n cng ngh 1

Nguyn Th Thy_11SHLT

Chng 3: ng dng kho st cc phng php tinh sch urease t u nnh 3.1 Urease - Urease l mt loi enzym thu phn urea hnh thnh NH 3 v CO 2, c ng dng rt nhiu trong Y hc v Cng nghip thc phm nh lng urea trong cc mu bnh phm v nc chm. Trc y, c rt nhiu nghin cu v enzyme urease v a ra nhiu phng php tinh sch urease, tuy nhin cha c nghin cu no so snh gia cc phng php tinh sch khc nhau. nc ta, cng c mt s nghin cu tinh sch enzym urease t u nnh nhng cng cha a c quy trnh tinh sch hiu qu v cha c ng dng trong sn xut ch phm urease. Ngnh Y hc v Thc Phm nc ta hin nay vn phi nhp ch phm urease v kit urease t nc ngoi vi gi thnh rt cao. - Do ti thc hin ti ny nhm tm ra mt quy trnh tinh sch urease hiu qu nht t u nnh, mt ngun nguyn liu r tin v d kim. ng thi s dng phng php in di trn gel acrylamide nghin cu thnh phn enzyme urease t u nnh. 1.5 Urease 1.5.1 Khi nim - Urea l sn phm chnh ca s trao i m thi ra vi nc tiu ngi, ng vt c v, b st v vi loi c. Urea c hnh thnh trong gan - Urease l mt protein c tm thy trong vi khun, nm men v mt s thc vt bc cao. 1.5.2 Tnh cht - Urease l mt enzyme xc tc thy phn urea thnh carbon dioxide v amoniac (NH2)2CO + H 2O CO 2 + 2NH 3 - Urease c bit c tm thy trong Helicobacter pylori, trong kt hp bn trong su enzyme thng xuyn trong mt tiu n v lp rp t din tn ti ca th 24 tiu n v
12

12

- C th tc dng vi kim loi nh: Nikel - Phn t khi: 480 kDa hoc 545 kDa - pH thun li: 60 C
o

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Nguyn Th Thy_11SHLT

Hnh 3.1 Cu to ca urea - Cht c ch: kim loi nng Pb v Pb

2+

- Enzyme c th: urease v hydroxyurease

Hnh 3.2 Trung tm hot ng ca urease Qu trnh thy phn urea qua tng bc sau:

Hnh 3.3 C ch phn hy urea - Sau cacbamil acid t ng phn hy to thnh amoniac v carbon dioxide

S dng thuc th phenolphtalein th dung dch va to ra s c mu do c s xut hin ion OH

-

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Nguyn Th Thy_11SHLT

- Ngi ta s dng urease xc nh hm lng urea trong mu. T phng trnh phn ng trn ta c th nh lng hm lng urea da vo sn phm to thnh l CO 2 hoc NH 3 hoc dng NH 3 tc dng vi cetoglutarate NH3 + NADH glutaric acid / NAD - nh lng glutaric acid hay NAD suy ra lng urea trong mu - Phng trnh tng qut

- Urea b thy phn bi enzyme urease hnh thnh nn amonia v carbon dioxide. T phn ng c th c kim sot bi s thay i dn in ca cc ion theo thi gian. dn in ny tng ng vi nng ion hnh thnh trong sut qu trnh phn ng. 3.2 Nguyn liu v phng php nghin cu 3.2.1 Nguyn liu - u nnh loi b cc ht xu, b su mt, xay mn, loi lipid, bo qun lnh. - Ho cht: Urease chun (urease Jack bean), Albumin bovine, Etherpetrolium, acetone, glycine, Tris(base), Coomassie Brilliant Blue G250 (Merck), Nessler, EDTA, L-Cystein.HCl, Acrylamide v bisacrylamide, SDS, TEMED, Amonium persulfate, Sephadex G 50, DEAE Cellulose (Merck) 3.2.2 Phng php nghin cu - Phng php ta sunfat amon + Phng php 1: ta phn on 2 nng 65% v 55% bo ho. + Phng php 2: ta phn on 2 nng 20% v 55% bo ho. - Phng php sc k: tch lin tc tng vi phn ca hn hp cht + Sc k ry phn t trn gel Cephadex G-50 (612cm) + Sc k trao i ion trn gel DEAE-Cellullose - Phng php Bradford: xc nh hm lng protein - Phng php Nessler: xc nh hot tnh urease

