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TINH SACH VA XAC DINH MQT S6 T ( N H C H A T C O A DAAO TAI TO HOP TO- TRIGONOPSIS VARIABILIS KCTC 0864
Phan Thj H6ng Thdo \ Dging Thj ThOy Duong \Ph( Quyet Ti^n \ Vu Thj H?nh Nguyen\ Quan LS Hi ^ Le Gia Hy' Wi$n Cdng nghi smh hgc, Viin Hin lam Khoa hgc vi Cdng ngh$ Wpf Nam
Win Cdng ngh$ smh hgc vi Cong nghi thiic phim. Tnrdng B^ htfc Bich Khoa Hi N6i T6M TAT
D-amino acid oxidase (DAAO, EC 1.4.3.3) ric tic jStia vmg oxy hoa lopi bo nhom amin cua cac D-amino acid t^o thinh a-keto acid tirong 6ng, ammonia va hydro peroxid. Enzym nay co ftfi tbu dupe tfc nhi&i ngudn goc khac nhau, tuy nhien c4c DAAO c6 ngufin g6c t i Tri^nopsis variabilis co khi nang (mg dyng cao trong diuy&i h6a sinh hpc cdng nghiSp. Troi^ nghiSn ciiu niy, DAAO tii Chung tii to hgp £. coli BL21(DE3)-rfflao dugrc tinh sach qua cot Ni - NTA co hogt do tang len 5,7 lan so vdi ban d^u. Enzyme tinh sach c6 h o ^ do riSng dgt 215,6 U/mg protein (co chit D-alanine) vdi hieu suit tiiii hhi enzym li 67,32 %. Trpng luong phan tii cua ezym tinh sgch la 39kDa tren gel SDS-PAGE 12,5%. Enzyme tii ^ bpp bfin trong viing nhiet d<i du6i 45°C vi pH 7,6-9,0. Trong khi do hogt tinh xuc lie cao nhit cua enzyme dgt dugc a 50°C va pH 8,5. Enzyme DAAO bi uc che boi pyruvic acid, Glutaric acid, benzoic acid 6 cac nong dg 10,20 va 30 mM. Enzyme tdi t6 hpp bt uc che manh bdi hai ion li Cu2+ vi Hg2+ ir n6ng d6 diip I mM va IjiM, tuong iing. Hai ion Mg2+ vi Zn2+ Um ling khi ning HIC lac ciia DAAO Ifin 117 - 122 % b ndng d§ ImM, trong khi do, Fe2+, Co2+ vi Mn2+ khong lim inh hudng den hoat tinh cua raizyroe. TTiong s6 dgng hgc cila DAAO vdi cic co chit D, DI^
alanine, D, DL-mediionine tin lugl nhu sau: Km: 5,25; 7,66; 0,15; 0,27 mM vi Vm: 0,15; 0,13; 0,1 vi0,12 (unol/s.
Keywords: D-amino acid oxidase, Trigonopsis variabilis, chat lie chd, enzym tai to hgp, BL2I(DE3)-dflao.
MdDAU
D-amino acid oxidase (DAAO, EC 1.4.3.3) ii m6t flavoenzym tham gia vao ph^n iing xuc tSc oxi h6a lo?i bo nhiSm amin ci^a c4c D-amino acid (D-AA) d l t$o thSnh a-keto acid hrerng t>ng. amonium vi hydro peroxide. D-amino acid owdase dmjFC ph^t hi#n tir nhikii ngu6n g6c khSc nhau vi ngufin g6c vi sinh vgt Id y4u t6 hang d^u quylt djnh tlnh d^c hi^u ca chit cOa c^c enzym: DAAO tCr F. oxysporum, C. parapsilosls vi C. bokiinii c6 tlnh d§c hi#u cao v6i D-atanine. trong khi cdc enzym ti> \ri sinh v#t kh^c hogt d^ng toi da vdi D-metfikinine. D-tr^Jtophan vi D-phenylalanine. D/\AO ti> T.
