TAP CH] SINH HQC, 2QI2,34(4): 473-478
PHAN LAP, TUYfeN CHQN VA XAC DINH HOAT TINH CUA VI KHUAN SINH T 6 N G HQfP ENZYME URICASE
Pham Thanh Hi", Trin Dinh Min, Trin Thj Hoa Vi?n Cdng nghij sinh hpc, *[email protected]
TOM TAT: Vi khuSn sinh tdng hpp unease dupe phfin l^p tir dat trfin ni6i tnrdng th^ich dTa chira axit uric, Cac chiing vi khuin dupe dinh gii ho^t tinh d^ra tr£n sif 190 v6ng phan giii tren b^ m{lt thach. Chiing vi khuin CN3 cd khi nfing I90 vong phin giii axit uric t6i da trong thdi gian ngin dupe tuyin chpn, O^ra Iren C3C dfic diem hinh thii, sinh li, sinh hda vi trinh tu gen I6S rRNA, chiing CN3 dupe xac djnh la Pseudomonasputida. Chiing P. puiida CN3 dupe tim Ih^y vira c6 khi ning sinh tflng hpp uricasc npi bao lai vira cd kha nitng sinh uncase ngo^i bio, Tren mdi trudng nuflt cAy djch th€ chira 0,5% axit uric, P. puiida CN3 sinh tdng hpp enzyme uricasc vdi ho^t tinh ngopi bio - 0,85 U/ml vi n^i bao ~ 0,45 U/ml.
Tu khda: Pseudomonas. axit uric, phfin t(ip, uricasc, vi khufin.
MO DAU
Uricase (ten mdi li urate oxidase) li mdt enzyme khu tru trong peroxisome, tham gia vio con dudng phin hiiy purine, xuc tic cho s\r oxyhda md vong purine cua axit uric khd tan diinh allantoin dl dang dd bii tilt [7]. Uricase dupe img dung chii yeu cho hda sinh lam sang nhu mpt chi thi chan doan dk xac dinli ham lupng axit uric trong miu vi nude tieu. Ngoai ra, no cdn dirpc dimg nhu thudc de ngin ngira va di^u tri bfnh gout, hdi chiing li giii khdi u vi mgt sd benh rdi loan ting cao axit uric mau.
Hifn nay, nhieu vi sinh vit mdi da dupe tim thiy cd khi ning sin sinh uricase vdi tinh chat tdt va ning suit cao. M$c dii uricase cd ngudn gdc vi sinh vat dugc nghien ciiu nhieu, nhung chi it loai dugc phat trien thanh san pham thirong mai [6, 10].
Viec nghien cim sin xuit uricase giup phdng vi diSu trj cac b?nh ting axit uric mau d Viet nam vin cdn li vin dk mdi. Trong nghieii cuu nay, chiing tdi tidn hanh phin l§p mgt s6 chiing vi khuin cd kha ning sinh tdng hgp enzyme uricase, chpn lgc mgt chQng tdt nhat vi xic dinh ho^t tinh sinh tdng hgp uricase ndi vi ngoai bio cua chung nay tren mdi trudng djch thi cd bd sung co chit cam ling.
V^T LI$U VA PHirONG PHAP NGHIEN ClTU Vi sinh v^t vi nudi cay
Cic chiing vi khuin cd boat tinh sinh tdng hpp enzyme uricase dupe phin I^p tir mOt s6 mlu
dit thu th^p t^i mpt sd dia difim tliupc Hi Ndi.
Mdi trudng phin l^p va giO: gidng (A) cd thanh phin (g/L): glucose I, cao nim men 1, axit uric 5, agar 20 theo Lehej Ckovi et al.
(1986) [5]. Moi trudng nudi ciy dich thS (B) chua (&'L): pepton 5, glucose 10, K2HPO4 1, MgS04.7H20 0,5; NaCI 0,5; axit uric 5; pH 7 theo Azab et al. (2005) [2].
Nudi ciy xac djnli hogt tinh enzyme- 1%
gidng tir nudi ciy dich thS 24h dupe chuyfn vao binh tam giic 500 ml chira 100 ml moi trudng (B). Nudi tren miy lie 200 rpm/30''C/48 h. Cudi giai doan nudi ciy do boat dp uricase npi va ngo^i bao.
Phan lap vi khuan
ciy gat dich huyln phu dit len dia pettri chda mdi trudng thach (A). Nudi nhilng dTa ciy d 30°C. Sau 3-5 ngay quan sat kha nSng phin giii axit uric ciia cic khuin lac tren hh mit dTa thach. S\r tao vdng trong ciia moi trudng quanh khuin lac xac djnh boat tinh uricase.