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Nguyn Th Thy_11SHLT

- Phng php in di: kim tra hiu qu tinh sch, in di trn gel 10% polyacrylamid vi tc dng 20mA, gel c nhum vi Coomassie Blue R-250. - Phng php siu lc: s dng thit b lc membrane Vivaflow. 3.2.3 Chun b mu nghin cu [20] - Chit xut ly enzyme: Nghin k bt u trong dung dch HCl 0,4% c thm EDTA (trilon B, complexon III) 5.10 M v L. cystein 5.10 M. Sau ly tm tch b ly dch chit. - X l nhit: Nng nhit nhanh n 60 C, gi trong 30 pht em ly tm tch b cn kt ta - Siu lc: Dch ly tm c lc qua mng siu lc loi b peptid v polypeptid c trng lng phn t b (M>500) - Kt ta bng aceton: Dch lc c x l bng aceton lnh (t l 1:1), ly tm tch kt ta. Phn kt ta em ha tan trong trisbuffer 0,1M, pH = 7 cha EDTA v L. cystein 5.10 M. - Sc k trao i ion: Dch enzyme c chy sc k trao i ion trn ct cha DEAE - cellulose vi gradien nng NaCl t 0-1M. Phn on cha enzyme c sy thng hoa (ng kh) vi cht saccharose lm cht n nh vi t l 2,5mg/1mg enzyme. Ch phm thu c c hot tnh tng 25 ln so vi ban u v hiu sut thu hi 43% 3.3 Kt qu 3.3.1 Kho st cc phng php tinh sch urease t u nnh 3.3.1.1 Kho st nng aceton kt ta enzyme - Kt qu so snh tinh sch v hiu sut thu hi enzym urease khi ta vi cc nng aceton t 30% - 60% c trnh by bng 3.1 Bng 3.1 Kt qu so snh hiu qu ta enzym cc nng aceton khc nhau
-3 o -3 -3

Giai on Trch ly Aceton

Vdch, ml mbt , mg 150.00 882.75

P, mg/ml (mg/mg) 19.69 0.21 Pt, mg

A, UI/ml (UI/mg)

Ap At, UI UI/mg Pr 15660.00 5.30 1200.54 6.48

tinh H, % sch 1.00 1.22 100 7.67

2953.50 104.40 185.38 1.36

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30% Aceton 40% Aceton 50% Aceton 60% 2976.00 0.46 3313.24 0.45 3788.25 0.44 1368.96 1.79 1490.96 1.87 1666.83 2.91

Nguyn Th Thy_11SHLT
5327.04 695.76 3.89 4.16 0.73 0.78 1.25 34.02 39.55 70.39

11023.81 6.61

- Kt qu bng 3.1 cho thy tinh sch ca mu ta 30% v 60% aceton gn bng nhau v ln hn 1, nhng hiu sut thu hi nng aceton 60% nhiu gp 10 ln so vi nng aceton 30%. mu ta 40% v 50% aceton c tinh sch thp nh hn 1 v hiu sut thu hi enzym cng khng cao. Kim tra 5 mu qua in di trn gel acrylamid 10%, kt qu c trnh by hnh 3.1

Hnh 3.4 Kt qu in di urease ta cc nng aceton khc nhau - in di trn hnh 3.4 cho thy: urease u ra d dng rt tinh khit vn xut hin 3 vch r rng, iu chng t urease ca u ra l mt loi enzyme heterogeneous. in di u nnh cha cc vt c trong in di u ra. Ngoi ra in di u nnh cn cha mt s vch khc cng rt r rng, c bit vch th 6 vi m tng dn t nng aceton 30% - 60%. Kt qu chy in di cho thy phng php ta bng aceton c hiu qu tinh sch cha cao, cn ln nhiu protein tp nn cc mu in di cha c s phn tch r rng. Nh vy t kt qu kim tra hiu qu tinh sch trn cho thy nng aceton 60% l nng dung mi lm ta enzyme urease c hiu qu nht v tinh sch cng nh hiu sut thu hi enzyme. 3.3.1.2 Kho st kt ta phn on bng (NH4)2SO4