variabilis vi R. gracilis hogt dOng tot tren cdc amino acki khong va nifoc. Tuy nhien, DAAO cua 7. variabilis c6 khd nang xOc tdc cao han vdri cdc amino acid phdn eye nhu- D-histidine ho$c D-aspagarine. V6i 00- chit cephalosporin C, DAAO ti> T. variabilis Id enzym hogt d^ng tot nhlt (Pollegioni et ai, 2008). Khd ndng duy tri hogt d$ng vd bn djnh vdri nhi^t d6 vd pH cCia DAAO biln doi theo ngu&n gen, vl dy DAAO tCr T. variabilis dirgrc coi Id enzym ben nhi^t nhat (bin if 45°C), frong khi d6 enzym tCr R.gracllis bat hoat if nhigt d? I6n hom 30''C (sau giai dOgn u 30 phut a 45''C, hogt tfnh mit hodn todn) (Pollegioni et ai. 2004). DAAO ti> A. protophormlae vi C. boidinii hogt tfnh bat diu gtam if nhi§t do ion hern 30°C vd mit hodn todn i> 50°C sau 30 phut u (Geueke et al., 2007). DAAO ti> cdc chflng nim n6i chung hogt dOng tnang viing pH kilm. R. gracilis vd T. variabilis bin trong khodng pH tCr 6,0 - 8,2, gidm nhg hogt tlnh or viing pH kilm cao (a10) (Pollegioni ef al., 2004). Cdc DAAO bj i>c chl bdi mOt sd edc sdn phim cOa qud blnh phdn img nhu-: a-keto acid, Glutaryl acid (Moreno et aL. 1996), hydns peroxide vd mgt so ca chit i>c ehi cgnh tranh khdc nhu benzoic acid. Ngoai ra, hai Ion Cu vd Hg^^gSy i>c chl hogt dOng cOa DAAO tCf T. variabilis du c6 mgt vdi mOt lu'ang rit nho (Schrader and Andreesen, 1996). Myc ti6u cua nghiSn ci>u nay Id tinh sgch enzyme DAAO tdi t l hgrp e6 ngu6n glc tir T. vanabilis KCTC 0864 vd xdc djnh mpt s6 tfnh chit cOa no nhim sir dung trong cdc nghi§n ciru chuyin hda sinh hpc.
NGUYSN LI^U VA PHUANG PHAP V|t li#u
Chung E. coli BL21 (DE3)-daao tdi to hp'p tCr Trigonopsis variabilis KCTC 0864 difprc lly tip bO sifu t$p gilng cOa phong C6ng ngh# iin men, vign Cong ngh§ sinh hpc. Cdc hda chat D, DL-alanine, D, DL-methionine, o-dianisidine.2HCL (3,3'- dimethoxy-biphenyl-4,4*-diamin), peroxidase (type 2) (Sigma) va cdc hda ehit tinh khilt khdc
Phirong phdp
Thu nh$n DAAO tif E. coli til t6 hgp
TebdoE. co//tdit6 hp'p thu dupe sau qud trinh bilu hi§n dupe ly tdm or 10000 vdng/phiit trong 15 phut 6'4"C, hda sinh khli trong dgm phosphate pH 8,0 vd sieu dm phd vd t l bdo. Tiep do ly tam d 13.000 vong/phut trong 20 phdt d l thu djch trong.
Tinft chi protein bing sac ky ai Iyc
DAAO tdi t l hpp dupe tinh sgch 1 budc bing kit ProBond TM Purification K850-0. Hon hpp enzyme tho dupe bo sung vdo cOt tinh sgch chua hpp chit Nickel - Nitriloacetk; acid (Ni-NTA) theo tf 10 1-5 mg protein/1ml Ni- UTA. DAAO tai to hpp dtrgrc thu bing dung djeh 500 mM imldazol. Protein thu dirpc sau moi phdn doan dupe kiem tra hogt tinh, xdc ^nh n6ng dO protein vd di|n di trdn gel polyacrylamide 12,5%.
