XC NH PHN T LNG V CC THNG S NG HC CA UREASE T U NNH HT VNG VIT NAM

L Th Ph, Nguyn Th Cm Vi Trng i hc Bn cng Tn c Thng 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. Trn th gii, c rt nhiu nh khoa hc nghin cu v enzym urease v v cc c tnh ca chng, tuy nhin cc bi nghin cu v urease t cc ngun nguyn liu u khc nhau cho cc kt qu khc nhau v c tnh urease. iu ny chng t urease t cc nguyn liu u khc nhau c th c mt s khc bit v tnh cht. Cn Vit Nam ch mi c cc nghin cu tinh sch urease, hon ton cha c tc gi no nghin cu no v cc c tnh urease t nguyn liu u nnh Vit Nam. c th ng dng urease trong Y hc v Thc Phm hiu qu rt cn thit phi nm c cc c tnh urease. 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 s c tnh urease u nnh ht vng Vit Nam: phn t lng ca urease v chui tiu n v, cc thng s ng hc ca urease. 2. NGUYN LIU V PHNG PHP NGHIN CU 2.1 Nguyn liu Urease u nnh ht vng Vit Nam: c tinh sch qua cc cng on: trch ly vi dm phosphate pH 7, 1/15M cha 0,05M EDTA v 0,05M L-cystein; ta phn on sunfat amon 65% v 55% bo ha; trao i ion trn DEAE cellulose; ng kh bo qun di dng bt trng, mn. Ho cht: dy protein chun 10 000 250 000 Da v 35 000 400 000 Da, Nessler (Merck), Glycine, Tris(base), EDTA, L-Cystein.HCl, Acrylamide v bisacrylamide, SDS, TEMED, Amonium persulfate, DEAE Cellulose (Merck). 2.2 Phng php nghin cu Phng php Nessler: xc nh hot tnh urease [4] Phng php in di: in di trn gel 7,5% v 10% polyacrylamid vi tc dng 20mA, gel c nhum vi Coomassie Blue R-250. [1] Xc nh phn t lng ca urease: lp th ng chun biu hin s tng quan gia phn t lng protein chun v khong di chuyn ca chng khi in di trn gel polyacrylamyd. T khong di chuyn ca urease, bng phng trnh ng chun trn chng ti s xc nh c phn t lng ca urease. [19] Xc nh vn tc phn ng thu phn ure ca enzym urease: da vo lng NH3 hnh thnh xc nh bng phng php Nessler, t tnh c lng ure phn hu theo thi gian. 3. KT QU 3.1 Xc nh phn t lng ca urease u nnh: Trong th nghim ny, chng ti s dng phng php in di trn gel poliarylamid 7,5% xc nh phn t lng cc mu urease u nnh ht vng Vit Nam tinh sch. Trong m mu chng ti khng s dng SDS v -mercaptoethanol nhm gi nguyn phn t lng urease chy trong in trng in di. so snh chng ti dng cc cht chun c phn t lng t 35

000 400 000 Da. Mu enzym urease ho tan trong m mu, x l nhit, sau qua in di trn gel polyacrylamid 7,5% vi m in di cng khng b sung SDS. Theo [19] mun xc nh phn t lng ca mt cht thng qua cc cht chun trn gel polyacrylamide trc ht phi lp phng trnh ng chun vi cc thng s tng quan tuyn tnh l phn t lng ca dy cht chun c khong cch di chuyn ca chng trn in di . Trong th nghim chng ti xc lp phng trnh ng chun vi dy protein chun c phn t lng t 35 000 400 000 Da. Kt qu hnh 1 v bng 1.

Hnh 3.1 in di phn tch phn t lng urease u nnh

Bng 3.1: Kt qu phn tch tng quan gia phn t lng dy protein chun v khong cch di chuyn ca chng trn in di gel 7.5% Phn t lng M (kDa) 400 250 160 105 75 50 5 Khong cch di chuyn d (cm) 0,3 1,3 2,1 2,9 3,5 4,6 5,1 5,99146 5,52146 5,07517 4,65396 4,31749 3,91202 3,55535 ln(M)

6.5 6.0 5.5 ln(M) 5.0 4.5 4.0 3.5 3.0 0 1 2 3 4 Khoa ng di chuye n (cm) 5 6 y = -0.5024x + 6.1392 R = 0.997
2

Hnh 3.2: th tng quan gia phn t lng protein chun v khong cch di chuyn ca chng khi in di trn gel polyacrylamide 7,5%

