mang X: Gc acid

OH R N2+X + Enzyme
cht mang ha

OH

RN=N Enzyme
Enzyme lin kt vi c hot

Enzyme lin kt vi cht mang cha c hot ha

Phn ng kt hp enzyme c tin hnh nhanh chng trong iu kin nhit thng v dung dch nc trung tnh. Nu cht mang c cha nhm amin c th hot ha bng cch cho tc dng vi phosgen hoc tiophosgen to thnh dn xut isosianat hoc izothyosianat. Cc nhm isosianat hoc izothyosianat pH trung tnh s lin kt d dng vi nhm -NH2 ca lysine. Cl

O=C
R NH2 + Cl Cl S =C Cl R N = C = O + H2N - E

R N = C = O + HCL

R N = C = S + HCl

R NH CO NH - E

Bng phng php ny ngi ta iu ch c cc dn xut enzyme c nh nh trypsin, chimotrypsin, -, -amylase, glucoamylase. Nu cht mang c cha nhm COOH nh CM-cellulose hoc nha tng hp th cn c hot ha trc khi gn kt vi enzyme bng cc phng php azit, carbodiimit.

GS.TS. Mai Xun Lng

Khoa Sinh hc

Enzyme Hot ha bng phng php azit: O

CM-cellulose

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H2N NH2
Hydrazin

O O CH2 C NH NH2
Hydrazit

- O CH2 C O CH3

N aN O 2 E

O - O CH C N = N- NH 2 +
Azit

E NH2

O - O CH2 C NH
Enzyme c

nh

Cc enzyme nh trypsin, DNA-ase, chimotrypsin, bromelin c c nh bng phng php ny. Hot ha bng phng php carbodiimit: - O CH2 COOH + -OH
CM-Cellucose

N=C=N N,N-Dicyclohexylcarbodiimide

+ H2N E
Enzyme

- O CH2 CO-NH E + NH - C O NH - OH Dixyclohexylure V phn ng xy ra trong mi trng acid yu (pH=5) nn phng php ny c bit thun li i vi cc enzyme c tnh acid nh pepsin. Kt hp ng ha tr gia cc phn t enzyme vi cht mang. C nh enzyme bng cch lin kt ng ha tr c thc hin bng nhiu cch khc nhau: 1/ Lin kt ng ha tr c to ra gia cc nhm phn ng ca vt mang v enzyme; trong mt s trng hp vt mang cn c hot ha s b; 2/ Cc phn t enzyme sau khi c hp ph trn cht mang hoc nm trong lng phn t cht mang lin kt vi nhau nh tnh lng chc hoc tnh a chc ca cht phn ng. Ngi ta thng dng aldehyde glutaric gn cc nhm amin ca lysine cc phn t enzyme. V d, trypsin c nh trypsin c iu ch bnh cch dng aldehyde glutaric 2% lin kt cc phn t enzyme v gn chng vo aminoethylcellulose. b/ Phng php gi enzyme trong khun gel.

GS.TS. Mai Xun Lng

Khoa Sinh hc

Enzyme

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gi enzyme vo khun gel ngi ta tin hnh trng hp ha hc cc gel khi c mt ng thi enzyme. Sau khi hon thnh qu trnh trng hp enzyme b gi chc trong gel. Gel c enzyme c th nghin nh bng cch p qua ry c l nh v sy kh nhit thp. Dn xut enzyme loi ny ln u tin do Bernfeld (1933) thu c bng cch trng hp acrylamide vi N,N-methylenbisacrylamide. Phng php ny thng dng do cht trng hp thu c d to thnh dng ht v ty thuc iu kin tin hnh m gel c th c xp khc nhau. Sau y l mt s phng php gi enzyme. Cc gel c th c hnh thnh t cc polymer tng hp nh acrylamide, hydroxyethyl-2-metacrylate, to phc cng cua bng ethyleneglycol-dimetacrylate, polyvinyl, polyuretan. Enzyme c ha tan hoc phn tn trong mt dung dch monomer v sau trng hp trong s c mt ca mt hay nhiu tc nhn to phc cng cua. Gel c th ct thnh mng t trn mt gi th rn, hoc cng c th nghin thnh bt sau khi loi tr nc. Alginate v caraghenan ly t rong bin thng c kh nng to gel rt tt v rt thun li bao gi cc enzyme hoc t bo nguyn vn. Cc enzyme cng c th b nht trong cc l nh ca cc si tng hp. Vi cch ny ngi ta cho dch lng chy bn trong si, do hn ch c s phn cc b mt v s bt lp thng gp vi cc mng. Mt nh tng ca cellulose triacetate trong methylene chlorua v enzyme trong dung dch m c cha glycerol c p qua mt khun lc di p sut nit. Cc si i ra khi khun c nhng vo mt ci b ng t c cha toluel, sau c lm kh trong chn khng. Cc si ny bn vi acid yu hoc kim yu, lc ion cao v chu c mt s dung mi hu c. Tuy nhin phng php ny ch thch hp i vi nhng enzyme kh b mt hot tnh trong cc dung mikhng ha ln vi nc. Phng php gi enzyme trong cc bao vi th (microcapsul). Enzyme c gi trong cc bao vi th c mng bn thm c to ra t cc polymer c kch thc l nh ngn cn s khuch tn ca enzyme ra ngoi v ln c cht i vo v sn phm phn ng i ra d dng. C nhiu phng php gi enzyme trong microcapsul. Sau y l mt trong cc phng php . Cho mt dung dch long cha enzyme v mt