Djnh tinh kha nSng phin giai axit uric ciia vi khuan
Cic chiing sau khi phin lip dupe ciy chim diSm len mdi trudng thach dia (A). Nudi nhung dTa ciy d 30°C. Sau 3, 6, 9, 12 ngay, quan sat hinh thii, mau sic khuin lac vi do dudng kinh cua vdng phan giai axit uric quanh khuin lac.
Dinh gii ho?it tinh cua cic chiing dua vao do Idn c\rc dai ciia vdng phin giai.
Chiing VK cd kha nSng phan giii axit uric
Pham Thanh Ha, Tran Dinh Man. Tran Thi Hoa t6t nhit tren mdi trudng thgch dTa dugc chgn lgc
va nudi lie trong binh tam giic chiia mdi trudng djch UiC' (B). Cu6i giai dopn nudi ciy, 50 pi djoh nudi di \o!}[ ti bio dugc nhd vio cic \h th^ch chira 0,5% axit uric, khdng chiia mdi trudng dinh dudng. Mdi trudng djch thi vd triing dirpc dimg nhu d6i chimg. DTa dugc ii d SCC trong 24 gid. Str t^o vdng phin giii axit uric quanh Id trfin th?ch dTa chimg minh s\r cd mjt ciia uricase ngo^i bio trong djch nudi ciy.
Phan lo^i %i khuin
O^c diem hinh thii hpc khuan l^c vi khuin, hinh thii hgc tc bio, klii ning bit miu Gram, t^o bio tu, khi ning hi^u klii, ho9t tinh catalase, klia ning di d$ng, khi ning len men theo Nguyin Lin DQng vi nnk. (1972) [3].
Cic d^c didm sinh hda dugc xic djnh bing kit chuin API 20NE (bioMerieux, Phip) vi phin tich bing phin mSm APILAB PLUS 3.3.3.
Phin lo^i vi khuin bing sinh hgc phin lir d\ra tren trinh t\r I6S rRNA: tich DNA tdng s6.
Gen 16S dugc tdng hpp diing c$p mdi PI: 5'- AGAGTTTGATCCTGGCTCAG-3' vi P2:5'- ACGGTTACCTTGTTACCGACTT- 3'. San pham PCR sau tinh sach dirge xic djnh trinh t^r tren may dpc t\r dpng Applied Biosystems 373A.
Phin tich phi h^ de xic djnh m6i quan h^ di truyin vdi cic chung vi khuin khic theo Sambrook & Russell (2000) [9].
Xic djnh ho^t tinh enzyme
Thu enzyme thd: sau khi nudi ciy kit thiic, li tam (12,000 vdng/I5 phuxJAX) tich rifing tl bao va djch nudi. (1) Thu enzyme ndi bio: tl bio dupe ddng nhit trong 1,2 ml dpm botate va xung di?n 10 x, mdi lin lOs, gitta cic dgt lim lanh Uong nude di 30s. Sau dd li tim (10.000 vdng/30 phiit d 4''C) lo^i xic tl bio, pha Uen dugc thu nhu enzyme n0i bio thd; (2) Thu enzyme ngo^ii bio: djch nudi di lo^i tl bio dupe thu nhu enzyme ngogi bio thd.
Phan tich ho^it tinh enzyme: s\r sin sinh enzyme cua vi khuin dupe do bing kit phin tich uricase (Invitrogen, MJ). Trong phan tich, uricase xiic tic cho vifc chuyin axit uric thinh allantoin, HzOj vi CO2. Sau dd H2O2 trong sv CO mSit ciia horseradish poroxidase (HRP), phan Ong vdi chi thj miu Amplex Red dl t(io san
phim oxyhoi hu^nh quang dd. Phin img dupe th\rc hi?n tren dTa ELISA 30 phiH/^7°C (Uinh inh sing). Do dO hip phy t6i uu tren miy dgc dTa ELISA (BioTek, MJ) d budc sdng 560 nm.
Tinh toin ndng dd uricase trong mlu d\ra trSn dudng chuin uricase.
KI;T QUA VA THAO LUAN
Phin I 9 P , tuyin chpn chiing vl khuin tfcb luf enzyme uricasc cao
Tir 18 miu dit di phin I^p dupe 50 chung vi khuin cd ho^t tinh sinh uricase tuong d6i cao (dudng kinh vdng phfln giii axit uric > 2,5 cm).
