CHUNG 7 K THUT SINH HA S DNG CH PHM ENZYME Enzyme l cc cht xc tc ca cc h thng sinh hc.

Chng c kh nng xc tc c bit, thng l mnh hn nhiu so vi cc cht xc tc tng hp. Tc dng xc tc ca chng mang tnh c hiu cao i vi c cht, lm tng ng k tc cc phn ng ha hc xy ra trong mi trng nc iu kin nhit v pH m du. Enzyme l mt trong cc cha kha hiu bit qu trnh hot ng sng ca t bo. Hot ng trong nhng trt t c tnh t chc cao, chng xc tc hng trm phn ng theo trt t xc nh trong cc con ng trao i cht m nh cc cht dinh dng b phn hy, nng lng ha hc c lu gi v bin i, cc i phn t sinh hc c to ra t cc cht tin thn n gin. Mt s enzyme tham gia trong qu trnh trao i cht l nhng enzyme iu ha, chu trch nhim i vi cc tn hiu trao i cht khc nhau bng cch thay i hot tnh xc tc ca chng mt cch thch hp. Thng qua hot ng ca cc enzyme iu ha cc h thng enzyme phi hp cht ch vi nhau to ra mi quan h hi ha gia cc hot tnh trao i cht cn thit cho vic duy tr s sng. 7.1. ngha ca vic nghin cu ng hc enzyme

Nghin cu ng hc enzyme l nghin cu nh hng ca cc yu t: nng c cht, enzyme, pH mi trng, nhit , cc cht km hm n tc phn ng do enzyme xc tc. Vic nghin cu ng hc enzyme s cho ta bit c cc vn sau y: - C th bit c c ch phn t ca s tc ng ca enzyme. - Cho php ta hiu bit c mi quan h v mt lng ca qu trnh enzyme. - Thy c vai tr quan trng c v mt l lun ln thc tin: khi la chn cc n v hot ng enzyme ngi ta cn phi bit nhng iu kin tt nht i vi hot ng ca enzyme, cng nh cn phi bit c cc yu t nh hng n hot ng ca chng. - L iu kin cn thit thc hin tt cc bc tinh ch enzyme, v ngi ta cn phi kim tra v mt lng bng cch xc nh c h thng hot ng ca ch phm enzyme trong cc giai on tinh ch.
7.2. ng hc cc phn ng enzyme

7.2.1. nh hng ca nng enzyme Trong iu kin d tha c cht, ngha l [S] >>[E] th tc phn ng ph thuc vo [E], v= K[E] c dng y=ax. Nh ngi ta o [E] bng cch o vn tc phn ng do enzyme xc tc. 114

C nhiu trng hp trong mi trng c cha cht km hm hay hot ha th vn tc phn ng do enzyme xc tc khng ph thuc tuyn tnh vi [E] .

Hnh 7.1. S ph thuc ca vn tc phn ng vo [E]

7.2.2. nh hng ca nng c cht [S] Ta kho st trng hp n gin nht: ch mt c cht

Gi v1 l vn tc ca phn ng to thnh phc cht ES. Gi v-1 l vn tc ca phn ng to phn ly phc cht ES to thnh E v S. Gi v2 l vn tc ca phn ng to thnh E v P (sn phm). v1 = k1[E][S] v-1 = k-1[ES] v2 = k2[ES] 115

Khi h thng t trng thi cn bng ta c: k-1[ES]+k2[ES] = k1[E][S] (k-1+k2)[ES] = k+1[E][S] (2) l nng ban u: Gi E0 [E0]=[E]+[ES]=>[E]=[E0]-[ES] (3) Thay tr s [E] t (3) vo (2) ta c: (k-1+k2)[ES] = k1([E0]-[ES]) [S] k1 [E0] [S] [ES] = -------------k-1+ k2+k1[S] Nu t Km= k-1+k2/ k1 (Km: gi l hng s Michalis Menten) Ta c: [ES] = [E0][S]/ Km+[S] Mt khc vn tc phn ng to thnh sn phm P l: V = k2[ES] Thay [ES] bng gi tr trn ta thu c: k2[E0] [S] v = ----------------- (4) Km + [S] Qua y ta thy nng enzyme cng cao th vn tc phn ng enzyme cng ln. Vn tc t cc i khi ton b enzyme lin kt vi c cht, ngha l: Vmax= k2[E0] Thay vo phng trnh (4) ta c: [S] v = Vmax ------------- (5) Km+ [S] Phng trnh (5) gi l phng trnh Michelis Menten Km gi l hng s Michelis Menten c trng cho mi enzyme. Km c trng cho i lc ca enzyme vi c cht, Km c tr s cng nh th i lc ca enzyme vi c cht cng ln, ngha l vn tc ca phn ng do enzyme xc tc cng ln.
ngha thc tin ca hng s Michaelis l ch n chnh l gi tr ca nng c cht khi tc phn ng bng tc ti a. Thay V v v bng cc con s tng ng 1 v

