HQI NGH! KHOA HUU U U N t i NGH^ SINH H("
KHAO SAT QUA TRlNH CO DINH ENZYME UREASE T R S N CHITOSAN
Lfi Thi M9 Ngoc', Huynh Ngpc Oanh^
' £>?( /lpc y duoc Thanh ph6 H6 Chi Mirih ' Tnrdng Dgf hgc Bach khoa Thinh pho Hd Chi Minh
T 6 M TAT
Urease duoc diing trong cac kit thii djnh hrong urea mau va nude tieu dl chSn doin va tiieo doi chdc nang tiign. T\ry nhi&i enzji nay ihmmg dugc nhap khau vdi gid tiiinh kha cao. Trong khi d6, b Vi?t Nam da so cac n ^ e n cuu chi tSp tnmg tSch chiet cnz)i ui4se tii dau ninh nhung dS tang khA nang tai sii dung san phim nay thi con tm che. Do do chiing tdi tien hinh nj^fin cuu cicdij kien c6 djnh enzyme urease trfin chitosan.
Cic khio sat dat ka qua tli mi vdi ti \& enzyme/chit mang la 2,46 mg/lg hat chitosan. Ho?! tinh enzyme urease c6 djnh trfin chitoji U 65% vdl s6 lan tai sii dyng IS 14 lan v6i hogt tinh enzyme duy tri trSn 50% so vdi ban dSu, Nhi|t dp vk pH tfii uu cila enzyi urease dang c6 dinh tren h?t chitosan la 70°C vi 7,5, dang hr do 14 eO'C vi 7,0. Cac thSng sfl dpng hgc V,^, Kn, cua enzyme c6 il vi tv do cung dugc xic djnh, lln lugt li 0,663 ((imol/mg.phiit), 225 mmol (enzyme co djnh tren hat chitosan) vi l^
{pmol/mg.phut), 92,5 mmol.
Sin phim enzyme urease c6 dinh tren chitosan dugc iing dyng phan tich urea trong huySt tiianh benh nhan vdi ket qua mong Aq vifri ket qua do dugc lit miy sinh hda X\f dgng
Tit khoa. ciak tach urease tfi d|u ninh, urease c6 dinh, chitosan, x6t nghi?m urea mau.
MtiroAu J Urease (EC 3.5.1.5) thuOc nhbm enzyme thuy phSn, dfing thfri ii mOt trong nh&ng enzyme du'Q'c li'ng dyng nhilu trQ|
hgc vi dc ITnh vyc khdc nhu thyc pham, mfii truong... En2yme urease thufi'ng duvc dCing Uong c^c kit thCr dinh lu||
urea mau vk nude tifiu hay h§ thfing chgy thgn nhSn tgo, Hlu het enzyme urease dup'c nh^p khau tii' nude ngoii ri gid thSnh khd dat. (3 nirdc ta, enzyme urease ti> d§u ndnh dS dugc nhieu tac gia nghifin cCru cdc phuong phdptN nh|n, dnh sgch (Nguyln Thi Vifit Huong, 1999; Nguyen Thj Clm Vi, 2007) nhung chua c6 nghien cuu ndo cho vi^J djnh sdn phfim enzyme ndy trfin chit mang chitosan d l ilng dyng trong y hpc. l/u dilm ciia chit mang ndy la re ti^n,i^
todn sinh hpc va cfi linh t^llt khdng khuin nfin thufi'ng dugc su dyng frong y hpc vd c6ng nghfi sinh hpc. Do 664 ngufin nguyen lifiu Id hat ddu ndnh Vifit Nam va chit mang chitosan, vi?c nghidn ci>u thu nhdn, tang kha nang tdli^
dyng cOa enzyme urease bing phuang phdp cfi djnh dfi ndng cao tinh li-ng dung Id cfin thifit. i NGUYEN LIEU VA PHl/aNG PHAP NGHI&N CCTU
NguySn li$u
- Hgt d^u ndnh (Xu3n Nfing) tdch bdo bing dung mfii diethyl ether.
