thu nhan enzyme

8/9/2019 Thu Nhan Enzyme

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Cng ngh thu ch phm enzyme


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Unit 4. Cng ngh thu nhn enzyme

4.1 Ngun thu enzyme4.2 Cc ph-ng php thu nhn enzyme th4.3 Ph-ng php thu nhn enzyme t thc vt v ng4.4 Cc ph-ng php thu nhn enzyme t vi sinh vt4.5 Cc ph-ng php nui cy vi sinh vt thu nhn e4.6 Cc ph-ng php ci to ging vi sinh vt thu enz


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1. Ngun nguyn liu thu enzyme.

Enzyme l nhng cht khng th iu ch-c bng ph-ng php tng hp ho hc.

Thu nhn chng t : ng vt (8%) Thc vt (4%)

Vi sinh vt (>90%)


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Enzyme thc vt

u : papain

Da : bromelin

Sung ; fixinMalt i mch: amylase

Gi u: Urease

Mm ht thu du : lipase

Ch : polyphenoxydase, protease, pectinase,

amylase, invertase


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E

nzyme vi sinh vt

Ngun enzyme rt phong ph, a dng, c

th thu nhn vi s l-ng rt ln.

H enzyme vi sinh vt c th thay i bng

cch thay i cc iu kin nui cy.

Vi sinh vt c kh nng pht trin nhanh v

sinh tng hp vi tc nhanh

Enzyme vi sinh vt c hot tnh mnh.


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Mt s quy trnh tch, tinh chenzyme thc vt & ng vt.


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Tch v tinh ch papain

Nha u

Ho tan nha

-a pH=9

Kt ta Ho tan kt ta Kt ta Sy kh

0,001M EDTA

14% (NH4)2SO40,02M Cystein

1M NaOH

40% (NH4)2SO4

0,1 photphat buffer 1M NACl


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Tch v tinh ch pepsin

Mng nhy d dy

Ra sch

Nghin

Phn hu Lc Kt ta Ch phm

5%HCl, 42oC, 42 g

22% NACl


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Cc ph-ng php thu ch phm enzyme vi

sinh vt

Qui trnh sn xut ch phm enzyme vi sinh vt

bao gm cc giai on ch yu sau :

Phn lp, tuyn chn v ci to ging vi sinh

vt

Nui cy vi sinh vt sinh tng hp enzyme

Tch v tinh ch enzyme


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Ci to ging vi sinh vt

t bin: ho lCc tc nhn vt l: UV, X-ray, to,Cc tc nhn ho hc ; cc cht gy t bi

t bin bng ph-ng php sinh hc phn tt bin imError-prone PCR

Suffling Ph-ng php bin np Ph-ng php tip hp gene Ph-ng php ti np


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Cc ph-ng php nui cy vi sinh vt

Ph-ng php nui cy trn mi tr-ng rn :Vi sinh vt pht trin trn b mt mi tr-ng

dinh d-ng th rn -c lm m v vtrng.

Ph-ng php nui cy trong mi tr-ng lng

:Vi sinh vt pht trin trong mi tr-ng dinhd-ng trng thi lng

Xem tip file khc


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Tch v tinh ch enzyme

Khu tr ca enzyme :

-Enzyme ngoi bo: enzyme -c tit rami tr-ng sng ca sinh vt.

- Enzyme ni bo : enzyme nm trong

t bo ca sinh vt- Enzyme periplasmic : enzyme nm

mng nguyn sinh cht ca t bo.


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Cc ph-ng php ph v t bo

1. Ph-ng php c hc :

Ph v bng sng siu m : 18 kHz 1MHz

Ph v bng my ng ho cao p : 150 Mpa

Ph v bng my nghin : vi cc ht c r = 0,1 0,2

Ph v bng ph-ng php lnh ng


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2. Ph-ng php phi c hc

Sc thm thu : -ng 20%

X l kim: pH 11,5 12,5

S dng cht ty ra : triton x100, tween 20, L

Phn gii bng enzyme


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Cc ph-ng php ng ho

T bo ng vt (cc t chc)

-N rt d ng ho v khng c v t bo (cell wall)- Cn phi loi b m v mng tr-c khi ng ho

Vi khun v nm mcCn phn hu v t bo bng enzyme (c th tot bo trn bng x l lysozyme hocchitinase/3-glucanase)

T

hc vt- Ph hu khng bo c th lm hng enzyme


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Cc ph-ng php tch

Kch th-c v khi l-ng

Siu ly tm (300 000g)-Mr l yu t chnh tch- Khng hiu qu tch enzyme t hn hpenzyme: th-ng tch cht bn

Lc gel (Mr ~ hng trm ngn)- Sephadex, Bio-gel P, Sephacryl, Sepharose- t v tn thi gian.- Th-ng trong cng on cui ca qu trnh tinh sch.


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Thm tch (Mr ~ hng chc nghn)

- Th-ng dng loi mui, dung mi hu c,

Siu lc

-Cc phn t nh -c lc qua nh p lc

- Dng c c protein


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Tnh phn cc

Sc k trao i ion

-Tnh cht tnh in-Chy qua nng mui thp v pH t-ng ng-Kh hp ph khi thay i nng mui v pH-Enzyme c th tch nh iu kin gradient-C th quy m ln-Th-ng tng tinh sch 10 ln.


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in di

-Tch nh chuyn ng ca cc phn t- tch in-in di mao dn (


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T

nh tan*Thay i pH

- Enzyme t tan pI bi v khng c lc y gia enzy- Enzyme khng b bt hot di pH

*Thay i trong lc ion- Cc phn t c in tch ln rt t tan trong n-c

tinh khit; b sung cc ion lm tng tnh ho tan(Salting in)- nh h-ng ca lc ion, cc phn t tch in b

kt ta nhanh (Salting out)- Ammonium sulphate -c -a dng- Tng 10 ln mc tinh sch


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Gim hng s l-ng in (dielectric constant)

-B sung dung mi hu c c th trn vi n-c(ethanol, acetone)-Gim hng s l-ng in

-i khi bt hot enzyme-Tin hnh nhit thp-Th-ng x dng PEG ~ Mr 4000 6000


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V tr lin kt c bit

-Sc k i lc- C cht hoc cht c ch lin kt vi gi th- Phn hp ph nh s y c cht hoc thay i pH, l

Sc k i lc

Cc protein hn hp (fusing protein)-Glutathione-S-transferase: glutathione- Maltose binding protein:maltose

-Hexahistidine:Ni2+ (tch ra nh imidazole hocthrombine v tr ct -c b sung sau His-tag.


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Cc loi sc k khc

i lc tch ra- Sau khi xut hin b-c phn hp th- C th gii quyt mt s vn ca sc k ilc v d nng cp.

Sc k dye ligand-Cibacron Blue F3G-A c th gn vi nhiu loi

dehydrogenase v kinase- Procion Red He-3B gn tt vi NADP+ -tuthuc vo dehydrogenase.

Sc k hp ph min dch- C nh khng th vo Sepharose x l CNBr- t -c tinh sch cao

Sc k covalent- Tch cysteine cha protein s dng thiol-Sepharose 4B


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Cc dng ch phm enzyme

Ch phm th: l ch phm ch-a -c tinh

ch.Ch phm k thut : l ch phm -c

tinh ch s b.

Ch phm enzyme tinh khit : loi protein

v enzyme tp, ch cn mt enzyme mong

mun.


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Phn on & tinh ch enzyme(xem ch-ng tinh ch)

thu nhan enzyme

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