Bi Bo Co Ha Sinh

GVHD: Nguyn Th Bch Hng

Trng: Cao ng T Thc c Tr Khoa: Cng Ngh Sinh Hc _ Mi trng Lp: 10MT

BI BO CO MN: HA SINH
TI: CC LU KHI THAO TC VI ENZIME V QUY TRNH TCH CHIT V LM SCH ENZIME

MC LC I. M U II. MT S LU KHI THAO TC VI ENZYME III. QUY TRNH TCH CHIT V LM SACH (Tinh Ch) ENZYME IV.KT LUN V. TI LIU THAM KHO VI. DANH SACH NHM

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I. M U
1.nh Ngha Trong c th sng (cc t bo) lun lun xy ra qu trnh trao i cht. S trao i cht ngng th s sng khng cn tn ti. Qu trnh trao i ca mt cht l tp hp cc quy lut ca rt nhiu cc phn ng ha hc khc nhau. Cc phn ng ha hc phc tp ny c lin quan cht ch vi nhau v iu chnh ln nhau. Enzyme l cc hp cht protein xc tc cho cc phn ng ha hc . Chng c kh nng xc tc c hiu cc phn ng ha hc nht nh v m bo cho cc phn ng xy ra theo mt chiu hng nht nh vi tc nhp nhng trong c th sng.Chng c trong hu ht cc loi t bo ca c th sng. Chnh do nhng tc nhn xc tc c ngun gc sinh hc nn enzyme cn c gi l cc cht xc tc sinh hc (biocatalysators) nhm phn bit vi cc cht xc tc ha hc.Enzyme hc l khoa hc nghin cu nhng cht xc tc sinh hc c bn cht protein. Hay ni cch khc, enzyme hc l khoa hc nghin cu nhng tnh cht chung, iu kin, c ch tc dng v tnh c hiu ca cc enzyme. II. MT S LU KHI THAO TC VI ENZYME
II.1. .Phng php Nghin Cu Enzyme C hai loi phng php dng nghin cu cc phn ng enzyme. Mt trong hai phng php l la chn cc mu sau nhng thi gian nht nh v o s bin i ca phn ng enzyme. Qua hng lot im ring bit nhn c s xy dng c ng biu din ca cc bc phn ng. Phng php khc l tin hnh quan st bn thn hn hp phn ng theo tin trnh xy ra v c th xy dng c mt s ln nhng thay i, hoc da vo cc phng php t ng ghi li.Chng ta s nhn c nhng ng biu din lin tc cc bc pht trin ca phn ng enzyme. phng php u, ngi ta thng o nng c cht hoc nng sn phm ca phn ng. Nu phn ng tng thi c hai cch va nu trn c th c s dng o hot ng ca enzyme.Trong mi trng hp xc nh tc , ngi ta phi nhn c t nht l 3 im: mt im thi im khng, im th hai khong thi gian nht nh tri qua, im th 3 khong

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thi gian ln gp hai ln khong trc. T c th kim tra c s ng n ca phn ng enzyme trong khong thi gian quan st. Ni chung, phn ln cc phng php c s dng nghin cu cc phn ng enzyme l loi phng php nghin cu lin tc v n c mi ngi a dng hn.

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II.2. . Nhng Nguyn Tc Chung Khi Nghin Cu Enzyme Nh phn u ni n, enzyme l nhng cht xc tc sinh hc c bn cht protein v rt khng n nh. Trong nhng iu kin bt li,chng rt khng bn, c th d dng b bin tnh (denaturation) v b mt hot . Do , khi lm vic vi enzyme, phi lun lun ch trnh nhng iu kin d lm mt hot ca n. Thng thng phn ln cc enzyme hot ng c vng pH trung tnh hoc gn nh trung tnh (pH =7). V vy cc yu t acid mnh, kim mnh d gy bin tnh enzyme. Nhng ion kim loi nng nh ch, ng, thy ngn... v cc iu kin v nhit cao cng thng lm mt hot enzyme. c bit l khi tch v lm sch enzyme, cn tin hnh nhit thp. Nhit thng dng cho cc cng vic ny thng thng t 0oC n 50C.i vi cc enzyme khng bn, cc cng on lm sch c th c tin hnh nhit thp hn (t - 50C n -200C). Trong cc trng hp ny, ngi ta hay s dng cc hn hp lnh nh nc vi CO2 hoc nc vi mui NaCl, hoc thm ch ngi ta dng c hn hp nc vi sulfuric acid m c... V d v mt s hn hp lm lnh c thnh phn hn hp t l nhit t c nh : Nc : mui 100:33 (3:1)- 21,30C Nc : H2SO4 m c 100: 25 (4:1)- 20,00C