Xac dinh ho?t tinh enzyme DAAO
Hogt tfnh D-AAO dupe xde djnh bang phuang phdp so mlu (Fischer, 1998) nhd viec tgo mau giO'a o-Dianisidine vd H202. trSn mdy do quang p h i HitachiU-1100 d budc sdng 540 nm. Mpt don v\ hogt tinh enzyme dupe xde Snh Id lupng enzyme ehuyen hda dupe 1 nmol D-alanine trong m^t phiit d dilu kign thf nghi|m.
Xic djnh him lirgng protein: Lugng protein cd frong dung djch enzym dupe dinh lupng theo phuang phdp Hradtdnl - Xic djnh inh hirdng eOa nhi$t dg, pH vi cic Ion kim loai lin tmfit (mA cua enzyme: Phdn dng xdc djnh dnh hudng nhi^ dd dli vdl hoat tinh DAAO dir(;fc xdc djnh khi u DAAO tdi to hpp tinh sach frumg d§m 50 mM phosphate, pH 8 &
cdc nhigt dO 10'C •*• 70''C. Anh hudng cua pH dli vdi hoat b'nh DAAO dupe xdc ffpih khi ttiay dfii pH cua a§m bdn d cdc dilu ki^n khde nhau pH 5 -^ 12. Anh hudng cOa edc ion kim logi nhu Fe**, Co**. Mn** Ca** vd Zn** lln hogt tinh DAAO dupe I d ^ fra bong hfin hpp phdn ung chira 1 vd 5 mM ion kim logi ridng thdy ngdn (Hg**) su dgng nfing d^ Id 1 pM. 10 pMvdlOOpM.
Xic dinh thdng so il^ng hgc Km va Vm
Enzyme DAAO dupe pha loang den nong dp protein 0,61 mg/ml (tuang dng vdi nfing dO DAAO 6.64 U/ml) vd cdc co ehit xac i^nh thdng so dpng hpc dirgre sd dung la D.DL-alanine, D. DL-mettiionine. Cdc thdng so d$ng hpc dupe xac djnh dua trdn ca sd nfing dp san phim H2O2 sinh ra sau phdn dng thay dfii theo nfing dp ca chit ban diu su dgng vd ttidi gian phan ung qua phuong trinh bde nhat Lineaweaver vd Burk: 1/v= KtraJ Vnex- 1/[sl + l/Vma. trong dd: v, Vmnlft vdn toe phdn ung va vin toe c ^ dgi tgi nfing dp dang xdt, [s]: ning dO ea ct^lL
K ^ QUA VA T H A O LUAN Tinh sgch DAAO bang sac kj/ di l i ^
Sdn phim protein ttiu duoc sau qud trinh tinh sgch chi xuat hi^n mpt bang ddm, cd khoi lupng xIp xi 39 kOa, tuang trr^
vdi khli lupng enzyme tdi tfi hpp trong miu protein tong trudc khi tinh sach (Hinh 1). Protein tinh sgch cd hogt d$ rifing 215,6 U/mg protein, tang 5.7 lln so vdi ban dau vd hieu suit thu hll enzyme Id 67,32 % (Bdng 1).
Bdng 1. H l ^ suit tlnh s^rah cua enzym DAAO til t6 hgp MSu
Dfch c h i ^ enzyme thd MSU sau tinh sgch qua cdt
H o ^ a e t d n g (U)
890,26 599,37
Piuteintdng (mg)
23,54 2,78
Hogt dd ri§ng (U/mg)
37,82 215.60
Tlnh sgch (1^)
1 5,70
Hi$usudtlhu h&i(%)
100 67,32
Krnh 1. EH$n di protein Idi to hg>p trudc va sau Unh sgdi DAAO bang c$t sac ky di l | ^
M: Thang protein chuin, 1: Mlu protein tfing trudc khi finh sgch, 2: San p h ^ protein tinh sgch.