- Hfia chat:
Chitosan (Dgi hpc Thuy sdn Nha Trang): chilt xuat ti> vd tfim.
Diethyl ether. NaOH, amonium sul^te, acid acetic (Trung Qufic), tris-aminomelhane (An Op); thuoc thip Bradford; tll|
thu Nessler (Merck), glutaraidehyde (Merck).
Hfia chit djnh lupng urea trong mau (Human), EDTA-Naj, trichloracetic acid ...
Phucmg phap nghi@n ciru
Logi lipid bang he thfing Soxhlet: khdo sdt nguon nguyfin lifiu fi'thoi gian tach b6o: 6h, 8h, lOh, 12h, 14h.
Thu nhdn enzyme urease; tua phdn doan 6' 2 nong dfi mufii (NH4)2S04 bao hba 65% vd 55%.
Xdc djnh ham lupng protein theo Bradford (Nguyin Vin Mui, 2002).
Xdc i^nh hoat tinh enzyme urease (Nguyln Van MCil, 2002).
Cfi i^nh enzyme urease trfin chitosan bing lien kfit cfing hfia trj:
7^0 h^t diitosan (Kumar vd cfing su, 2009)
Dung djch chitosan 2% (w/v) dupc pha trong acid acetic 1,5% cho tan d nhifit dfi khodng 60°C bing bfip khuly tif- iM mdt dung djch (4-8 C). Tgo hgt chitosan bang cdch nhfi dung djch chitosan 2%vdo NaOH 1M. Hgt tao thdnh duvcw nhilu lln voi nufic cfit vd tifin hdnh hoat hoa vfi'i glutaraidehyde 1 % trong 6h 6' 37°C. Hgt tgo thanh duoc rua bling dfl djch d§m his 0,2 M pH 7,0, EDTA-Na2 ImM va tru d 10 ignh 4-8°C trong dung djdi d§m cho din khi su dyng,
Cij dinh enzyme len h^t chitosan da hogf hda i Hat chitosan sau khi da hogt h6a u vol dung djch enzyme urease 3mg/ml theo ti l# I g hgt vdi cdc the tich enzymel|
luiTt Id 2,5, 3; 3,5; 4; 4,5; 5 mi. D l h5n hop qua dfim trong tu lanh. Sau dfi n>a hgt bing dpm tris 0,2 M pH 7,0, EDi
Naz 1 mM '
„ ^ H Q C C O N G N G H E S I N H H O C T O A N Q U O C 2 0 1 3
O j n h l u g m g urea m d u t i l i n g kit t h u f i c thi> u r e a c u a h a n g H u m a n tren m d y sinh h o a tg d ^ n g Hitachi 9 1 7 .
K t r Q U A V A T H A O L U A N
K h d o s a t n g u d n n g u y f i n l i S u fi* c a c thfiri g i a n t a c h b d o k h d c n h a u B a n g 1 . A n h h u d n g cua t h M gian tach b t o d i n h i f u suat t h u nhan enzyme
T h d n g s f i A n .
(U/ml) I^ An. (U) Acp (U/mg c h l pham) Hi$u s u i t thu nh9n
enzyme {%) KhOng tdch b t o
6h 8h lOh 12h 14h
7,3 7.7 9-0 9,5 10,9 10,3
805,7 843,7 992,2 1041,7 1199,0 1130,8
0,86 1,02 1,36 1,15
193,5 205,5 245,8 270,2 312,8 2G6,8
24,0 24.4 24,8 25,9
Ghi diu: ATC tdng hoat tinh djch trich ly; ACP: tdng ho^t tinh chi p/iam
K i t q u d BSng 1 c h o t h i y k h a n a n g thu n h § n e n z y m e u r e a s e t a n g theo thcri gian tdch b f i o v a d g t gid h i c u e dgi tgi m a u t d c h bfio 1 2 h vcri h o g t t f n h Id 3 1 2 , 8 U . H o a t tfnh c f i a die p h a m e n z y m e s o v f i i dich c h i l t dgt 26,1 % c a o hem nghign c u u t r u d c d a y dfii v 6 i hgt d ^ u n a n h V i ^ t N a m c f i cCing thd'i gian tach b e o 12h (19,38%) ( N g u y l n T h j C i m V i , 2 0 0 7 ) . N h u v g y hogt l i n h c u a d u n g d j c h c h i l t v a c h l p h i m e n z y m e thu d u p c tang d a n khi tdch bfio tO 6 - 1 2 h , dgt gia trj c a o n h l t 6- m l u t d c h bfio 1 2 h l l n l u g l Id 1 1 9 9 U vi 3 1 2 , 8 U ( B d n g 1), c a o h o n h i n s o v f i i Wiong tach b f i o . Hifiu s u i t thu n h d n e n z y m e c u n g t a n g ( 2 6 . 1 % ) . D d y la d o t o n g q u a trlnh chiet n i t lipid, l u p n g lipid s e d u p c logi b f i d a n t h e o thd'i gian nfin l u g n g e n z y m e t h u d u ^ r c cQng t d n g d i n .