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Nh trn ni, nhiu enzyme b mt hot tnh cc dung dch c pH < 5 hoc pH > 9, tuy rng c mt s ngoi l nh pepsin bn trong acid. Do , ty thuc mi loi enzyme, song nn ch trnh pH qu acid hoc qu kim. Khi iu chnh pH ca dung dch m c cha enzyme cn phi thm t t v rt thn trng cc acid hoc kim. V khi thm ha cht iu chnh pH th nn tin hnh 0oC. Khi lm vic vi enzyme cng cn ch trnh to bt v nhiu enzyme b bin tnh (mt hot tnh) mt phn cch hai pha nc v kh. trnh vic to bt c th xy ra, ngi ta thng rt dung dch enzyme theo thnh ng thy tinh v khng c lc. C khi vic tch tng phn enzyme bng bt d lm mt hot tnh enzyme. V vy, khc phc tnh trng ny, ngi ta thng thm ammonium sulfate di dng dung dch bo ha ca n.Trong khi x l cc mu th nghim nh ct, thi, xay nh cc mu thc vt v ng vt (v d l cy, tht, cc c quan ni tng...) khng dng cc dng c dao ko dng c xay han r trnh tc dng ca cc ion kim loi nng nh (Cu, Pb, Fe...) m dng dng c inox..Khi dng cc dung mi hu c nh aceton, alcol kt ta enzyme cn tin hnh nhit thp. Tch kt ta enzyme bng cch ly tm lnh tt hn lc lnh v tin hnh nhanh hn. mt s trng hp , khi tch v lm sch enzyme c hin tng gim dn hot , v vy cn phi lm th nghim nhanh. Tt nht l thc hin th nghim lin tc,khng ngt qung. V d tch chit cc enzyme chng oxy ha(antioxidant enzyme) ty th trong vng 6h v o lun nu khng th mt hot tnh. Cn microsome th tin trnh c th ko di hn vn

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khng nh hng n hot cc enzyme chng oxy ha v ccenzyme oxy ha kh.Mt iu cn ch na l trong khi tin hnh xc nh hot ca cc enzyme, nu xc nh trong khong nhit no th tt c cc thnh phn ca hn hp phn ng phi c gi nhit y. Lc ny nht thit phi dng my n nhit ( thermostate). Khi hn hp phn ng t c nhit cn thit th mi tin hnh o pH trong qu trnh ny cng phi c gi n nh bng dung dch m v phi m bo chnh xc ca pH: nhng phn ng to acid th phi thm kim vo v ngc li. m bo kt qu tin cy, trnh sai s nhiu, phi ly tht chnh xc lng dch enzyme. Ngi ta thng dng loi pipet khng chia hoc sau ny dng cc loi micropipet. Trong khi th nghim cn ch trnh nh ri enzyme vo dung dch nghin cu. V d ang lm th nghim vi amylase chng hn th khng ni chuyn nhiu. Khi c ch phm enzyme, cn bo qun chng nhit thp. Mt s enzyme n nh dung dch m c ca ammonium sulfate. Trong trng hp ny, ngi ta gi cc kt ta dng huyn ph trong dung dch ammoni sulphate boha v ly ch phm ra bng cch ly tm. Trong iu kin phng th nghim, vic sy kh ch phm enzyme s lm mt hot enzyme hon ton. Nhng iu kin chn khng nu sy kh nhit thp hoc dng phng php ng kh (lyophilization) th c th duy tr c hot ng bnh thng ca chng. III. QUY TRNH TCH CHIT V LM SACH (Tinh Ch) ENZYME Phng php tch chit v tinh sch enzyme: Tch chic enzim theo cc cng on sau:
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Ph v mng t bo gii phng enzyme: c th dng cc phng Dng dung mi chit enzyme: thng dng nc, NaCl, dng dch Lc , ly tm;

