TP CH PHT TRIN KH&CN, TP 9, S 7 -2006

NGHIN CU THU NHN, TINH SCH UREASE T U NNH

L Th Ph, Nguyn Th Cm Vi, Nguyn Tn t Trng i hc Bn cng Tn c Thng
(Bi nhn ngy 28 thng 02 nm 2006, hon chnh sa cha ngy 14 thng 08 nm 2006)

TM TT: Trong nghin cu ny chng ti so snh cc phng php tinh sch s b enzym 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 enzym 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 enzym urease ng vi nng mui 0,3 M. Thu nhn peak enzym, 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 chng ti xy dng tinh sch c enzym urease. 1.M U
Urease l mt loi enzym thu phn ure hnh thnh NH3 v CO2, c ng dng rt nhiu trong Y hc v Cng nghip thc phm nh lng ure trong cc mu bnh phm v nc chm. Trc y, c rt nhiu nghin cu v enzym 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 chng 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 acrylamid nghin cu thnh phn enzym urease t u nnh.

2.NGUYN LIU V PHNG PHP NGHIN CU 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, Ether petrolium, 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), 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 [6] 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 [4] - Phng php Nessler: xc nh hot tnh urease [4] - 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. [1] - Phng php siu lc: s dng thit b lc membrane Vivaflow. Trang 57

Science & Technology Development, Vol 9, No.7- 2006

3.KT QU 3.1 Kho st cc phng php tinh sch urease t u nnh 3.1.1. Kho st nng aceton ta enzym 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 1. Bng 1.Kt qu so snh hiu qu ta enzym cc nng aceton khc nhau
Giai on Trch ly Aceton 30 % Vdch , ml mbt , mg 150.00 882.75 P, mg/ml (mg/mg) 19.69 0.21 0.46 0.45 0.44 Pt , mg 2953.50 185.38 1368.96 1490.96 1666.83 A, UI/ml (UI/mg) 104.40 1.36 1.79 1.87 2.91 At , UI 15660.00 1200.54 5327.04 6.95.76 11023.81 Ap, UI/mg pr 5.30 6.48 3.89 4.16 6.61 tinh H, % sch 1.00 1.22 0.73 0.78 1.25 100 7.67 34.02 39.55 70.39

Aceton 40 % 2976.00 Aceton 50 % 3313.24 Aceton 60 % 3788.25

Kt qu bng 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 %0% 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 1.

Vch 6

Urease Aceton Jackbean 30%

Aceton 40%

Aceton 50%

Aceton 60%

Hnh 1. Kt qu in di urease ta cc nng aceton khc nhau

in di trn hnh 1 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 enzym 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 enzym urease c hiu qu nht v tinh sch cng nh hiu sut thu hi enzym.

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3.1.2 Kho st ta phn on bng (NH4)2SO4 (theo phng php mc 2,2) Kt qu xc nh tinh sch v hiu sut thu hi ca hai phng php ta phn on sunfat amon c trnh by bng 2. Bng 2. So snh hiu qu tinh sch ca 2 phng php ta phn on (NH4)2SO4 Giai on
Trch ly P.Php 1 P.Php 2

A, Vdch ,ml P, mg/ml Tinh Pt , mg UI/ml At ,UI Ap UI/mg pr sch mbt ,mg (mg/mg) (UI/mg)
150.00 1896.72 1214.25 19.69 0.19 0.19 2953.50 104.40 15660.00 360.38 230.71 1.60 1.36 3034.75 1651.38 5.30 8.42 7.16 1.00 1.59 1.35

H,%
100 19.38 10.55

Cc s liu thc nghim cho thy phng php c tinh sch enzym v hiu sut thu hi enzym u cao hn so vi phng php 2. Tuy nhin hiu sut thu hi enzym 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 5.
1 2 3 4 5 6 7 Urease Jackbean Trch ly Ta PP1 Ta PP2

Hnh 2. 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 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.

3.1.3 Kho st qu trnh tinh sch urease bng phng php sc k trn ct Sephadex G50
Ly 0,5ml dch trch ly iu chnh v pH 7 cho ln ct Sephadex G-50). Ra ct bng m phosphate pH7; 1/15M. Kim tra hm lng protein v hot tnh enzym mi phn on.

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Sc k trn Sephadex G - 100
OD 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 2 4 6 8 10 12 14 16 18 20 ng
Protein Hot tnh

Urease jackbean Trch ly Qua Cephadex

Hnh 3. Sc k ca dch trch ly t u nnh Hnh 4.Kt qu in di mu qua ct khi qua ct SephadexG-50 Sephadex, phn on 4

T sc k cho thu v tr cc i ca peak protein cng chnh l v tr cc i ca peak enzym 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 4. 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 cephadex. 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.