N g o d i r a hifiu s u i t t h u n h g n fi' thd'i gian 1 2 h t h i hogt t i n h c h l p h i m dgt mOc c a o n h l t c h u n g tfl l u p n g lipid c f i t h f i d a dirge chiet n i t d f i n mi>c t o j d a . T u y nhifin khi t a n g t h o i g i a n tdch b d o Ifin 14h t h i hogt tinh c f i p h i n g i d m d i n d i n hifiu s u i t thu n h $ n c u n g g i a m d a n . D o d f i c h u n g tfii c h p n t h d i gian t d c h bfio Id 12h d e t h u nhfin e n z y m e tot n h a t C h l p h a m u r e a s e t i p d o
B i n g 2. Hofit tinh cua c h l p h i m urease t ^ d o
Hogt tlnh(U/mg chfi p h i m ) Hdm lupng protein
(mg protein/mg c h l p h i m ) Hogt tlnh n6ng{U/mg protein)
K h d o s a t d n h h u d ' n g c u a t i lfi e n z y m e / v g t l i # u d i n h i e u s u i t c f i ^ n h p r o t e i n B&ng 3. A n h huo-ng cda ti I f enzyme/chltosan d i n hl^u s u i t c o djnh protein
Thdng s f i Vdd enzyme (ml) I PBD (mg) I PcB (mg) Hco(%)
2,5 1.55 1.31 84.5
3.0 i,8e 1.6C
86,C 87,1
2,48 2,18 87,9
4,5 2,79 2,46 88,2
3,10 2,43 78,4 GW chl); Vdd: the tidi dung djch; PBD-- lugng protein tian diu; Pc^- lugng pmtein c6 djnh: Hce: hiiu suit a K h a o s a t d n h h u f l ' n g c f i a t i 1^ e n z y m e / v g t l i g u d i n h i f i u s u i t c o d j n h e n z y m e Bang 4. A n h h u o m g cua ti I f enzyme/chttosan d e n h l f u suat c o dinh enzyme
K i t q u i M a u l M l u 2 M l u 3 M l u 4
Vdd enzyme (ml) I A M ( U ) 5Aco(U) H.(%)
2.5 10,23 5,46 53,4
3,0 12,27 7,16 58,3
3,5 14,32 8,79 61,4
4,0 16,36 10.41 63,6
20,45 12.02 58.8 Ghi did: Ago. hoat tlnh enzyme ban diu; Aa>: hoat tlnh enzyme c6 dnh; H^ diu sua! c6 djnh enzyme
K i t q u d k h d o sdt (BSng 3, H i n h 1) cho t h i y v d i t h f i tich e n z y m e c d n g tang thi l u p n g protein c f i djnh c u n g tang theo ( m l u 1-5) v d l u v n g e n z y m e t i i d a khi g a n len hat c h i t o s a n id 2 , 4 6 m g ( m l u 5), s a u d f i d u c f i tang l u p n g e n z y m e thi c u n g k h f i n g t h i lifin kfit vfi'i hgt chitosan d u p c nOa, d o n g t h a i hogt t l n h enzyme cfi djnh s e g i a m (Bang 4 , H i n h 2 ) . Ngoai r a hifiu s u i t c f i djnh e n z y m e urease dgt gld h i c a o nhat la 6 5 % . K i t q u d ndy t u o n g d u o n g vcri nghifin ci>u c 6 djnh e n z y m e u r e a s e trfin c h i t o s a n c u a Kayastha v d c f i n g s y (2001) ( 6 4 % ) n h u n g t h a p h o n nghifin c t f u cfia K u m a r v d cfing s y (2009) ( 7 7 % ) . Mfit s f i nghifin c d u khdc v l c o flnh e n