php vt l, ha hc, c hc
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m c pH n nh v ph hp vi enzyme;
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Kt ta enzyme: c th dng dung mi hu c ( ethanol, acetol), Ly tm, tc ta: Sy kt ta cho n kh.

dng mui, im ng in pH=pI; -

Qua cc bc nh trn ta thu c enzyme cha tinh khit, cn qua giai on tinh sch enzyme c enzyme tinh khit. C nhiu phng php tinh sch nh sau: +Phng php thm tch Dialise: l phng php s dng mng lc bn thm ch cho nhng cht c khi lng phn t nh i qua; +Phng php kt ta phn loi : thay i nng cht kt ta; +Phng php in ly; +Phng php lc gel ( gen sephadex); +Phng php sc k. Cc bc chi tit:

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III.1. Chn Ngun Nguyn Liu

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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 enzymecng 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: cc m v c quan ng vt, m v c quan thc vt, t bo vi sinh 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. 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 cnhiu - 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
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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.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 - 100gi) 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, gir, 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.Vi sinh vt rt nhy cm i vi tc ng ca mi trng, thnh phn dinh dng nui
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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 xctc 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.

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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 nuicy 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

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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.

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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 cavi sinh vt. Trong thnh phn mi trng phi c cc cht m bo c s sinh trng bnh thng ca vi sinh vt v tng hp enzyme.c bit lu l tng s tng hp enzyme ngi ta thng da vo hin tng cm ng. V nu nh trong thnh phn mi trng c cc cht cm ng th cht hay sn phm phn gii ca n s km hm hoc lm yu tc dng km to ca cht km hm nhm bo m kh nng sinh tng hp enzyme cho khng b cn tr. Cht cm ng tng hp enzyme cho thm vo mi trng nui thng l c cht tng ng ca enzyme cn tng hp.V d: Mun tch - amylase nm mc (Asp. Oryzae), ngi ta cho vo mi trng nui cy tinh bt, maltose, isomaltose,oligosaccharid... c cha lin kt - 1,6 glucozid. Mun tch pectinase Asp. Niger, ngi ta cho thm vo mi trng pectin. i vi hemicellulase th cht cm ng l hemicellulose; cn i vi proteinase cht cm ng c hiu lc l protein, bt u nnh, lng, sng nghin nh( Actinomyces fradiae). Cht cm ng cng c th l nhng cht ging c cht v nhng sn phm thy phn ca chng. V d: thay cho protein th peptid v thay cho tinh bt th erithrodextrin u c tc dng cm ng.C nhiu yu t nh hng i vi mi trng nui cy. Nhit nui cy thng thng t 25 30oC. Tr s pH ban u ca mi trng(ch yu mi trng nc) cng c th gy nh hng no n s to thnh enzyme, nhng khi cng cn tnh n kh nng bin i nhanh chng ch s bi vi sinh vt. Thng thng i vi -amylase, pH ti u cho s sinh tng hp (pH = 7 - 8) khc vi pH ti u cho hot ng can (pH = 4,7 - 4,9). Cc enzyme ng ha khc ca nm mc nh glucoamylase th pH ti u cho s sinh tng hp v cho hot ng l chung nhau (4,5 - 5,0). thng kh cng rt cn thit cho vic sinh tng hp enzyme. V vy mi trng b mt ngi ta thng thm cht xp nh tru vo, cn mi trng b su (mi trng dch th) , th ngi ta thng lc (nu enzyme cn lc th vic ny cc k quan trng). m cng rt quan trng (ch c tc dng nui cy b mt), ph thuc vo thnh phn mi trng b
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mt.Mt iu cn ni thm na l enzyme thng cha cc t bo sinh vt gi l cc enzyme trong t bo (intracellular), nhng n cng c th c cc sinh vt tit ra mi trng sng. l cc enzyme ngoi t bo (extracellular). Enzyme vi sinh vt thng chit l enzyme ngoi bo III.2. Chit Rt Enzyme