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Nguyn Th Thy_11SHLT

- Kt qu xc nh tinh sch v hiu sut thu hi ca hai phng php ta phn on sunfat amon c trnh by bng 3.2 Bng 3.2 So snh hiu qu tinh sch ca 2 phng php ta phn on (NH4)2SO4

Giai on

Vdch, ml mbt , mg

P, mg/ml (mg/mg

Pt, mg

A, UI/ml (UI/mg

A t, UI

Ap, UI/mg pr

Tinh sch

H, %

Trch

150.00

) 19.69 0.19 0.19

) 2953 104.40 .50 360. 38 230. 1.60 1.36

15660. 00 3034.7 5 1651.3

5.30 8.42 7.16

1.00 1.59 1.35

100 19.38 10.55

ly P. Php 1896.72 1 P. Php 1214.25

2 71 8 - Cc s liu thc nghim cho thy phng php 1 c tinh sch enzyme v hiu sut thu hi enzyme u cao hn so vi phng php 2. Tuy nhin hiu sut thu hi enzyme ca c 2 phng php ny u thp, ch 10 20%. Kim tra 4 mu qua in di trn gel acrylamid 10%, kt qu c trnh by hnh 3.5

Hnh 3.5 Kt qu chy in di cc mu urease thu t hai phng php ta phn on sunfat amon - Kt qu in di cho thy, c hai mu ta Sunfate amonium u xut hin cc vch tng ng vi mu chun v cha t vch hn so vi mu trch ly. mu ta

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Nguyn Th Thy_11SHLT

theo phng php 2 tuy loi bt protein tp vch s 2 v s 4, tuy nhin vn cn st mt t v th hin thnh cc vch m. mu ta sunfat amon theo phng php 1 loi c hon ton cc protein tp cc vch 2, 4 v 7 so vi mu trch ly. in di ca mu ta theo phng php 1 ch cn li 4 vch. - Nh vy, phng php ta phn on Sunfate amonium theo phng php 1 em li hiu qu tinh sch tt hn phng php 2. Do chng ti chn ta bng Sunfate amonium theo phng php 1 tip tc cc bc nghin cu sau. 3.3.1.3 Kho st qu trnh tinh sch urease bng phng php sc k trn ct Sephadex G-50 - Ly 0,5ml dch trch ly iu chnh v pH = 7 cho ln ct Sephadex G-50). Ra ct bng m phosphate pH = 7; 1/15M. Kim tra hm lng protein v hot tnh enzyme mi phn on.

Biu 3.1 Sc k ca dch trch ly t u nnh khi qua ct Sephadex G - 100

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Nguyn Th Thy_11SHLT

Hnh 3.6 Kt qu in di mu qua ct khi qua ct SephadexG-50Sephadex, phn on 4 - T sc k cho thy v tr cc i ca peak protein cng chnh l v tr cc i ca peak enzyme phn on 4. kim tra hiu qu tinh sch, chng ti tin hnh chy in di trn gel acrylamide 10% vi cht chun l urease t u ra. Kt qu trnh by hnh 3.6 - Sc k trn gel acrylamide cho thy hu nh tt c cc vch trn mu trch ly u xut hin mu qua tinh sch trn Sephadex. iu c ngha trc v sau khi chy sc k lc gel trn ct Sephadex G-50 cha c hiu qu lm v mt tinh sch, c th do Sephadex G-50 khng ph hp nn cha th loi b mt s tp cht. 3.3.1.4 Siu lc - Dch trch ly c cho qua thit b lc mng membrane c kch c l 10000 Da, p sut lc 2.5 bar, lc trong 2h. Sau khi qua siu lc, nng enzyme trong mu rt cao. Bng 3.3 Kt qu nh gi hiu qu tinh sch ca phng php siu lc Giai on Trch ly Dung dch siu lc Siu 6876.40 0.43 2956.85 0.76 5226.06 1.77 0.71 50.94 Vdch, ml P, mbt , mg mg/ml 150.00 90.00 Pt, mg A, UI/ml At , UI Ap, UI/mg tinh sch 1.00 1.06 100 68.54 H, %

(mg/mg) (UI/mg) pr 27.28 4029.00 68.40 10260.00 2.51 29.34 2640.60 78.14 7032.60 2.66

lc k - Khi s dng mng lc 10000 Da l qu nh so vi phn t Urease nn hiu qu tinh sch khng cao, ch lm dch enzyme m c hn. Dch siu lc sau ng kh b gim hot tnh, c th do mt s cht n nh hot tnh urease nh L-Cystein, EDTANa 2 b loi khi siu lc. 3.3.2 So snh cc phng php tinh sch - Kt qu so snh cc phng php tinh sch s b enzym urease trnh by bng 3.4.