Hi$u qui tinh sgch cde DAAO tai to hpp ti> T. variabilis vi m^t so chung khde bilu hien ti^n E. cdi ttiay d l i ft nhilu theo phuang phdp sdc kf di i\fc sir dung. Cdng bo cCia Ma et al., 2009 cho ttily higu suit tinh sgch qua cOt sac kf di li/c kim logi (Ni ) IMAC khd eao, dgt 89% vdi hogt tinh ddc hi§u cao han eua chting tdi 1,27 lan, dgt 272.97U/mg protein. Ttoni khi dd, Dib et al. 2007 sir dyng ept sic kf di lye Sfrep - Tactin cho higu qua tnh sgch 80%, vdi hogt tfnh d|c hl|u ciia mlu enzym sau tinh sgch dgt 160 U/mg protein, tdng 2,75 lan so vdi miu enzym thd.
Nhl^t dp xuc tac va d$ ben ciia DAAO vdi nhift do
Sv bien ttiien hogt tfnh cua DAAO cho Uily, hogt tinh tang din Uieo sy tang ciia nhiet dO ti> 10 + 50°C vd dgt gid frj cao nhlt d SO'C (thip han nhidt dd toi uu cdng bo bdi Wang va dfing tdc gid (2008) Id 5°C), gidm mgnh ti> 55 + TQ-C vd hlu nhu mat boat tlnh d 70 C. Nhu vgy, DAAO ed thfi xdc tdc phdn ung chuyin hda D-alanine trong ddl nhigt dd rpng ti> 25
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Hinh 2. Anh hudng ciia nhiet d$ den boat tfnh xuc tic cda DAAO Hlnh 3. Dp ben cua DAAO d m$t so nhift d$ khac nhau h i ^' ! i " ^ ? ^ ° ^ ° ' ^ ^ ' ^ '^"^ *^' ' ^ ' "^^* * ^ ' "^ ^°°^- S3" '' 20 Pfiut giir enzym d 37 + 40''C thi hoat tfnh eOa DAAO hdu nhu khong dfii. Hogt tinh enzyme gidm mgnh (40%) sau 30 phut giu- enzym d 50''C. Hogt tinh DAAO gidm nh? v6i
t^uiNLj iNDni^ oimn n y u i U A N U U U U Ztird
H A ? ^^ ^ *^'^'" ^^"^ ^ " ^ ° ^^ " ' ("'"'' ^)- ^ ' ^ ™ ' ^*°?* *'"^ ' ^ ^ " ^ *^^ °^^^ *^"'*' (ac dyng nhi§t la do DAAO ii indt enzyrne oxy hda khir ed chira FAD vd kha nang lidn k i t giua phin apoenzym vdi phin coenzym (FAD) phy u u «^A"^1 ° "^'^* ^^ ^^ P" *™"9 dung djeh. Enzyme d l dang bj mit phan coenzym (mat hoat dd xdc tac khi hg pH ttilp hode tdng nhidt dO) (Hefti et al.. 2(»3).
0$ pH hoat d$ng vd do bin cda DAAO vdi pH
I ? 7 * ' ^ t ' i ' i " ' ^ - i t ' * ° - r - * ^ f e ' ° ^ ° ^ * ^ ^^ ^^ P ^ ^ " " ^ ' ^ " y ^ " ^ " ^ [ > ^ I a n i n e frong d d i p H kiem v d i bidn d d k h d r d n g . Z.f hV." > ^ ° Tishkov v d K h o r o n e n k o v a , D A A O tir T. variaMs hoat dOng tfit n h a t frong mdi frudng k i l m , d a c b i ^ t vdl c a c h a t s u d y n g Id D-alanine, p H hogt d p n g tfii u u c u a e n z y m e ndy £ 7,5 (Tishkov, Khoronenkova. 2006). W a n g v d ddng t a c gia (21^08) cpng d u a ra k i t ludn t u a n g t y v e v d n g p H hoat dpng cCia D A A O tdi t f i h p p tCr T. variabilis vi R.
gracilis vdl gia fri pH tdi u u d g t 8,5.