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Mun tm hiu ton b hot ng sng ca c th sinh vt, chng ta phi bit bn cht ca nhng bin i ha hc xy ra trong tng m t bo. iu ch thc hin c khi chng ta tch c cc t bo ra khi cc m v chit rt cng nh lm sch cc enzyme cha trong chng. T cc dng enzyme tinh khit thu c chng ta c th nghin cu su scc ch tc dng, tnh c hiu trong hot ng xc tc ca chng. Tytheo nhng c tnh ring bit ca tng loi enzyme m la chn phng php lm sch cho thch hp. Trong qu trnh tinh ch enzyme, mc du trnh t v cc th thut cc bc c th thay i , song vn c nhng nguyn tc chung. Nh chng ta bit, trong c th sinh vt, enzyme c trong t bo cht v cc cu t (nhn, microsome, ty th, lysosome...) ca t bo.T bo c bao bc bng mt lp mng. Lp mng ny vi khun i khi rt bn v dy. Ngi ta cn thy nhiu enzyme lin kt rt cht ch vi cc cu t ca t bo. Cc phn t enzyme khng c kh nng i qua mng ca t bo v mng ca cc cu t ca t bo. Do c th chit rt cc enzyme ni bo, bc u tin l phi ph v cu trc ca cc t bo c cha enzyme v chuyn chng vo dung dch. C th ph v cu trc ca cc t bo bng cc bin php c hc nh nghin vi bt thy tinh hoc ct thch anh, lm ng ha bng thit b ng ha (homogenizator). Thit b c chy thy tinh gn vi mt mt quay v c th iu chnh c tc quay theo yu cu. Cc t bo gia chy thy tinh v thnh ci s b ph hy. vic ph v c hiu qu m thc vt, trc khi nghin ngi ta thng thi nh mu vo ngn hoc cho trng nc (v d nh i vi mu ht kh). Cn cc m ca ng vt nh gan hoc thn, khi chit enzyme ngi ta cn ct b cc m lin kt. Mun tch c cc enzyme trong cc cu t ca t bo, ngi ta cn phi dng cc yu t vt l v ha hc khc nhau nh sng siu m, dng cc dung mi hu c nh butanol, aceton, glycerin, ethyl acetate...v cht detergent. Cc ha cht c tc dng tt cho vic ph v cc cu t ca t bo v trong cc c quan ny thng cha m. Sau khi ph v cc cu trc ca t bo, enzyme c chit bng nc ct, bng cc dung dch m thch hp hoc cc dung dch mui trung tnh.
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C mt s yu t nh hng n qu trnh chit rt cn lu .Trc ht l nhit . trnh mt hot tnh hoc thm ch v hot,cn chit rt v tin hnh kt ta enzyme nhit thp (t 3 n 5oC).Cc thao tc phi nhanh. Mt s cht in ly lm tng qu trnh chit rt enzyme nh NaCl, ZnCl2, CaCl2. Tc dng ca chng cn ph thuc vo phng php dng khi chit rt. V d nh nu dng my nung th c ba cht trn u c tc dng. Nu ch lng th ch NaCl c tc dng. V vy cn dng cht in ly thch hp. V d khi chit rt amylase, nu cho thm NaCl 0,1 - 0,2 % vo dung dch chit rt th hiu sut chit rt tng ln 30%. Ngi ta cn nhn thy, nu thm vo dch chit CaCl2 0,2% s lm cho kt ta enzyme tt hn v cu trc ca kt ta cng tt hn.Trong qu trnh chit rt enzyme cc i tng ng, thc vt,c trng hp cn c mt cht mu lm nh hng n vic lm sch hoc xc nh hot enzyme. Trong trng hp ny ngi ta cn cho thm vo cht kh loi mu. Mu ca hemoglobin hng cu hoc cachlorophyll v mt s cht mu khc l c th b loi tr bi hn hp ethanol, chloroform vi t l thch hp. Hot enzyme superoxidedismutase (SOD) - mt enzyme chng xy ha c th xc nh sau khi loi mu khi dch chit enzyme. cc mu t ng vt c sc t melanin mu nu. Ngi ta thng loi mu trn ct nha trao i ion bng cch cho dch enzyme qua ct hoc lc. Khi qua ct, cht mu b gi li v enzyme khng b gi. V d ngi ta hay dng DEAE - cellulose(Diethylamino ethyl - cellulose) hoc than hot tnh. Trong qu trnh ny phi ch kim tra pH. Sau khi loi mu cn kim tra li hot ca enzyme.Trong dch chit, ngoi enzyme cn c cc protein cu trc, cc cht cao phn t khc nhau nh polysaccharid nucleic acid, cc cht c phn t nh nh ng monose, cc cht lipid, mui khong... loi chng phi s dng phi hp nhiu bin php khc nhau. loi b mui khong v cc loi ng... l cc tp cht c phn t lng thp, ngi ta thng dng phng php thm tch(dialysis) i nc hay i cc dung dch m long hoc bng cch lc qua gel sephadex. Cch lm thm tch nh sau: cho dung dch enzyme vo ti colodion hoc cellophane (thng thng ngi ta dng cellophane tt hn), sau t c ti vo
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nc ct hoc dung dch m pha long (nh m phosphate c pH = 7, nng 0,01M chng hn). Mng cellophane l mng bn thm, c kch thc l cho cc cht c phn t nh xuyn v i qua vo cc dung dch m long theo nh lut khuch tn. Cn li trong mng l cc cht protein c phn t ln. (Hnh 2.1)