Da vo kt qu phn tch bng 1, chng ti xy dng c ng chun xc nh phn t lng vi trc honh l khong di chuyn protein chun v trc tung l Ln(phn t lng protein chun). ng chun do chng ti xy dng c chnh xc cao vi phng trnh y = - 0,5024x + 6,1392. iu ny chng t chng ti hon ton c th da vo ng chun trn xc nh phn t lng ca urease u nnh Vit Nam. Trn in di thy r khong di chuyn ca urease u nnh l d = 0,1 cm. T phng trnh ng chun va tm xc nh c phn t lng ca urease u nnh vo khong 440 000 Da. 3.2 Xc nh trng lng ca cc chui tiu n v Trong th nghim ny, vi mc ch xc nh trng lng cc tiu n v trong phn t urease, chng ti ng thi s dng cc tc nhn SDS, - mercaptoethanol ct cu trc bc 4 urease u nnh thnh cc tiu n v mang in tch m, v qu trnh in di c tin hnh trn gel polyacrylamide nng 10% vi cng dng 20 mA. Cc cht chun l nhng protein c phn t lng 10 000 250 000 Da. Kt qu thu c nh sau

Vch xut pht

Hnh 3.3: in di xc nh trng lng chui tiu n v ca urease u nnh (Mu x l bng SDS, - mercaptoethanol; gel 10% , dng in 20mA)

Bng 3.2: Kt qu phn tch tng quan gia phn t lng protein chun v khong di chuyn ca chng khi in di trn gel acrylamide 10% Phn t lng M (kDa) 250 160 105 75 50 35 30 25 15 10
6.0 5.5 5.0 4.5 ln(M) 4.0 3.5 3.0 2.5 2.0 0 1 2 3 4 5 Khoang di chuyen (cm) 6 7 y = -0.5158x + 5.3109 R = 0.9651
2

Khong di chuyn d(cm) 0,35 0,65 1,1 1,5 2,3 3,1 3,6 4 5,4 6

ln(M) 5,52146 5,07517 4,65396 4,31749 3,91202 3,55535 3,40120 3,21888 2,70805 2,30259

Hnh 3.4: th tng quan gia phn t lng protein v khong cch di chuyn ca chng khi in di trn gel acrylamide 10%

Trn in di hnh 4 cho thy urease u nnh sau khi x l bng SDS, -mercaptoethanol; gel polyacrylamide 10% xut hin mt vch ca cc tiu n v ca phn t urease, khong cch t im xut pht ti vch d = 4,8 cm. iu ny chng t cc tiu n v ca urease u nnh ht vng Vit Nam c cng trng lng, Da vo phng trnh ng chun y = - 0,5158x + 5,3109 ( th hnh 3.12) tm khi lng tng ng vi khong cch 4.8cm, s l phn t lng ca tiu n v urease u nnh. Trn ng chun trong th nghim ca chng ti, ng vi khong cch 4,8 cm chng ti xc nh c trng lng ca tiu n v urease l 17000Da.

3.3 Xc nh cc thng s ng hc ca urease u ra v u nnh: Thng s ng hc ca enzym l tc phn ng Vmax v hng s Michaelis Km. Tc ca phn ng enzym trong mt gii hn no ph thuc vo nng c cht c trong mi trng. Chng no enzym cha b bo ho bi c cht th tc phn ng vn t l thun vi nng c cht. Do chng ti kho st bin thin vn tc phn ng thu phn ure ca urease u nnh v u ra bng cch thay i nng ure t 1 100 mM tm khong nng ure m trong enzym cha b bo ho bi c cht. Qu trnh xc nh ng hc enzym urease c tin hnh nh sau: ho tan bt enzym urease u ra (Merck) v urease u nnh tinh sch bng m phosphate 1/15M, pH7 c dung dch enzym hot lc 20UI Summer/1ml (n v hot lc UI Summer c nh ngha l lng enzym c kh nng thu phn ure to thnh 1g NH3 trong 1 pht iu kin tiu chun).B sung 0.5ml dch urease 20 UI Summer/1ml vo cc ng nghim c sn ure vi nng tng dn t 1 100 mM, 300C trong 5 pht. Lng NH3 to thnh c xc nh theo phng php Nessler, qua tnh ton tm c lng c cht ure b thu phn ri tnh c vn tc phn ng enzym urease. Kt qu thu c nh sau:
8 7

V (mM/phut)

6 5 4 3 2 1 0 0 10 20 30 40 50 60 70 80 90 100

Nong o urea (mM)

Hnh 3.5: th biu din s bin i vn tc thu phn ca enzym urease u ra khi thay i nng c cht ure
7 6

V (mM/phut)

5 4 3 2 1 0 0 10 20 30 40 50 60 70 80 90 100

Nong o urea (mM)

Hnh 3.6: th biu din s bin i vn tc thu phn ca urease u nnh khi thay i nng c cht ure.