Cic vi khuin phfln Iflp dugc ciy chim diim len mdi trudng th^ch dTa chita 5 g/L axit uric. Vi^
sin sinh uricase dupe xic djnh bdi s^ t^o vdng phfln giii axit uric. Sau 12 ngiy nudi ciy, quan sit thiy cd 6 chimg CN3; RCl; SH3; DVI;
DHI vi DB4 t^o vdng phin giai axit uric rit cao (phin giii hit axit uric trong mdi trudng th^ch dTa). Anh khufln I9C ciia mOt sd chiing vi khuin t^o vdng phin giii axit uric Uen mdi trudng th^ch dia sau 12 ngiy nudi ciy dugc trinh bay d hinh 1.
Trong sd cac chiing vi khuin cd ho^t tinh sinh uricase cao, chung CN3 cd khi ning phan giai hit axit uric Uen mdi tnrdng th^ch dia trong thdi gian ngin (5 ngiy) dupe chiing tdi 1 ^ chpn cho nhDng nghien ciru tilp theo.
Phin I09I chung dugc tuyen chpn D\ra tren cdc d^c diSm hinh thdi
Kit qua quan sit d^c diim hinh thii khuin I9C trSn th^ch dia, hinh thii te bio vi khuin dirdi kinh hiln vi di^n tu, khi ning bit miu Gram, khi ning hinh thinh bao tu vi mOt sd die diim sinh li, sinh hgc dugc trinh bay d bing I.
Theo cic djc diim hinh thai, sinh li chiing CN3 cd nhieu d$c diim gi6ng vdi vi khuan thu^ chi Pseudomonas [4].
Phdn IOQI vi khudn theo kit chudn sinh hod API 20NE
Chung CN3 thudc lo^i tri^c khuin Gram (-) vi cd nhiiu d$c diim gi6ng vi khuin thu^c chi Pseudomonas nen dugc phan lo^i theo kit API 20NE. Trong qui trinh kiem tra, cic phan img dugc n h ^ bilt bing sv ddi miu hoi chit. Ket qui dirge trinh biy d bing 2.
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Pham Thanh Ha. Tran Dinh Man. Tran Thi Hoa Bdng 2. Cic phiip thir sinh li. sinh hda theo kit API 20 NE ciia chiing CN3
r~Nd7 I TRP 1 GU£i + I IMNHl I IMANl I INA(
D6i chi^u vdi dXt li$u phin mim cho thiy chimg CN3 luong ding vdi i\ khuin Pseudomonas putida (% id) > 95% (xic djnh t6t). Ket hgp vdi khda phin lo^i vi khuin ciia Bcrgey (1994) [4] cd thi so bd djnh l6n chiing CN3 la P. putida D I khing djnh them vl vj tr(
phin logi cua chiing nay, chiing tdi tilp t^ic tiln hinh xic djnh trinh ttr gen 16S rARN.
Phdn loi.ii dira tren trinh Hr gene lOSrARN
ADN t6ng s6 tir chiing CN3 di dupe chilt nit (hinh 3)= Tir khudn ADN tdng sd, bing ky thu^t PCR sir dung c9p mdi, chiing tdi di thu dugc gen mS hoi I6S rARN ciia chiing CN3.
Kit qui di$n di d6 trdn hinh 4 cho thiy sin phim PCR thu dugc 1 bang rit d^c hi^u. Kich thudc phfln tur vio khoing - \ ,5 kb tuong duong vdi tinh toin It thuylt. Sin phim PCR sau tinh S9ch dupe diing tryc tilp dl xic djnh trinh Wf nucleotit ciia gen 16S rARN.
Hinh 3 ADN tdng Hinh 4. San phim
M"> PCR
Hinh 5. Mdi quan h$ di tmyen ciia chiing CN3 qua phin tich gene I6S rARN Phan tich tren miy xic djnh trinh t\r ADN tg
dpng. xir li bing chuong trinh PC/GENE. Truy cap ngan hing gen de tim nhihig loai di ding ky cd trinh tg tucmg ddng.
Trinh tg cda do^n gen 16S rARN ciia chiing CN3 so sirUi vdi chiing cd mire dp tuong ddng cao nhit trong ngan hang gen. Kit qua cho thiy, chung CN3 the hi^n mire dg sai khic vl nucleotit ciia dogn gen 16S rARN rit it, chi sai khac d I vj tri. Mi>c dd tuong ddng vdi P. putida J312 rit cao tdi 99%. Dl bilt m6i quan h? di truyin giOa cic chiing dugc phSn tich, cay phi h$ (hinh 5) d^ra tren phan tich phan do^n gen 16S rARN di dupe IhiSt I|p. Cay pha h§ cho thiy, chimg CN3 phan I|;p t^i Vi^t Nam ciing nhdm phy vdi P putida33l2.