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0,5 vo phng trnh trn, ta s thy r iu . Nh vy, Km c o bng n v nng , tc mol/l. Hng s Michaelis l mt hng s rt quan trng. N xc nh i lc ca enzyme vi c cht. Km cng nh th i lc ny cng ln, tc phn ng cng cao v tc ti a V t gi tr nng c cht cng thp. Trn c s phng trnh Michaelis-Menten, bng cch xy dng ng biu din s ph thuc ca v vo [S] v bng th xc nh tc ti a V ta c th tm thy gi tr ca [S], v = V/2, tc gi tr ca Km (hnh 7.2).

Khi tng [S] th v phn ng tng, tng [S] n mt gi tr no th v t n gi tr vmax v s khng tng na nu ta vn tip tc tng [S]. Khi Km = [S] th v0 =1/2 Vmax Nm 1934. Lineweaver v Burk, trn c s ca phng trnh (5) nghch o bin thnh dng ng thng y = ax+b, n c ngha ln i vi vic nghin cu km hm enzyme.

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Trong nhiu phn ng do enzyme xc tc c 2 hay nhiu c cht, v d hexokinase xc tc phn ng: ATP + glucose hexokinase ADP + glucose 6 phosphate C ch enzyme xc tc cho phn ng 2 c cht c th nh sau: a/ C ch to phc 3 thnh phn

b/ C ch khng to phc 3 thnh phn

y l trng hp c cht th 2 (S2) ch kt hp vo enzyme ( trng thi E) sau khi P1 c to thnh.

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Vn tc ca phn ng trong trng hp ny c th c phn bit qua hng s Michalis-Menten i vi mi c cht. Qua nghin cu ng hc cho thy:

Phn ng 2 c cht (bisubstrate) thng vn chuyn 1 nguyn t hay 1 nhm chc t c cht ny n c cht khc. Khi cho S2 khng i, ng biu din tc trong c hai trng hp

7.2.3. nh hng ca cht km hm (inhibitior) L cht c tc dng lm gim hot hay lm enzyme khng cn kh nng xc tc bin c cht thnh sn phm. * Cch 1: Km hm cnh tranh (Competitive inhibition)

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Trong trng hp km hm cnh tranh l c cht v cht km hm u tc dung ln trung tm hot ng ca enzyme, Cht km hm chon ch ca c cht enzyme.

Khi c cht d tha, nng cht km hm thp th c th loi b tc dng ca cht km hm, cn nng c cht thp v nng cht km hm cao th li c tc dng km hm hon ton.

Ngi ta thy km hm nh vy phn ln gia cht km hm v c cht c s tng ng v mt ha hc. v d: malic acid c cu trc gn ging vi succinic acid nn km hm cnh tranh enzyme succinatdehydrogenase, l enzyme xc tc cho s bin i succinic acid thnh fumaric acid.

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Trng hp c bit ca km hm cnh tranh l km hm bng sn phm. Trng hp ny xy ra khi mt sn phm phn ng tc dng tr li enzyme v chon v tr hot ng phn t enzyme. ng thng c cht km hm th c xin ln hn v ct trc tung mt im l 1/Vmax * Cch 2: Km hm phi cnh tranh (Uncompetitive inhibition) c trng ca kiu km hm ny l cht km hm ch lin kt vi phc hp ES, m khng lin kt vi enzyme t do.

* Cch 3: Km hm hn tp (Mixed inhibition) 121

Trong , cht km hm khng nhng lin kt vi enzyme t do m cn lin kt vi c phc hp ES to thnh phc hp EIS khng to c sn phm P. Hin tng km hm ch ph thuc vo nng cht km hm. Tc cc i o c khi khng c mt cht km hm l cao hn khi c mt cht km hm. Gi tr Km thay i khng ging nh trong trng hp cnh tranh. Tng t nh trn ta c phng trnh :

Cc gi tr , c nh ngha nh trn. Trng hp = gi l khng cnh tranh (noncompetitive).