Hnh 2.1. Thm tch loi mui (NH4)2SO4 trong kt ta protein. loi b cc protein tp (protein cu trc, protein tr) v cc chtc phn t lng cao khc ngi ta hay dng kt hp cc phng php khc nhau: phng php bin tch chn lc nh tc dng ca nhit hoc pH ca mi trng, phng php kt ta phn on bng mui trung tnh hoc cc dung mi hu c, cc phng php sc k (sc k hp ph, sck trao i ion), in di, phng php lc gel.Phng php bin tch chn lc nh tc dng ca nhit hoc pH ca mi trng ch dng i vi trng hp cc enzyme bn vi nhit hoc bn vi acid.Th thut c tin hnh nh sau: dch enzyme c gi 50 -700C hay pH = 5 trong mt thi gian xc nh. Protein b bin tnh c loi b bng cch lc hoc ly tm

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III.3. Cc Phng Php Tch Tng Phn Protein Enzyme Protein l cc cht lng tnh,v vy trong cc dung dch acid v kim chng s b phn ly nh sau:kim Protein - COOH protein COOH - acid Protein NH 2 protein NH 3 + kim. mt ch s pH xc nh, mi phn t protein c mt in tch tng s no y m ln ca n ph thuc vo s lng cc nhm tch in dng v tch in m. Kt qu l ch s nng ion hydro c nh, cc protein khc nhau trong hn hp s c tng in tch khc nhau. Nhiu phng php dng tch cc hn hp protein u da vo ctnh ny. Cc phn t protein mang in tch tng s (dng hoc m)cng du y nhau ra xa nn d tan vo dung dch. Mi mt protein cmt tr s pH nht nh m tng s in tch m v in tch dngtrong phn t bng khng. Tr s pH gi l im ng in. im ng in, ha tan ca protein l thp nht, protein rt d b kt ta.Da vo tnh cht ny, ngi ta c th tch tng phn cc protein enzyme trong hn hp... cc phng php tch tng phn ny, ngi ta c th s dng phng php kt ta thun nghch bng mui hoc cc dung mi hu c, phng php sc k ct. Ni chung t kt qu tt, ngi ta thng phi hp hai phng php vi nhau. 3.1. Dng Mui (NH4)2SO4 Tch Enzyme