Hlnh 4. Anh h i r d n g cda pH t d i hogt d^ng xdc tdc cua DAAO " ' ' ^ ^ ^- ^ ^ " "^^ DAAO d cAc pH khac nhau Hogt tfnh D A A O d u p e d u y fri tfit n h l t theo t h d i gian frong d $ m pH 8,0; g i d m nhg d cac p H khde ti> 7 * 9 v d g i a m nhanh trong v d n g p H k i l m c a o (pH £ 1 0 ) ( H i n h 5). 6 p H 10 v d 1 1 , hogt Onh c d a e n z y m e l l n luprt edn 6 4 % v d 5 0 % , s a u 150 phut. B d c bidt v d i p H 12. h o g t tfnh e n z y m e g i d m r i t n h a n h vd m i t h o a n todn sau 6 0 p h i i t u. T h e o mdt s l n g h i i n c d u , pH hogt d d n g cGng n h u d p b i n p H eua D A A O tdi t l h p p ttiay dfii ft nhieu ttieo n g u d n g e n daao. E n z y m e D A A O tir R.
gracilis v d T. variabilis c d d o b i n v d i pH h r a n g t y n h a u : fin ^ n h tix)ng khodng pH t d 6,0 + 8.2; gidm d i n d pH c a o h a n (tgi pH 10, hogt tfnh c d n Igi s a u Ci 30 phut Id k h o d n g 5 0 %) (Pollegioni et al.. 2008). D A A O t d A. protophormlae hogt ddng tfit v d b i n v d i pH 6.0 + 9,0 (Geueke ef a/., 2007). D A A O t d C. boidinii ben h a n v d i p H , dgc bi§t tgi cdc g i d fri pH kiem v d fin d j n h d pH fren 10,5 (Pollegioni etai, 2008).
A n h h u d n g c i i a m ^ t s f i c h i t 6<c c h e va i o n l d m logi
Hz02 g d y CFC c h l mdt p h i n h o g t tfnh e n z y m e d n i n g do 10 m M (Bdng 2), sau 2 0 . 3 0 phdt O hogt tinh cOa e n z y m e gidm cdn 5 1 , 8 % v d 5 0 % . K i t q u d t u a n g t y cQng d u p e chf ra trong nghien c d u ciia W a n g vd c d n g s y . khi dng k h d o sdt s y bien ddng c d a h o g t tfnh D A A O tdi tfi h p p ed ngufin g o c t u T. variabilis. D § c bidt khi t d n g nfing d p khdo sdt Idn 100 m M sau 3 0 phiS d ttii e n z y m e m i t h o g t t f n h h o d n todn ( W a n g et al., 2008). Pyruvic acid ldm g i d m mgnh hogt tfnh D A A O , dge bidt td d n f i n g d d 30 m M , s a u 10 phdt hogt tfnh e n z y m e bj m i t hodn t o d n . (J nfing d 5 10 m M , hogt t i n h g i l m m a n h sau 10 phdt ii, s a u d d gidm n h g d t h d i gian k h a o sdt t i l p theo.
Bdng 2. Anh h u d n g cda m$t s d c h i t d c che d4n hogt tinh cda DAAO
Thdi gian (phat)
0 10 20 30
E)di chdng 100.0 100,0 100,0 100,0
Pyruvic acid 10 mM 100,0
56,7 50.3 41.3
Pymvic acid 30 mM 100,0
Hogt linh brong doi {%) Benzoic add
10 mM 100,0 78.8 66,2 50,8
Senzoic acid 30 mM
100,0 68,5 1.0 0
H2O2 10 mM 100,0 100,0 51,8 51,8
GLA 10 mM 100,0 77,6 68,5 29,4
GLA 20 mM 100,0 55,3 24,8 "
0 Ghi chO: Dfii chdng: M 3 U enzyme khdng bd sung cdc co c h i t Pyruvic, Benzoic. HiOz vd Glutaryl 7-ACA (GLA).
B i n g 3. Anh h u ^ n g cda m$t s f i ion kim loai den hogt tfnh DAAO tdi t f i h c ^ Hogt tinh tuong dfii (%)
Dfii chdng F e S O , CUSO4 MnSO.