z y m e u r e a s e c u n g c h d n g tfi hieu qua c u a qua trinh c f i djnh h f i n m d n g c h i t o s a n rat c a o , hogt tfnh e n z y m e g i d dirge d i n 9 4 % s o v d i d g n g t y d o (Krajewska et ai. 1990), c a o h o n c o flnh trfin hgt gelatin (68%) ( K u m a r ef ai. 2 0 0 5 ) . N h u vfiy v d i I g hgt chitosan s e c f i flnh d u p c tfii d a la 2,46 m g e n z y m e
B v d i hogt t l n h e n z y m e c f i djnh Id 6 5 % K i t q u i nay c f i h i l n v o n g cho cdc k h a o sdt t i l p theo.
IllIII. ' lUIII
M l u l M k i ] M l u 3 M 1 U 4 M J U ^ M i u 6 M ) u l M l u j M S U I u S u a M K U S Mii>« _ _
Hinh 1 Anh huwng eua tl If enzyme/chltosan den hlfu sua! Hlnh 2. Anh hud'ng cua ti If enzyme/chltosan din hEfutt|
cfi dinh pratein <» djnh enzyme ^
Khao sat dnh h u ^ g cfia nhifit dfi d i n hogt tinh enzyme urease i Khi nhifit dfi tdng thi hogt tlnh urease dang ty do vd co djnh deu tdng vd khi dgt d i n SO^C dfii vdi enzyme ty do vd 70^
dfii vdi enzyme cfi flnh (Hinh 3) thi hogt tinh dat eye dgi, sau dfi neu tang nhifit dfi thi hogt tfnh sfi giim.Kit qui ni cung phu hgp vdi nhilu nghifin cdu truwc day. Chen va Chiu (1999) da nghien cuu co flnh enzyme urease ti> d^u ^ tren b l mgt hgt chitosan vd nh§n thay ring d cung nhifit dfi Id 70°C tht dgng t y do hi m i t di mfit nua hogt tfnh ban dl|
sau 70 phiit, trong khi dfi dfii vdi enzyme cfi dinh trfin chitosan thi cin din 175 phiit mdi bj mat di mfit ni>a hogttfnhj^
vdi ban dfiu. Nghifin cdu cOa Kayastha vd cfing sy (2001) cung cho thiy enzyme urease tCr dfiu Hd Lan khi cfi djnh trSu alginate cfi dfi bin nhifit Ifin dfin 77°C nhung enzyme ty do chl cfi 47°C. Trong khi dfi enzyme urease tir ddu ndnh d nhifit dfi tfii uu Id 65''c, d n ndng luo'ng hogt hoa Id 3,68 kcal.mol" nhung khi co djnh tren hgt chitosan thi cfi thi diji dupc nhifit dfi 75°C vdi ndng lupng 1^ 5,02 kcal.mol"' (Rejikumar vd cfing sy, 1998). Nhu v§y enzyme urease ty do^i cfi flnh cfi dfi bin nhifit cao nfin cfi the ung dung d nhung quy mfi ldn.
Anh tiuvng cua pH
Hlnh 3. Anh hirong cua nhift df din hogt tinh enzyme ty do Hlnh 4. Anh hudng cua pH den hogt tfnh enzyme ty do vli d vd cfi dinti djnh Khdo sdt dnh hufi'ng cua pH d i n hogt tinh enzyme urease
TCr kit qud khao sdt trfin cho thl)^ hogt tinh cOa enzyme urease ty do dgt gia tri cao nhlt tgi pH 7.0, trong khi dfi doi v4 enzyme cfi flnh hogt tinh cao nhat tgi pH 7,5 (Hinh 4). Dilu ndy chdng to enzyme co flnh hoat dfing tfii uu d pH kill hdn enzyme ty do.