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Khoa Sinh hc

Enzyme

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monomer a nc trong mt dung mi hu c khng ha ln trong nc to nh tng, sau thm mt monomer k nc gy phn ng trng hp to ra mt mng bao quanh nhng git lng nh. Thng phi thm vo mt tc nhn hot ng b mt lm bn nh tng cng nh iu chnh cc capsul c kch thc mong mun t 1 n 100 m. Phng php tin polymer gi cc cht xc tc sinh hc. C nh enzyme bng phng php ny c nhng u im nh sau: - Qu trnh gi rt n gin trong nhng iu kin nh nhng; - Cc cht tin trng hp khng cha cc monomer vn c th gy nh hng xu n enzyme c gi; - Cu trc li ca gel c th c iu chnh bng cch dng cc tin polymer c chiu di chui bt k; - Cc tnh cht ha l ca gel (nh s cn bng gia tnh ho nc v tnh k nc, bn cht ion) c th nh hng trc bng cch chn cc tin polymer thch hp vn c tng hp ha hc trong iu kin vng mt enzyme. Di y l mt v d v phng php tin polymer. l phng php to lin kt cho gia cc tin polymer bng cch chiu tia cc tm gi enzyme. Khi chiu tia cc tm gn ln dung dch c cha cht tin polymer v enzyme th s to ra cc lin kt cho gia cc gc ca tin polymer v mt gel c hnh thnh bao ly enzyme. S gi enzyme bng phng php ny c th tin hnh iu kin nh nhng, trnh c cc thay i pH qu kim hoc qu acid, thi gian li rt nhanh, ch trong vng t 3-5 pht. Vi phng php ny c th gi enzyme, t bo vi sinh vt hoc cc bo quan. c/ C nh enzyme bng phng php hp ph trn cc cht mang c hoc khng c in tch. Vi phng php ny enzyme c hp ph trn cc cht c hot tnh b mt nh than hot tnh, cellulose, tinh bt, dextran, collagen, albumin, agarose, chitin, polyacrylamide, nilon, polystyrol v.v...v mt s nha trao i ion, silicagen, thy tinh. Trong trng hp cht mang khng c l enzyme c nh vo cht mang thnh tng lp trn b mt; khi cht mang c l th cc phn t enzyme s xm nhp vo trong cc l. 3. Mt s c tnh ca enzyme c nh. Vic c nh lm thay i ng k nhiu tnh cht ca enzyme nh bn, tnh c hiu, c im ng hc. Thng thng vic c nh lm tng GS.TS. Mai Xun Lng Khoa Sinh hc

Enzyme ng k bn ca enzyme. V d, c nh protease lm tng bn vi nhit gp chc nghn ln.