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Phng php kt ta phn on bng (NH4)2SO4 da trn c s s khc nhau v kh nng kt ta ca cc protein enzyme mt nng mui (tnh theo phn % nng bo ha) xc nh c dng ph bin loi b bc u protein tp ca cc dch enzyme. Cc loi mui c thc dng l (NH4)2SO4,Na2SO4 , MgSO4... ngi ta nhn thy mui (NH4)2SO4 l tt nht v n khng lm hi m lm n nh (lm bn) hu ht cc loi enzyme. Loi mui ny li r v ph bin. ha tan ca n li rt ln (bo ha 767g/l 250C). Ngoi ra nng (NH4)2SO4 cn thit kt ta enzyme khc nhau th khc nhau nhiu. V d: Protease ca nm mc d b kt ta 70% ca (NH4)2SO4 bo ha hon ton, cn amylase ca mm la b kt ta 50% bo ha ca dung dch mui ny. iu ni ln tnh kt ta la chn ca (NH4)2SO4 cao hn cc mui khc.Thng dng hai dng bt hoc bo ha - Khi dng bt: Ngi ta cho tng t mt vo dch chit enzyme. Cch cho cng nh hng ln n lng kt ta ban u ca enzyme. Khi cho mui vo dch chit cn phi c my khuy t m bo s ha tan ca mui. - Khi dng dung dch bo ha:

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Trong nhiu sch v phng php nghin cu ngi ta a ra bng tnh s lng mui cn thit pha cc dung dch c bo ha khc nhau nhng nhit nht nh. Khi nim v s phn trm ca bo. Ha hon ton c cp n. Nh v d trn ni, enzyme c th b kt ta 50% (0,5) hoc 70% (0,7) ca bo ha hon ton ca (NH4)2SO4 . Khi cho dung dch vo dch chit enzyme th nng (NH4)2SO4 khng tng t ngt.Sau khi kt ta xong ngi ta thng lng khong 2h hoc qua m, mc ch l to kt ta hon ton ( phng php dng dung mi hu c th khng cn lu). Kt ta c ly ra bng cch ly tm hoc lc qua phu Buckner. Khi ha tan kt ta li ngi ta thng thm ion Ca2+ lm bn (CaCl2) hoc Ca(COOH)2 . giai on loi mui, ngi ta dng phng php thm tch nh c trnh by phn trc. Thi gian thm tch thng l 24 - 28h, nc thay cng nhiu cng nhanh cng tt.C th loi mui bng cch lc qua gel sephadex G25 l dn sut ca dextran. u th ca phng php ny l tin hnh vi thi gian ngn(khong 30 '), nn khng lm mt hot enzyme. Mui c trng lng phn t b b gi li, cc enzyme c trng lng phn t ln xung trc(xem phng php lc gel 2.2.3.4.a)Giai on tip theo l lm ng kh thnh bt trng. Chuyn trng thi t dch nc sang trng thi kh m khng qua trng thi lng . tin li ngi ta a ra cng thc cch tnh lng (NH4)2SO4 cho vo dung dch c bo ha cho trc (S1) t n mt bo ha cn thit (S2). Ty theo trng thi (NH4)2SO4 cho thm vo dung dch chitenzyme, m c cng thc tnh ton khc nhau.- i vi (NH4)2SO4 dng bt 0,515 x V (S2-S1) x (g) =1 - 0,272 S2. Trong V l th tch dung dch S1, S2 l bo ha cho trc v bo ha cn t v d S1= 0,5 v S2 = 0,7 chng hn.Ngi ta cng c th dng bn ton (nomogram) chiu v xc nh c lng (NH4)2SO4 thm vo dung dch enzyme t c mt bo ha nht nh. Hoc c th i chiu bng c sn. Lng(NH4)2SO4 a vo dung dch c bo ha nht nh c khc nhau ty thuc nhit th nghim. i vi (NH4)2SO4 dng dung dch bo ha. Th tch (tnhtheo ml) ca dung dch bo ha cn cho vo 100ml dung dch c bo ha ban u S1 t n
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mt bo ha S2 cn thit c tnh theo cngthc sau:100 (S2-S1) V (ml) =1 S2 3.2. Dng Dung Mi Hu C Phng php ny c tin hnh da trn c s: ha tan ca protein ph thuc vo s tng tc ca cc nhm tch in trong phn t protein vi cc phn t nc.S tng tc (cn gi l s hydrate ha) s b gim xung khi thm vo dung dch enzyme cc dung mi hu c. Dung mi hu c thng dng l ethanol, isopropanol, acetone hoc hn hp cc loi ru. phng php ny cng ch tin hnh nhit thp (t 5 0 Ctr xung). Dng dung mi hu c c th tin hnh tch phn on di00C v c th n 20 0 C, nh vy n c tc dng tt n n nh ca protein enzyme.Khi c kt ta, ch ly nhanh kt ta ra khi dung mi bng cch dng my li tm. Phng php ny c li th l khng cn loi mui,nhng c nhc im l hay c mu. 3.3. Dng Nhit Cng c th dng nhit loi b cc protein enzyme tp ra khi dch chit enzyme. Tuy vy phng php ny t c dng v him enzyme bn vi nhit. 3.4. Cc K Thut Sc K Ct Dch chit enzyme c loi b phn ln cc protein tp nhng vn cha m bo ng nht cn thit. Do dch chit enzyme c tip tc lm sch bng phng php sc k ct. Phng php sc k(chromatography) l do hai ch "chroma" l mu sc v " grapho" l vit, ngha l "vit bng mu". Thu ban u, ngi ta s dng phng php sc k tch cc cht mu v ch sau ny ngi ta mi p dng cho vic tch cc cht khng mu. 3.5.Phng Php Dng Cht Ry Phn T (lc gel - gel filtration)