Chitosan cfi cdc nhfim amin nfin mang tinh chat cua chat da Ion duong {pKa = 6,5). Trong mfii hirdng kifim thi chilosa se gidm mfit dfi flfin tich duong do c6 cac nhfim OH" bao xung quanh Idm duy hi muc pH thip han b'ong mfii truoT?
phdn ung din dfin sy thay dfii pH tfii uu ve phfa kiem hon. Ket qui ndy cung phu hgp vdi cdc nghiSn cdu tnJ^c dij nhu cua tdc gid Kayastha vd cfing su (2001) vdi pH tfii uu cho enzyme urease td ddu Hd Lan khi cfi flnh trfin chitosai la 8,5 cao hon dgng ty do Id 7,3; hay nghien ciiu cua Kumar vd cfing sy (2009) vfii pH tfii uu cOa enzyme cfi flnh trij chitosan id 7,9 cao hon dgng ty do Id 7,0.
Kfit qud khdo sdt cung da cho thay khoang pH cua enzyme cfi flnh (7 - 8) rfing han enzyme ty do (7 - 7,5) (Hlnh 41 dfing thdi tgi pH 7,5 thi hogt tlnh cCia enzyme cfi flnh vd ty do cfi gid trj gan nhu nhau chtrng tfi enzyme cfi flnh l:6n vi It bi anh hudng bfl'i sy thay dli pH so vdi enzyme ty do.
Khao sat tinh chit dfing hpc cua enzyme urease
Qua khao sdt 3nh h i ^ g cua nfing dfi ccr chit tu 0--l% den hogt ttnh cCia enzyme urease ty do vd cfi djnh, chiing nhgn thiy khi tdng nfing dfi co chit thi toe dp phan ung tdng, tuong ung vdi nfing dfi ca chit lln lupt Id 2% dli enzyme ty do vd 2.5% dli vfii c6 flnh. Tuy nhien khi ldng nong dfi ca chat v u d qua gld hi eye dgi thi v§n tfie phdn j' do enzyme ty do cOng nhu cfi flnh xiic tac deu giam. Dieu ndy dupc gidi thich la do cdc vj fri' lien kfit vdi co chiti enzyme da dupc gdn ket din mdc tfii da, nfin neu tilp tyc tdng ning do co chat thi eo chat cd thfi gin vdo pf enzyme-ca t ^ l t Idm gidm khd ndng xiic tde cua enzyme.
Td nong dp co chit vd toe dfi phdn dng enzyme chung tfii xdy dyng dupc bilu d l Lineweaver-Burk (Hlnh 5).
Td bieu dfi Lineweaver-Burk chung tfli de dang xdc dinh dupc cdc thfing so dfing hgc ciia enzyme urease dgnq tydov*
CO flnh (Bdng 5).
HOC C O N G NGHE SINH HOC TOAN QUOC 2013
mfl -OOIl -DUOS OGOO OUS ODD 0 0 6 01131
12000 1 10000
•b J 8000 u „ 6 0 00 3 o 4 0 00
£ ? 2 0 00 r 0.00
-
1 * * 1 i . j . ~ "
- — —1. ^ * * S t . ^ ^ ^ =
~ ^ ^ ^ ^ ^ r ' ~
z i i : „ : „
— ~"' ~~
I 0 5 10 15 20 S6 l i n 5i> dMng
Hinh 5. Bilu do Lineweaver-Burk bilu diin mil quan hf glira Hinh 6. Khi ning tai 5i> dgng cua enzyme urease co djnh [1/V] vi [1/S] cda enzyme t y do vd cd iflnh
Bdng 5 Cdc thdng s l d f n g hpc Urease V , M
T y do C l d j n h
zia enzyme t y d o (pmol/mg.phl]t)
1,202 0,663
v a o d)nh Km (mmol)
92,5 225,0 Ghi chii: V™: tic dO phdn dng eye dgi; K,,: hing sfi Michaelis-Menten
Qua khdo sdt ehiing tfii xdc djnh dup'c gid trj Kn, cfia enzyme urease cfi flnh gap enzj^e ty do hon 2 lln chung tfi enzyme cfi djnh co di Iyc vdi ca chat tfilp hon enzyme ty do. Kit qud ndy cung phu hpp vcri nghifin edu cfi djnh enzyme urease trfin chitosan cua Kumar vd cpng sy (2009) vdi gia hi Km cCia enzyme co flnh gip gin 1,5 lln so vfii enzyme ty do, hay nghifin cdu ctia Kayastha vd cfing sy (2001) cung dio thiy Km cfia enzyme co flnh cao han enzyme ty do gin 3 lln.