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Nguyn Th Thy_11SHLT

Bng 3.4 So snh hiu qu tinh sch ca cc phng php

Giai on

V dch , ml m bt,

P, mg/ml (mg/mg) 27.28 29.34 7.88

Pt, mg

A, UI/ml (UI/mg)

A t, UI

Ap,

H, %

UI/mg tinh pr 10260.00 2.51 7032.60 2.66 6924.00 2820.88 7110.58 2.93 3.79 3.19 sch 1.00 1.06 1.17 1.51 1.27 100 68.54 57.88 27.49 69.30

Trch ly Dung dch lc SK. Cephadex (NH4)2SO 4 65% Aceton 60%

mg 150.00 90.00 300.00

4029.00 68.40 2640.60 78.14 2362.50 23.08 743.34 1.48

1906.00 0.39 4837.13 0.46

2225.08 1.47

- Kt qu cho thy ta phn on (NH 4)2SO 4 theo phng php 1 cho tinh sch cao nht (1,51), nhng hiu sut thu hi li tng i thp (18,01%). phng php siu lc v sc k trn Cephadex hiu sut thu hi rt cao, nhng hiu qu tinh sch li khng cao. Phng php ta aceton 69% thc hin rt n gin, thi gian ta ngn v rt d thu hi, nhng qu trnh ny khng n nh, enzyme rt d bin tnh. Hn na, bt sau khi ng kh ho tan rt km. Bt ng kh thu c t mu ta sunfat amon c tan rt tt. Kim tra 4 mu qua in di trn gel acrylamid 10%, kt qu c trnh by hnh 3.7

Hnh 3.7 Kt qu in di urease qua 4 phng php tinh sch

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Nguyn Th Thy_11SHLT

- Kt qu in di hon ton ph hp vi s liu thc nghim bng 3.4. Cc phng php sc k ry phn t, siu lc, ta aceton khng em li hiu qu tinh sch cao, iu th hin qua in di cn rt nhiu vch tp cht. mu ta sunfat amon theo phng php 1 c hiu qu tinh sch cao nht, in di ch cn 4 vch rt r rng, ngoi 3 vch tng ng vi cc vch trn in di ca urease u ra cn mt vch th 4 rt m. T nhng nhn xt trn, ti quyt nh chn phng php ta phn on sunfat amon theo phng php 1 tip tc qua sc k trao i ion. 3.3.3 Kt qu tinh sch trn ct sc k trao i ion DEAE cellulose - Trong phn ny, ti thc hin 2 th nghim song song: 1) Mu enzyme urease u nnh sau khi tinh sch bng phng php ta phn on sunfat amon qua thm tch i nc v qua ct trao i ion DEAE cellulose, dng h m phosphat pH = 7 1/15M, gradient bng mui NaCl nng t 0 1M. Sc k c trnh by biu 3.2 2) Dung dch sau thm tch c ng kh v chy sc k trao i ion trn ct DEAE cellulose nh trn. Sc k c trnh by biu 3.3

Biu trn ca (NH 4)2 1

3.2 Sc k DEAE-Cellullose dch ta bng SO 4 phng php

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Nguyn Th Thy_11SHLT

Biu 3.3 Sc k DEAE-cellulose bt ta (NH 4)2SO 4 phng php 1 ng kh - Sc k cho thy khi tch phn on dung dch enzyme sau ta (NH 4)2SO 4 nh sc k trao i ion thu c 3 peak protein v 1 peak enzyme. V tr cc i ca peak enzyme trng vi v tr cc i ca peak protein th 3 nng mui 0,3M. Enzyme ta phn on (NH 4)2SO 4 sau ng kh qua sc k trao i ion c sc k tng t sc k mu cha ng kh, gm 3 peak protein v 1 peak enzyme. Peak enzyme cng trng vi peak protein th 3, nhng hot tnh enzyme ch tp trung 2 ng 11 v 12, trong ng 12 c hot tnh cao nht. Nh vy, c 2 mu dung dch ta (NH4)2SO 4 v ta (NH4)2SO 4 qua ng kh, enzyme urease c y ra nng mui 0,3M. Peak cha enzyme urease thu c sau sc k trao i ion c kim tra tinh sch qua in di, kt qu trnh by hnh 3.8