T ng bin thin tc phn ng enzym c 2 th cho thy c urease u nnh v u ra hot tnh 10 UI Summer u b bo ho c cht khi nng ure 30 mM (tc tc phn

ng khng tng khi nng c cht tng). nng ure 30 mM, vn tc thu phn ure ca enzym urease u nnh v u ra tng dn khi tng nng c cht ure. Nh vy, cc thng s ng hc ca urease c th c xc nh khi thay i khong nng c cht 30 mM ng vi 10 UI Summer urease. Cc thng s ng hc ca urease u ra v urease u nnh thu c t qu trnh tinh sch trn c xc nh da trn tng quan vn tc thu phn ca enzym urease v nng c cht. lp ng thngn th hin mi tng quan gia vn tc phn ng thu phn v nng c cht chng ti xc nh vn tc thu phn ca enzym urease nng c cht t 1 10 mM vi hot tnh enzym 10 UI Summer. Chng ti xy dng ng thng tng quan gia vn tc thu phn ca enzym v nng c cht theo phng trnh Lineweaver Burk: Km 1 1

v
Kt qu phn tch nh sau:

V max

[s]

+ V max

Bng 3.3: Kt qu xc nh vn tc thu phn ca urease u ra [S] (mM) 1 2 3 4 5 6 7 8 9 10 [V] (M/pht) 0,971 1,606 2,065 2,524 2,700 2,771 3,194 3,265 3,406 3,441 1/[S] (mM)-1 1 0,5 0,333 0,25 0,2 0,167 0,143 0,125 0,111 0,1 1/[V} (mM/phut)-1 1030,30 622,71 484,33 396,27 370,37 360,93 313,08 306,31 293,61 290,60

Bng 3.4: Kt qu xc nh vn tc thu phn ca urease u nnh [S] (mM) 1 2 3 4 5 6 7 8 [V] (M/pht) 1,076 1,818 2,382 3,053 3,265 3,512 4,006 4,112 1/[S] (mM)-1 1 0,5 0,333 0,25 0,2 0,167 0,143 0,125 1/[V} (mM/phut)-1 928,962 550,162 419,753 327,553 306,306 284,757 249,633 243,205

9 10

4,218 4,324

0,111 0,1

237,099 231,293

1200 1000 1/V ( mM/pht) -1 800 600 400 200 0 0.1 0.3 0.5 0.7 1/[S] (m M ) -1 0.9 1.1 y = 827.14x + 204.58 R2 = 0.9985

Hnh 3.7: th biu din s ph thuc gia 1/V v 1/[S] ca urease u ra

1000 800 1/V ( m M/pht) -1 600 400 200 0 0.1 0.3 0.5 0.7 1/[S] (m M)-1 0.9 1.1 y = 785.29x + 147.86 R2 = 0.9984

Hnh 3.8: th biu din s ph thuc gia 1/V v 1/[S] ca urease u nnh

Bng 3.5: Cc thng s ng hc ca urease u ra v u nnh u u ra u nnh Vmax (mM/pht) 4,89.10-3 6,77.10-3 K m (mM) 3,84 5,3

T kt qu phn tch cho thy gi tr Km ca urease u ra thp hn u nnh, iu ny c th gip chng ta kt lun urease u ra c i lc vi c cht ure ln hn urease u nnh nhng s khc bit ny khng ln lm. Kt qu tnh gi tr Vmax cho thy urease u nnh c gi tr Vmax cao hn urease u ra.

4. KT LUN Sau qu trnh nghin cu, chng ti xc nh c mt s c tnh urease u nnh v u ra nh sau: c tnh Trng lng phn t (Da) Trng lng chui tiu n v (Da) Km (mM) Vmax (mM/pht/10UI summer) u nnh 440 000 17 000 5,3 6,77 u ra 3,84 4,89

y l mt s c tnh quan trng h tr cho nhng nghin cu tip theo a urease u nnh vo ng dng trong Y hc v Thc Phm.

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