Ngodi ra v$n tdc phdn dng eye dgi do enzyme co djnh xuc tdc cOng thip hon ty do cfi thfi Id do enzyme cfi flnh dup'c gdn chgt Ifin bfi mgt hgt chitosan vd chim xufing ddy dng nghifim Idm cho co chit urea phdi mit nhieu thdi gian ban d l gin vdo tmng tdm hogt dfing eua enzyme. Trong khi dfi enzyme ty do phdn phfii deu trong dung djch nfin khd nang tilp xfic vdi CO chit de ddng nen Idm cho tfie dfi phan dng x i y ra nhanh hon. Tuy nhifin ta efi t h i khic phgc nlu diing cfit ehda enzyme efi flnh vd cho eo chit chdy vdo chdy vdo thi khd ndng xiic tdc ciia e n z ^ e se cao han.
Khdo sdt khd ndng tdi s u dyng cua enzyme urease
Kit qud khdo sdt eho thiy enzyme urease efi flnh frfin chitosan efi so lln tdi su dyng rit cao. Hon nda enzyme efi flnh duy tri dupe tren 70% hogt tinh so vdi ban d i u sau 12 lln su dgng va gida cde l l n sd dyng thi hogt tfnh gidm rat IL Din lln thd 14 enzyme efi flnh van giu duyc frfin 50% (Hlnh 6). Ngodi ra qua thyc nghifim chiing tfii nhgn frily hgt enzyme cfi flnh khfing cfi d i u hifiu truong nd, hgt enzyme bin do duoc gan chgt vdi hgt chitosan thfing qua tay gin Id glutaraidehyde. Cdc nghifin cdu trud'c dfiy cung eho thiy enzyme urease efi flnh frfin hgt chitosan cfi khd ndng tdi sd dgng tu 6 - 14 lan (Ngfi Thj Ty Na, 2009; Sandeep e( al., 2009).
liTng dung urease co djnh trong xet nghifm urea mdu
Bing 6: So sdnh kit qud urea mdu khi dung urease cfi djnh vdi kit qud td mdy sinh h6a tg df ng Hitachi 917 M i s d b f nh
nhan ID10-01 ID10-02 ID10-03 IDI 0-04 ID1(M)5 lOICHie ID1(M17 IDI 0-08 ID10-09 ID10-10 ID10-11 ID10-12 ID10-13 ID10-14 ID10-15 IDI 0-16 tD10-17 ID10-18 ID10-19 ID11-20
A O D T B
0,090 0,224 0.167 0,237 0.134 0,301 0.188 0,163 0.100 0.123 0,060 0,241 0.203 0.155 0.132 0.311 0.113 0.327 0,219 0.121
K i t qud dung urease cfi flnh (n 17,66 48,11 35,16 51,07 27.66 65.61 39.93 34,25 19,93 25.16 10.84 51.98 43,34 32,43 27.20 67,89 22.89 71,52 46.98 24.70
ig/dL)
K i t qud tiF mdy sinh h6a t y d f ng (mg/dL) 18
47 33 55 29 61 38 32 19 22 13 50 46 34 25 63 20 72 43 21
HOI NGH! KHOA HOC C G N G NGHE SINH HOC
X d W k i t Qud nfing d f i urea t d p h u o n g phap dClng e n z y m e u r e a s e c 6 flnh v d d o d u p c t d m a y sinh h f i a t y d f i n g (Hitacf AIT ly "x »i _ _a_ j ^ . _^^^ „ K A n tA tAnn\ia\ r h i ' i n n Wi n h s n t h i n »«., „ i A t ^ 4..A^ fhort nhSn nhfii Flsher Vdl W 917) bang p
h / d o ( k - 1 , r t ^ n g m d ^ s nghTa t h o n g k f i . K ^ L U A N
N h u viy. q u a q u d frinh nghifin c d u c h i i n g tfii d u a ra cac ket ludn s a u :
Enzyme urease tdch chiet t d d f i u nanh v d l t h d i gian tdch b e o 12h c f i hifiu s u i t c a o n h l t .