Cin cOt vao cic d^c diim hmh thii, sinh H,
sinh hoi, kit chuin API 20NE, trinh t\r gen 16S rARN kit hgp vdi hf thdng phan lo^ vi khuin cua Bergey chiing CN3 dirpc phan lo^i la P. puiida. Trinh t^r gen I6S cua chimg P. putida CN3 dfl dugc chimg tdi ding ki tren GenBank vdi mi s6 GU967502. Ho?t tinh uricase chua dupe tic gii nio cdng bd tim thiy d P. putida.
Vi v§y, cd Ihl diy li mdt phit hifn mdi.
P. putida li m0t vi khuin khdng gay bpnh cho ngudi ciing nhu d$ng th\rc v|t, do dd chiing P. puiida CN3 cd thi sii dgng cho nhOng nghien cdn san xuit enzyme uricase lam nguyen lieu sin xuit thu6c phdng vi dilu trj cic b?nh ting axit uric Uong miu.
Nghien cthi cich thih: sin sinh uricase ciia P. putida CN3
M^t s6 vi sinh v§t nhu Microbacterium
ZZJ4-I va Streptomyces cyanogenus sinh uricase n0i bio vi cin phi vd tl bio dl thu enzyme [II]. Cdn mdt so vi sinh v§t nhu Bacillus fastidious, Mucor hiemalis va Pseudomonas aeruginosa \^i sinh uricase ngogi bao [2, 8]. PhSn Idn cic chiing vi sinh v$t t\r nhien tich luj uricase ngi bio, tuy nhiSn cung CO m$t sd it vi sinh v§t dupe thong bio vCra cd khi ning tich luj uricase ndi bio l^i vira cd kha ning sinh tdng hgp uricase ngo^i bio [6].
//;/;/; 6 llo.il liiih >inh King hnp uricasc ii ni;o,ii \\io eua cluin-/' n""^'" C>J--
Hinh 7- Kiem tra kha nang sinh uricase ngoai bao cua CN3
P. putida CN3 sau khi nudi ciy tren mdi trudng djch thi dupe li tam tich sinh khdi. Hoat tinh uricase di dugc do trong ci hai dich ndi va sinh khdi. Kit qui (hinh 6) cho thiy, uricase thd dugc tim thiy trong ca dich ndi di lo^ii sinh khdi (~ 0,85 U/ml) vi trong sinh khdi (~ 0,45 U/ml). Tren th&ch dia khdng dinh dudng chira 0,5% axit uric (hinh 7) uricase ngoai bio chuyin axit uric khdng tan thinh allantoin hoi tan trong nude the hifn bing vdng trong (dirdng
TAP CHi SINH HQC, 2012,34(4): 473-478 kinh vdng phin giii ~ 1,8 ± 0,08 cm). Djch mdi trudng vd trimg khdng cd uricase khdng thiy t?o vdng phin giii. Nhu viy, P. putida CN3 li vi kliuin vira cd khi ning tich luj uricase nOi bio, l^i vira cd khi ning tilt uricase ngogi bio.
Da s6 vi sinh vit thudng tich lOy uricase ben trong tl bio, vi thi, vifc tich enzyme thudng qua cic budc nghiin, phi vd tl bio. Do dd, vifc sin xuit uricase bdi nhOng vi sinh v(lt niy dira h^n qui trinh 3 budc, gdm m0t budc nudi tl bio, mdt birdc cam img sin xuit enzyme trong su c6 m$t ciia axit uric sau khi tliu tl bio nudi ciy va mdt birdc tich uricase sinh ra trong tl bio. Theo qui trinli 3 budc, vifc tich vi tinh s^ch uricase dupe xem Ii phdc t^p vdi nhiiu lo^ii protein vi enzyme khic nhau lin Uong dd vdi lugng Idn lim cho vifc san xuit cho hifu qua thip [1].