Hnh 3.8 Kt qu tinh sch qua cc giai on tinh sch

11

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Nguyn Th Thy_11SHLT

- Kt qu in di cho thy, hiu qu tinh sch cng nh kt qu tch phn on qua ct sc k rt cao, loi b gn ton b cc protein tp. Nh vy, trn in di ch cn mt vch rt r. Kt qu ny hon ton ph hp vi kt qu ca mt s nghin cu trc y. T , chng ti c th kt lun: urease u nnh (Glycine max) v u ra (jackbean) mc d c hot lc thu phn nguyn liu c hiu nh nhau nhng khc nhau v phn t lng cng nh thnh phn cu to, c th: kt qu trn sc k v in di trong th nghim ca chng ti cng nh trong cc cng trnh nghin cu trc y th urease u ra (jackbean) l loi enzym heterogeneous, cn urease u nnh (Glycine max) l homogeneous. Bng 3.5 Kt qu tng kt qua cc giai on tinh sch Giai on Vdch, ml P, mbt , mg mg/ml Trch ly Dung dch (NH4)2SO 4 DEAE -Cell dung dch (NH4)2SO 4 k DEAE 1897.23 0.35 379.45 0.98 664.03 371.86 1.34 5.07 2542.29 3.83 1923.81 5.17 1.50 2.03 27.13 20.53 150.00 75.00 225.00 Pt, mg A, UI/ml At , UI Ap, UI/mg tinh sch 1.00 1.52 1.62 100 44.25 24.32 H, %

(mg/mg) (UI/mg) pr 24.66 3669.00 62.47 9370.50 2.55 14.25 1068.75 55.29 4146.75 3.88 2.46 553.50 10.13 2279.25 4.12

Cell k - Nh vy, qua mi cp tinh sch th urease dn dn c tch ra khi cc protein tp khc. mu ta (NH 4)2SO 4 qua ng kh khi qua ct DEAE-cellulose th hot tnh ch yu tp trung trong 2 ng, do tinh sch ca n cao hn mu dch ta (NH 4)2SO 4 v t 2,03 vi hiu sut thu hi enzym l 20,53. 3.4 Kt lun Qua qu trnh nghin cu chng ti gii quyt c vn sau: - So snh cc phng php tinh sch s b enzyme urease v kim tra tinh sch bng in di trn gel acrylamide v nhn thy phng php ta phn on sunfat amon 65% v 55% l hiu qu nht so vi cc phng php siu lc, ta aceton, sc k ry phn t.

12

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Nguyn Th Thy_11SHLT

- Sc k trao i ion trn DEAE-cellulose thu c 3 peak protein v 1 peak enzyme urease ng vi nng mui 0,3M. - xy dng c mt quy trnh thu nhn v tinh sch enzyme urease t u nnh hiu qu, loi b gn ton b cc protein tp, tinh sch t c l 2,03 v hiu sut thu hi enzyme l 20,53. - S dng in di kim tra tinh sch enzyme v xc nh c urease u nnh l loi homogeneous. Trong nghin cu ny chng ti so snh cc phng php tinh sch s b enzyme urease: ta phn on sunfat amon, ta bng aceton, sc k ry phn t v siu lc. Da vo kt qu xc nh tinh sch, hiu sut tinh sch enzyme v kt qu kim tra tinh sch bng in di trn gel acrylamid chng ti nhn thy phng php ta phn on sunfat amon nng 65% v 55% bo ho hiu qu nht. Tip tc thc hin tinh sch qua sc k trao i ion DEAE cellulose, sc k cho thy c 3 peak protein v ch c mt peak enzyme urease ng vi nng mui 0,3 M. Thu nhn peak enzyme, kim tra trn in di gel acrylamid xc nh c urease u nnh l mt loi homogeneous. Kt qu in di cng chng minh quy trnh tinh sch do ti xy dng tinh sch c enzyme urease.

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