Bng 7.1. nh hng ca kiu km hm ln Vmax v Km

Cnh tranh
(Competitive)

Phi cnh tranh

(Uncompetitive)

Hn tp

Khng cnh tranh

(Mixed)

(Noncompetitive)

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Trng hp km hm enzyme bng nng cao ca c cht gi l km hm c cht nh km hm urease khi nng ure cao, ngoi ra cn c cc enzyme khc nh lactatdehydrogenase, carbonxypeptidase, lipase, pyrophotphatase, photphofructokinase (i vi ATP). Nguyn nhn ca nhng hin tng ny cn cha c bit r. c th l: + Tn ti nhiu trung tm lin kt vi c cht bng cc i lc khc nhau. Khi nng c cht thp th enzyme c th ch lin kt vi mt phn t c cht, cn khi nng c cht cao n lin kt vi nhiu c cht dn n hnh thnh phc hp ES khng hot ng. + C cht cng c th c lin kt nh nhng v tr c bit ca enzyme. l mt nhm enzyme quan trng (enzyme d lp th) bn cnh trung tm xc tc cn c trung tm iu chnh. + C cht c th lin kt vi mt cht hot ha v bng cch ny n tch khi E. + C cht c th chon ch (ngn cn) mt cofactor hay mt coenzyme. + C cht c th nh hng n ion lc ca mi trng v qua lm mt i tnh chuyn ha ca enzyme. 7.2.4. nh hng ca cht hot ha (activator) L cht lm tng kh nng xc tc chuyn ha c cht thnh sn phm. Thng thng l nhng cation kim loi hay nhng hp cht hu c nh cc vitamin tan trong nc. V d: Mg++ hot ha cc enzyme m c cht c phosphoryl ha nh pyrophosphatase (c cht l pyrophosphate), adenosintriphosphatase (c cht l ATP). Cc cation kim loi c th c tnh c hiu, tnh i khng v tc dng cn tu thuc vo nng . Tnh cht hot ha ca cc cation kim loi: + Mi cation kim loi hot ha cho mt kiu phn ng nht nh. + Cation kim loi c tnh c hiu tng i hay tuyt i. + Cation kim loi c th c s i khng ion. + Ph thuc nng cation kim loi . + Cation kim loi lm thay i pH ti thch. 123

+ Ph thuc bn cht cation kim loi. 7.2.5. nh hng ca nhit Ta c th tng vn tc ca mt phn ng ha hc bng cch tng nhit mi trng, hin tng ny tun theo quy lut Vant -Hoff. iu ny c ngha khi tng nhit ln 100C th tc phn ng tng ln 2 ln.

i vi phn ng do enzyme xc tc cng c th p dng c quy lut ny nhng ch trong mt phm vi nht nh,v bn cht enzyme l protein.Khi ta tng nhit ln trn 40-500C xy ra qu trnh ph hy cht xc tc. Sau nhit ti thch tc phn ng do enzyme xc tc s gim. Nh tn ti nhit ti u ngi ta phn bit phn ng ha sinh vi cc phn ng v c thng thng. Mi enzyme c mt nhit ti thch khc nhau, phn ln ph thuc ngun cung cp enzyme, thng thng trong khong t 40-600C, cng c enzyme c nhit ti thch rt cao nh nhng enzyme ca nhng chng a nhit. 7.2.6. nh hng ca pH S phn li khc nhau ca mt phn t protein cc gi tr pH khc nhau lm thay i tnh cht ca trung tm lin kt c cht v hot ng phn t enzyme, dn n gi tr xc tc khc nhau ph thuc vo gi tr pH. Nh bit mi enzyme c mt pH ti thch, mi enzyme c ng biu din nh hng pH ln vn tc ca phn ng do enzyme xc tc c dng nh hnh 7.9:

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nh hng ca gi tr pH n tc dng enzyme c th do cc c s sau: a/ Enzyme c s thay i khng thun nghch phm vi pH cc hp. b/ hai sn ca pH ti thch c th xy ra s phn ly nhm prosthetic hay coenzyme. c/ Lm thay i mc ion ha hay phn ly c cht. d/ Lm thay i mc ion ha nhm chc nht nh trn phn t enzyme dn n lm thay i i lc lin kt ca enzyme vi c cht v thay i hot tnh cc i. Nh xc nh Vmax v Km ph thuc gi tr pH cho php nhn nh li bn cht ca cc nhm tham gia vo lin kt c cht v qu trnh t xc tc. 7.2.7. Cc yu t khc + nh sng: C nh hng khc nhau n tng loi enzyme, cc bc sng khc nhau c nh hng khc nhau, thng nh sng trng c tc ng mnh nht, nh sng c tc ng yu nht. nh sng vng t ngoi cng c th gy nn nhng bt li, enzyme trng thi dung dch bn hn khi c kt tinh dng tinh th, nng enzyme trong dung dch cng thp th cng km bn, tc ng ca tia t ngoi s tng ln khi nhit . V d: di tc ng ca tia t ngoi nhit cao, enzyme amylase nhanh chng mt hot tnh. + S chiu in: in chiu vi cng cng cao th tc ng ph hy cng mnh. Tc ng s mnh hn i vi dch enzyme c nng thp. C th do to thnh nhng gc t do, t tn cng vo phn ng enzyme. + Sng siu m: Tc ng rt khc nhau i vi tng loi enzyme, c enzyme b mt hot tnh, c enzyme li khng chu nh hng. * Nhn xt chung: bn ph thuc vo trang thi ca tn ti enzyme, cng tinh khit th enzyme cng km bn, dch cng long th bn cng km, tc ng ca mt s ion kim loi trong dch vi nng khong 10-3M nh Ca++ lm tng tnh bn. Enzyme allosteric (d lp th, d khng gian) 125

Cho n nay, ngi ta m t enzyme m hot tnh enzyme ph thuc nng c cht khng c dng hyperbol m c dng sigmoid l enzyme d lp th (hnh 7.10). i vi enzyme ny, khi nng c cht thp th tc phn ng tng chm, sau tip tc tng nng th tc nhanh chng t gi tr cc i. Nh ta bit, enzyme tun theo ng hc Michalis-Menten th 1 hay nhiu c cht cng ch lin kt vo 1 v tr trn phn t enzyme, iu ny s dn n enzyme bo ha c cht. Cn enzyme c ng cong tc sigmoid ch xut hin khi enzyme l mt oligomer, nn c th lin kt vi nhiu phn t c cht. iu ny c ngha l trn enzyme d lp th c nhiu trung tm lin kt, mi monomer c 1 trung tm lin kt.

Hnh 7.10. Bin thin vn tc phn ng theo nng c cht

Ngi ta cho rng, trong trng hp ny c tnh hp tc gia cc v tr lin kt c cht trong phn t enzyme oligomer. Cc enzyme oligomer ny c Monod gi l enzyme d lp th (allosteric). ng cong tc sigmoid c th b cht iu ha (modulator) y v pha tri hay phi. Cht iu ha dng tc lm tng i lc ca enzyme allosteric vi c cht, ngc li l cht iu ha m. Cc cht iu ha c th lm nh hng khc nhau n cc thng s ng hc, lm thay i gi tr ring l mt trong hai gi tr Km hay Vmax .

Hnh 7.11. Minh ha khi c modulator

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7.3. Enzym c nh 7.3.1. Gii thiu chung Enzyme khng ho tan hay cn c gi l enzyme c nh (immobilized enzyme) thng l nhng enzyme ho tan c gn vo mt cht mang bng nhiu k thut khc nhau. Nh qu trnh gn ny m enzyme t trng thi ho tan chuyn sang khng ho tan. Khi chuyn t trng thi ho tan sang trng thi khng ho tan, enzyme khng ho tan c nhng u im nh sau: Enzyme khng ho tan c th c s dng nhiu ln, hot tnh ca enzyme khng ho tan t thay i trong nhng ln ti s dng. Nh ta c th ti s dng nhiu ln v s lm gimchi ph cho vic sn xut enzyme. y l u im ln nht ca vc thu nhn v ng dng enzyme khng ho tan. Enzyme khng ho tan khng ln vo sn phm cui ca phn ng, do chng ta khng phi chi ph cho vic tch enzyme ra khi sn phm. Sn phm cui cng thu c s c coi nh l sn phm tng i sch. Dng enzyme c nh c th dng nhanh chng phn ng khi cn thit bng cch tch n ra khi c cht. C IM CA ENZYM KHNG TAN Khi gn enzyme ho tan vo mt s cht mang, enzyme ny c nhng c im khc hn enzyme ho tan. 1- Hot tnh ca enzyme khng ho tan thng nh hn hot tnh ring ca enzyme ho tan cng loi. S d c s thay i v hot tnh ring ca enzyme khng ho tan l do nhng nguyn nhn sau: - 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 xy ra s 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. - Do enzyme b nht vo mt gel. Khi ta nht 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 nhiu kh khn hn so vi s tip xc gia c cht v enzyme t do. 2- Enzyme khng ho tan hon ton tun theo nh lut Michaelis-Menten. Tuy nhin, trong phn ng gia c cht vi enzyme khng ho tan c nhng sai khc nht nh: - C th xy ra hin tng cnh tranh c cht vi enzyme v cht mang.