Chiing P. putida CN3 cd uu diim cd khi ning san sinh uricase ngoai bao vi vgy enzjmie luicase tCr chimg niy cd the dupe tinh sach true tilp tir djch nudi khdng cin qua budc phi vd tl bio phiic t9p I?i cho enzyme cd hogt tinh va dd tinh s?ch cao hon so vdi enzyme nfi bio. Vi v^y, tilp theo chiing tdi sg tap trung nghiSn cOn vg ho^t tinh sinh enzyme uricase ngogi bao tir chiing vi khuan nay.
Trong sd 50 chung vi khuan cd khi ning sinh tdng hpp uricase phan lip tir 18 miu dit, chiing Pseudomonas puiida CN3 ed khi ning sin sinh miic dp cao enzyme trong thdi gian ngin da dupc chpn lgc. Tren mdi trudng dinh dudng dich thi chda 0,5% chit cim faig axit uric, chiing P. putida CN3 sinh tdng hgp uricase ndi bao vdi ho^t tinh ~ 0,45 U/ml vi uricase ngoai bio vdi ho?t tinh ~ 0,85 U/ml.
L&i cam ffn: Cdng trinh dupe th^rc hifn vdi su hd trp kinh phi ciia Dd tai cip Vifn Khoa hpc va Cdng nghf Vift nam 2010 -2011.
TAI LlEU THA M KHAO
1. Adamek V., Kralova B , Suchova M., Valentova O., Demnerova K., 1989.
Purification of microbial uricase.
J. Chromatography, 497: 268-275.
2. Azab E. A., Ali M. M., Fareed M. P., 2005.
Pham Thanh Ha. Tran Dinh Man. Tran Thi Hoa Studies on uricasc induction in certain
bacteria. Egyptian J. Biol. 7: 44-54.
3. NguySn Lun DDng, Doin Xu5n Mugii, Nguyin Phiing Tiln, Dgng Diic Tr^ich, Ph^im van Ty, 1972. MOt sd phuang phip nghifn cim VSV hpc, 85 trang, 4. John G. H., Noel R. K., Peter H. A. S„
James T. S., Stanley T. W., 1994, Bergey's manual of determinative bacteriol. 9*
Edition. The Williams and Wilkin, Co., Baltimore: 94-125.
5. Lehej Ckovi R., K. Demnerovi, B. Krilovi., 1986. Screening of microorganisms with uricase activity. Biotechnol. Letters. 8(5):
341-342.
6. Lotfy W. j-i. 2008. Production of thermostable uricase by a novel Bacillus thermocalenulatus strain. Biores. Tech, 99 (4): 699-702.
7. Nelson D, L., Cox M. M., 2004. Lehnmger
Principles of Biochemistry. Freeman, New York: 75-86.
8. Saeed H. M., Abdel-Fattah Y. R., Gohar Y.
M., Elbaz M. A., 2004. Purification and characterization of extracellular Pseudomonas aeruginosa urate oxidase enzyme. Pol, J. Microb., 53; 45-52.
9. Sambrook J. and Russell D., 2001:
Molecular Cloning: A Laboratory Manual (3rd ed.) Cold Springs Harbor Press, Cold Springs Harbor N.Y, 76 pages.
10. Yazdi M. T., Zarrini G., Mohit E., Faramarzi M. A., Setayesh N., Mohseni F.
A., 2006. Mucorhiemalis: a new soiuce for uricase production. World J. Microbiol.
Biotechnol. 22: 325-330.
11. Zhou X.. Ma X., Sun G., Li X., Guo K, 2005. Isolation of a thermostable uricase producing bacterium and study on its enzyme production conditions. Process Biochemistry 40 (12): 3749-3753.
ISOLATION, SELECTION AND ACTIVE DETERMINATION OF BACTERIA PRODUCE URICASE
Pham Thanh Ha, Tran Dinh Man, Tran Thi Hoa Institute of Biotechnology, VAST SUMMARY
Uricase producing bacteria was isolated from soil with a nutrient agar media containing uric acid. The isolation of bacteria was based on uricolytic activity on agar surface. A bacterial strain CN3 showed a maximum zone of clearance in short time had been chosen Based on morphological and biochemical tests, as well as 16S rRNA sequence and phylogenetic Ucc analysis, the bacterial isolated was identified as Pseudomonas putida. The uricase was present as intracellular and extracellular enzyme in this strain. When the strain was cultured at SCC and pH7 for 48h on nutrient broth media containing uric acid 0.5%, tiie extracellular uricase activity peaked at - 0.85 U/ml and inUacellular uricasc activity peaked at - 0.45 U/ml.
Keywords: Pseudomonas, isolation, uric acid, uricase.
Ngay nhdn bai: 14-3-2012