Chitosan Id chat m a n g d e m Igi hifiu q u d kinh t f i trong c f i flnh e n z y m e u r e a s e d o g i d t h d n h r e t i l n v d kha nh tdi s u d g n g d u g t : 14 lan v d i t i n g hogt tfnh frfin 5 0 % s o v d i b a n d i u .
D i l u kifin tfii u u c h o c f i dinh e n z y m e urease tren hgt chitosan: 2.46 m g e n z y m e / l g h g t .
Enzyme urease o6 flnh frfin chitosan c f i n h i e u u u flim n h u : d f i bfin nhifit, chiu duge p H c a o h o n enzyme H d o ft bi d n h h u f i n g b d i s y thay doi ciia p H s o v d i e n z y m e t u d o .
Enzyme urease d g n g h j d o c f i V™« = 1,202 pmol/mg.phiit, Kn, = 9 2 , 5 m m o l , d g n g c f i flnh c f i Vm« = 0,66 umol/mg.phiiL Km = 2 2 5 m m o l .
K i t q u d d n g d g n g e n z y m e urease cfi flnh tren chitosan d l xac flnh n f l n g d f i u r e a t r o n g huyet t h a n h bfinh nhi khflng cfi s y khde bifit d d n g k l s o v d l k i t q u d d o d u p c t d m d y s i n h h f i a t y d f i n g .
T A I L I E U T H A M K H A O
Nguyln Van Mill (2002). Thyc hdnh sinh hfla Whd xuit bin Khoa hgc-ky lhu$t Ha N^i.
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INVESTIGATE PROCESS OF IMMOBILZING ENZYME UREASE ONTO CHITOSAN
Le Thi M y Ngoc \ Huynh Ngoc O a n h ^
' Ho Chi Minh City Universdy of Medicine and Pharmacy ' Ho Chi Minh City University of Technology
SUMMARY
Urease is an enzyme which applied in lots of fields, especially in medicine. Urease is used in many reagent kits to determine bla and urine urea concentration for diagnostic and monitoring kidneys fiinction. However, this enzyme is usually inported with Iq cost. In Vietnam, most of researches only focus on extracting urease ih>m soya bean, but researches to increase reusable capiciiyi this product still limited Consequently, we carried out a research about some immobilzing urease conditions onto chitosan, a n * support.
The investigations obtained optunum results with enzyme/chitosan rate that were 2,46 mg / I g of activated-chitosan t>eads; wih * total activity was 65%. The chitosan-immobilized ureases could reuse for 14 times with their acivity maintained above 50%.Ti optimum temperature and pH of chitosan-immobilized ureases were 70''C and 7,5; solube ureases were fiO^C and 7,0. The kiii«
parameters (V^,,, k^) of non immobilized and immobilized enzyme were determined, respectively, 0,663 (jimol/mg.min)and21 mmol (chitosan-immobilized ureases); 1,202 (nmol/mg.min) and 92,5 mmol (solube ureases).
Chitosan-immobilized ureases were applied to analyzeserum urea concentration of patients with the similar results measunng*«
the auiomaiicaly biochemistry machine.
Keywords: extract urease from soybeans, immobilized urease, chitosan. blood urea assay.
" Author for corresspondence. Tel' +84-918684472; Email; lethj mvnQOf:(avahoo com