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Sau khi c c nh khng nhng bn vi nhit ca enzyme tng ln m phm vi nhit ti thch ca enzyme cng c m rng. Nhit cao lm cho enzyme b bin tnh, cu trc bc ba ca enzyme b ph v v enzyme b mt hot tnh. Khi enzyme c c nh nh nhiu lin kt gn vi cht mang qu trnh bin tnh s b ngn cn, lm cho enzyme chu c nhit cao. Trong trng hp i vi cc enzyme thy phn protein, v d papain v trypsin s c nh lm ngn cn mt phn hoc hon ton hin tng t phn. iu ny rt c ngha thc t v n gip lm tng thi gian lm vic ca enzyme. S bin i c im ng hc ca enzyme do vic c nh gy ra c th c minh ha qua v d i vi enzyme glutamate dehydrogenase: i vi enzyme c nh trn mng collagen hng s Michaelis dnh cho acid glutamic tng 6 ln, tc dng hot ha ca ADP cng tng ln ng k. Mt trong cc nguyn nhn lm bin i tnh cht ca enzyme khi chng c c nh l s thay i ca phn ng mi trng. Enzyme thng c c nh trn cc cht mang c in tch. V d mt cht trao i anion mang in tch m c bao quanh bi mt mi trng giu proton, v v th pH trong cc lp nm k st vi enzyme c c nh s thp hn so vi pH ca mi trng dung dch. iu lm cho ng cong ph thuc pH ca hot tnh enzyme c nh trn cc cht mang anionit, cationit v trung tnh s khc nhau. 4. ng dng ca enzyme c nh. a/ Trong cng nghip. - T nm 1969 Wilson xy dng thnh cng mt xng thc nghim sn xut lin tc glucose bng glucoamylase c nh; - T 1971 ngi ta thnh cng trong vic dng chimotrypsin c nh trn carboxymethylcellulose lm ng t sa thay cho renin t tin; - Enzyme rasemase c nh c s dng chuyn ton b Daminoacid thnh L- aminoacid tng tng, lm tng gi tr dinh dng ca sn phm ln gp i. - Lm trong nc tri cy bng cch x l enzyme phn gii pectin hay thy phn protein trong bia bng protease hon ton c th c thc hin trong cc ct cha cc enzyme c nh tng ng.

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Khoa Sinh hc

Enzyme

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- C nh enzyme glucoisomerase trong ct hon ton c hiu qu i vi vic ngn nga s chuyn ha glucose thnh fructose, lm cho ngt ca nc gii kht b gim. Nh c nh enzyme glucoisomerase vic thu nhn xir gluco-fructose ngy nay tr thnh mt quy trnh cng nghip quan trng v ph bin. chu nhit cao ca enzyme ny cho php thc hin qu trnh sn xut nhit 60-70 gim ng k kh nng nhim o, lm khun ca thit b enzyme. b/ Trong y hc. - Urease gn trong vi tiu cu c s dng c hiu qu loi tr urea ca mu trong thn nhn to; - Vi tiu cu cha catalase c th thay th mt cch c hiu qu s catalase b thiu trong c. - a vi tiu cu c gn enzyme L-asparaginase vo c th c kh nng c ch s pht trin ca mt s u c tnh v s pht trin ca nhng u ny ph thuc vo s c mt ca L-asparagin. c/ Trong phn tch ha sinh. Glucoamylase gn ng ha tr vi polystyrol c dng xc nh t ng glucose; in cc urease c nh dng xc nh t ng urea trn dng lin t c; in cc alcoholoxydoreductase c nh c dng xc nh methanol, ethanol trong dung dch nc. C nh enzyme to ra kh nng nghin cu hng lot cc vn l thuyt ca enzyme hc. Vi d c nh enzyme c th c s dng ngn cn s phi hp ngu nhin ca cc phn di n v ca cc enzyme oligomer. S dng vic c nh c th gii quyt vn liu mt enzyme dng cc phn di n v c hot tnh hay khng. Nu cc phn di n v trng thi c nh c hot tnh th qua so snh hot tnh ca enzyme vi hot tnh ca cc oligomer c nh s thu c nhng thng tin c ga tr v vai tr ca s tng tc gia cc phn di n v trong vic thc hin chc nng ca enzyme. Ngy nay ta bit r rng phn ln enzyme ni bo hot ng trong mi trng tng t nh gel hoc chng c gi trong mng ti th, lc lp hoc cc c quan t khc. V vy, cc enzyme c nh c th l m hnh tt nghin cu hot ng ca enzyme. K thut c nh khng ch dng cho cc ch phm enzyme m c th dng cho cc t bo vi sinh vt nguyn vn. c nh cc t bo vi sinh vt GS.TS. Mai Xun Lng Khoa Sinh hc

Enzyme

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ngi ta thng dng gel polyacrylamide, cellulose, caraganine v cc vt liu khc. ng lu l trong nhiu trng hp hot tnh enzyme ca cc t bo c nh cao hn nhiu so vi cc enzyme t do. Cc t bo c nh thm ch c th thc hin c cc qu trnh sinh ha tinh vi v phc tp. V d cc t boRhisobium lupini nt sn cy lupin vng gi c rt lu hot ca tnh c nh m ca chng.

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