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- Hin tng cn tr s khuch tn c cht v cc sn phm ca phn ng lm gim tc phn ng. 3- Enzyme khng ho tan c tnh bn nhit hn enzyme ho tan cng loi v chng c bo v bi cht mang. 4- Enzyme khng ho tan c xu hng chuyn dch pH ti u sang kim hoc acid so vi pH ti u ca enzyme ho tan ch khng trng vi pH ti u ca enzyme ho tan cng loi. 5- Enzyme khng ho tan c kh nng bo qun tt hn enzyme ho tan cng loi 6- C th ti s dng enzyme khng ho tan nhiu ln, trong khi enzyme ho tan ch c th s dng mt ln. c im ny rt c ngha v mt kinh t khi ta tin hnh cc phn ng theo quy m cng nghip Cht mang dng c nh enzym a) Nhng yu cu ca mt cht mang l tng. Mt cht mang l tng cn c nhng tnh cht sau: - Cht mang phi r tin - Phi c tnh cht c l bn vng, n nh - Phi bn vng, khng tan trong mi trng phn ng, khng lm mt hot tnh enzyme, khng gy ra nhng phn ng hp ph khng c hiu. - Cht mang phi c tnh khng khun cao. - Phi c trng tt, c din tch b mt tip xc ln - Cht mang c th c cu trc l xp, siu l, dng ht, dng mng b) Phn loi cht mang Cht mang dng c nh enzyme c chia lm hai nhm: cht mang polymer hu c v cht mang v c. Cht mang polymer hu c gm hai nhm: polymer tng hp v polymer t nhin. 7.3.2. Cc phng php c nh enzym Theo k thut ch to enzyme c nh, enzyme c gn vo mt vt mang no

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. 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. - 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 ph 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).

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

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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).

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:

Enzyme lin kt vi cht mang cha c hot ha

Enzyme lin kt vi cht mang c hot ha

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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.

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. -Hot ha bng phng php azit:

Azit

Enzyme c nh

Cc enzyme nh trypsin, DNA-ase, chimotrypsin, bromelin c c nh bng phng php ny. -Hot ha bng phng php carbodiimit:

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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 bng cch dng aldehyde glutaric 2% lin kt cc phn t enzyme v gn chng vo aminoethylcellulose. b/ Phng php gi enzyme trong khun gel. 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, hydroxyethyl2-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

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 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 mi khng 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 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.

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

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hot tnh enzyme c nh trn cc cht mang anionit, cationit v trung tnh s khc nhau.

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ng dng ca enzyme c nh. / 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 carboxymethylcellulose lm ng t sa thay cho renin t tin; trn

- Enzyme rasemase c nh c s dng chuyn ton b D- aminoacid thnh Laminoacid tng ng, 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. - 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 60-70o, lm gim ng k kh nng nhim khun ca thit b enzyme. 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. 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 tc;

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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

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Filename: Chng 7.doc Directory: D:\DH CNTP\Mon Giang day\Mon K thut thc phm 3\Soan\Mon KTTP 3\Giao ttrinh in nop Template: C:\Documents and Settings\welcome\Application Data\Microsoft\Templates\Normal.dot Title: Chng 7: K thut ha sinh s dng ch phm enzyme Subject: Author: User Keywords: Comments: Creation Date: 9/5/2010 11:27:00 PM Change Number: 13 Last Saved On: 3/13/2011 3:07:00 PM Last Saved By: User Total Editing Time: 1,406 Minutes Last Printed On: 10/16/2011 10:55:00 PM As of Last Complete Printing Number of Pages: 25 Number of Words: 5,336 (approx.) Number of Characters: 30,420 (approx.)