3.1.4 Siu lc Dch trch ly c cho qua thit b lc mng membrane c kch c l 10 000 Da, p sut lc 2.5 bar, lc trong 2h. Sau khi qua siu lc, nng enzym trong mu rt cao. Bng 3. Kt qu nh gi hiu qu tinh sch ca phng php siu lc Giai on
Trch ly Dung dch siu lc Siu lc k

Vdch,ml P, mg/ml P , mg mbt ,mg (mg/mg) t

150.00 90.00 27.28 29.34 4092.00 2640.60

A, UI/ml (UI/mg)
68.40 78.14

Ap , tinh H,% At ,UI UI/mg pr sch

10260.00 7032.60 2.51 2.66 1.00 100 1.06 68.54

6876.40 0.43 2956.85 0.76 5226.06 1.77 0.71 50.94 Khi s dng mng lc 10 000 Da l qu nh so vi phn t Urease nn hiu qu tinh sch khng cao, ch lm dch enzym 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, EDTA-Na2 b loi khi siu lc.

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 4. Bng 4. So snh hiu qu tinh sch ca cc phng php Giai on
Trch ly Dung dch siu lc SK.Cephadex

Ap, VTr.ly ,ml P, mg/ml A, UI/ml Tinh At ,UI H,% P , mg (UI/mg) mbt , mg (mg/mg) t UI/mg pr sch
150.00 90.00 300.00 27.28 29.34 7.88 4092.00 2640.60 2362.50 68.40 78.14 23.08 10260.00 7032.60 6924.00 2.51 2.66 2.93 1.00 100

1.06 68.54 1.17 57.88

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(NH)4SO4 65% Aceton 60%

1906.00 4837.13

0.39 0.46

743.34 2225.08

1.48 1.47

2820.88 7110.58

3.79 3.19

1.51 27.49 1.27 69.30

Kt qu cho thy ta phn on (NH4)2SO4 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, enzym rt d bin tnh. Hn na, bt sau khi ng kh ho tan rt km. Bt ng kh thu c t mu ta sun fat amon c tan rt tt. Kim tra 4 mu qua in di trn gel acrylamid 10%, kt qu c trnh by hnh 5.

1 2 3

4 Urease jackbean Siu lc Qua Cehadex Ta (NH4)2SO4 PP1 Ta Aceton 60%

Hnh 5. Kt qu in di urease qua 4 phng php tinh sch

Kt qu in di hon ton ph hp vi s liu thc nghim bng 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, chng 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 Kt qu tinh sch trn ct sc k trao i ion DEAE - cellulose Trong phn ny, chng ti thc hin 2 th nghim song song: 1) Mu enzym 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 pH7 1/15M, gradient bng mui NaCl nng t 0 1M. Sc k c trnh by hnh 6. 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 hnh 7.

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O D 0.25 0 .2 0.15 0 .1 0.05 0 1 2 3 4 5 6 7 8 9 101112131415 161718
P ro tein Nng mui Ho at tnh

N ng mu i 0.6 0.5 0.4 0.3 0.2 0.1 0

ng

OD 0.2 0.15 0.1 0.05 0

P rotein Nng mui

Hoat tnh

N ng mu i 0.6 0.5 0.4 0.3 0.2 0.1 0

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8On g

Hnh 6. Sc k trn DEAE-Cellullose Hnh7. Sc k DEAE-cellulose bt ta ca dch ta bng (NH4)2SO4 phg php 1 (NH4)2SO4 phng php 1 ng kh Sc k cho thy khi tch phn on dung dch enzym sau ta (NH4)2SO4 nh sc k trao i ion thu c 3 peak protein v 1 peak enzym. V tr cc i ca peak enzym trng vi v tr cc i ca peak protein th 3 nng mui 0,3M. Enzym ta phn on (NH4)2SO4 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 enzym. Peak enzym cng trng vi peak protein th 3, nhng hot tnh enzym 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)2SO4 v ta (NH4)2SO4 qua ng kh, enzym urease c y ra nng mui 0,3M. Peak cha enzym urease thu c sau sc k trao i ion c kim tra tinh sch qua in di, kt qu trnh by hnh 8.

Urease jackbean

Trch ly

Ta (NH4)2SO4

Sau DEAE-cellulose

Hnh 8.Kt qu in di qua cc giai on tinh sch

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 [18]. 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 5. Kt qu tng kt qua cc giai on tinh sch

Giai on Trch ly Dung dch (NH4)2SO4 DEAE-Cell Ddch (NH)4SO4 k DEAE-Cell k Vdch,ml mbt ,mg 150.00 75.00 225.00 1897.23 379.45 P, mg/ml (mg/mg) 24.66 14.25 2.46 0.35 0.98 Pt , mg 3669.00 1068.75 553.50 664.03 371.86 A, UI/ml (UI/mg) 62.47 55.29 10.13 1.34 5.07 At ,UI 9370.50 4146.75 2279.25 2542.29 1923.81 Ap UI/mg tinh pr sch 2.55 1.00 3.88 1.52 4.12 1.62 3.83 1.50 5.17 2.03 H,% 100 44.25 24.32 27.13 20.53

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Nh vy, qua mi cp tinh sch th urease dn dn c tch ra khi cc protein tp khc. mu ta (NH4)2SO4 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 (NH4)2SO4 v t 2,03 vi hiu sut thu hi enzym l 20,53.