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C s ca phng php lc gel l da vo s khc nhau v kch thc hnh dng v phn t lng ca enzyme c trong hn hp tch chng ra. m bo cho vic tch enzyme c tt, cht ry phn t phi l cht tr, khng phn ng vi protein enzyme. Cht ny cng khng ha tan v tng i bn vi cc yu t v c hc cng nh sinh hc. Ngoi ra cht c s dng cho mc ch lc phn t phi l cht khng c tnh n hi (khng c) v phi l cht a nc (hydrophyl).Gel sephadex l cht tho mn cc yu t trn. Sephadex l ch phm dextran do cc loi vi sinh vt khc nhau l Leuconostoc to ra khi chng c nui cy trn mi trng cha saccharose. Trng lng phn t ca dextran c th t ti hng triu v ln hn. Phn t dextran bao gm cc chui do cc gc glucose to thnh cc lin kt glucsid 1,6.Sephadex nhn t dextran bng cch x l ha hc (do tc dng ca epichlohidrin) to ra cc li phn nhnh c lin kt ngang gi l "sng phn t" v cht ny tr thnh khng tan trong nc. S lin kt ngang to ra cng nhiu, kch thc ca l sng phn t cng nh.Phng php lc trn sephadex c tin hnh nh sau: chosephadex vo ct thy tinh di v cn bng bng dung dch m c pH nht nh. Sau cho dung dch enzyme ln ct. Khi lc v chit bng dung mi thch hp, cc phn t c trng lng phn t nh ( y l cc mui) s khuych tn chm chp qua cc l nh ca cc ht Sephadex b trng phng, cn cht c trng lng phn t ln hn ( trng hp nyl protein enzyme ) khng c kh nng i vo m lch nhanh qua cc ht sephadex v s ri xung trc, s c chit nhanh ra khi ct (Hnh 2.2) V vy ta c th tch c cht c trng lng phn t cao hn ra khi cht c phn t lng nh. Hng Sephadex (Pharmacia) caThu in tung ra th trng cc loi sephadex c kch thc khc nhau c k hiu t G10 n G200. S k hiu nhm ch ra mc nhn(ht) nc ca chng. V d G10 ch khi trng phng th 1g gel kh nhn 1ml nc (1ml/g) Hnh 2.2. Hot ng ca lc phn t sephadex

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Quy trnh tch chit enzym proteaza

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