4.KT LUN
Qua qu trnh nghin cu chng ti gii quyt c vn sau: - So snh cc phng php tinh sch s b enzym 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. - Sc k trao i ion trn DEAE-cellulose thu c 3 peak protein v 1 peak enzym urease ng vi nng mui 0,3M. - xy dng c mt quy trnh thu nhn v tinh sch enzym 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 enzym l 20,53. - S dng in di kim tra tinh sch enzym v xc nh c urease u nnh l loi homogeneous.

EXTRACTION AND PURIFICATION OF UREASE FROM SOYBEAN

Le Thi Phu, Nguyen Thi Cam Vi, Nguyen Tan Dat Ton Duc Thang University ABSTRACT: In this work, we compared some methods of urease preliminary purification: ammonium sulfate fractionations, acetone precipitation, gel filtration chromatography and ultrafilter. Determination of enzym fold pury and recovery yield, acrylamid-gel electrophoresis of urease from each method showed amonium sulfate fractionations with 65% and 55% saturated concentrations was the most effective. Purification was carried out continuously by DEAEcellulose column, the chromatograph showed three peak protein and only one peak urease at 0,3 M salt concentration. Acrylamidgel electrophoresis of urease of the highest specific activity peak indicated that soybean urease was homogeneous enzym. The result of electrophoresis indicated our purification process was effective and pure urease was obtained. TI LIU THAM KHO
[1]. Phm Th nh Hng, K thut sinh ha, NXB HQG Tp.HCM, 2003. [2]. Nguyn Thi Vit Hng, Tch chit urease t u nnh v ng dng xc nh urease trong bnh phm, Lun n thc s HKHTN, 1999. [3]. Nguyn c Lng v cc tc gi, Cng ngh enzyme, NXB HQG Tp.HCM, 2004. [4]. Nguyn Vn Mi, Thc hnh sinh ha, NXB KH&KT H Ni, 2002. [5]. L Th Ph, Bi ging cc phng php nghin cu ha sinh hin i, HBC Tn c Thng. [6]. o Hu Vinh v cc tc gi, Cc phng php sc k, NXB KH&KT H Ni, 1985. [7]. Cristian Follmer v cc tc gi, Jackbean, soybean and bacillus pasteurii urease biological effects unrelated to ureolytic activity, Eur. J. Biochem. 271, 1357-1363, Cambridge, 2004.

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[8]. E. G. Schmidt, The inactivation of urease, The department of biological chemistry of the university of Maryland schoolof medicine, Baltimore, 1928. [9]. Frederick J. Dechow, Separation and purification technique in biotechnology, Noyes publications, New Jersey, 1989. [10]. Henry Tauber, Israel S. Kleiner, The toxicity of crystalline urease, medical college and flower hospital, J. Biol. Chem. 92, 177, New York, 1931. [11]. Irwin W. Sier, The activation energy of urea hydrolysis catalyzed by soybean urease, Eur. J. Biochem. 132, 209, Cambridge, 1939. [12]. J. G. Mateer, E. K. Marshall, The urease content of certain beans, with special referenace to the jack bean, John Hopkins University, Baltimore, 1916. [13]. Jack Peterson v cc tc gi, The dependence of the specific activity of urease upon the chemistry, California Institute of Technology, Pasadena, 1970. [14]. James B. Summer, The isolation and crystallization of the enzym urease, J. Biol. Chem. 69, 435 - 441, New York, 1926. [15]. K. Takisnima, T. Suga, The structure of jack bean urease, the complete amino acid sequence, limited proteolysis and reactive cystein residues, Eur. J. Biochem. 175, 151 165, Cambridge, 1988. [16]. Stacey F. Howell, James B. Sumner, The specific effects of buffer upon urease activity, J. Biol. Chem. 104, 619, New York, 1934. [17]. Isobel J. Rosenstein, Jeremy M. Hamilton - Miller and William Brufitt, Role of urease in the formation of infection stones: comparision of urease from different sources, 1981. [18]. Joseph C. Polacco v Evelyn A. Havir, Comparisions of soybean urease isolated from seed and tissue culture, www.biomedcetral.com, 1979. [19]. Prem P. Sehgal and Aubrey W.Naylor, Purication and properties of urease derived from hydrated seeds of jack bean, Canavalia ensiformis (L), Plant Physiology. 4, 567 572, Horsham, 1966.

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