MgSO, ZnSO, CaCij C0CI3 HgClj
100,00 100,00 70,70 108,02 122,70 117.10 106.40 103,23 7.00
100,00 99,70 29,50 100,01 102.90 106,00 103,41 99.50 3,00
Gh/c/)u.O DAAO vdi cdc kim logi d nfing d?H mM. ngng vdi H g O i nfing dg khao sdt Id I p M ( c d t A ) ; 0 D/\AO vdi cdc kim logi d nong
HOI NUMj K.MUA MyU L;UIN^J iNoni? o i i i n n\
ao 5 mM, rieng vdi HgCI? nfing dd khdo sdt ii 10 pM thdi gian khdo sdt cdc miu Id 60 phirt (cdt B).
Glutaryl 7^CA (GLA) cung gdy anh hudng Widng tfit din hogt tfnh xdc tdc enzyme tdi tfi h^rp. Khi u DAAO vdi GLA din nfing dd cufii Id 10 mM hogt tfnh gilm edn 77,6% sau 10 phiit tilp xdc. Ddc bidt, hogt dO enzyme gidm mfit nda %u 10 phdt vd mit hodn todn sau 30 phdt n l u tdng nfing dd GLA len 20 mM. Ket qud tucmg bjr cung dupe cfing bfi bdi Moreno vd dfing tde gid (2004), Iheo dd pymvic acid, Id a- keto add tuan^ dng ciia D-alanine. cung nhu mfit sfi edc a-keto acid khde Id chat dc chl cgnh to^nh vdi D-amino acid tirang dng. O nong dO cdng cao, khd ndng Crc che cda eae a-keto acid cdng xdy ra nhanh ehdng vd fridt d l . Vdri benzoic aeid ttii sy bit hogt enzyme ti 1$ ttiu^n vdi nfing dO vd ttidi gian bip xue 0 nfing d010 mM, hogt tinh gidm din diu ttieo ttidi gian tilp xde. Hogt ttnh DAAO sau 30 phut d vdi dung djch acid ndy d nfing dd 10 mM cdn 37,3% vd mit hodn todn d nfing dO 30 mM sau 20 phut.
Cdc ton kim logi cd Inh hudng khdc nhau tdi hogt dd cua DAAO, trong dd Cu^, Hg^ gdy tdc ddng uc ehe rd r$t nhlt d nwig dd 1 mM. CuS04 lam gi^m hogt tinh enzyme cdn 70,7% sau 1 gid vd edn^25,5% d nfing dd 5 mM. Ridng dfii viii Hff^ hogt tfnh gdn nhu mit hoan todn sau 1 gid u d nfing dd IpM. Mg vd Zn cd tdc dyng tdng cudng Idid ndng njc tdc ciia DAAO Idn 117 - 122 % d nfing dO nhfi (ImM), d nfing dp 5 mM Igi khdng cd tde dflng den DAAO. MOt sfi ion nhu Fe^. Co^' va Mn^* khdng Idm dnh hudng din hogt tinh eua enzyme (bdng 3). Ticng nghidn edu eiia Schrader vS Andreesen (1996) Fe^ khdng lam gidm hoat tinh ciia DAAO h> T. variabilis. Hai ion Cu vd Hg Idm gidm nhanh ch6ng hoat Iyc cua DAAO d ham luang r i t nhd. Khi ed mgt 20 pM CUCI2, tgi pH 6,0, enzyme s§ mit mfit nda hogt tlnh cW bong ba phut vd nlu bfi sung ttiem mot lupng rit nhd Hga2 sS lam bit hogt enzyme frong vdng 8 phdt. Vd d l frdnh bit hogt enzyme cd ttil bfi sung cdc chit nhu EDTA, aeid citric, L-histidine va mdt sfi chat chelat khdc d nfing d01 mM.
Dpng hpc DAAO vdi mdt s l c a chat
Nghidn cdu dfing hpe cua DAAO frdn ca ehat D-alanine eho thiy, khi tdng d i n nfing dp D-alanine tu 0,25 gli din 1,5 g/l, trong thdi gian diu cua phdn irng, nfing dp sdn phim tgo thdnh tdng dan theo thdi gian va theo nfing d$ ca chit ban diu sd dyng. Cy ttil. vgn tfie tdng d i n td 0,05; 0,08; 0,1; 0,12 din 0,14 pM/s khi ldng nfing dfi ca chit tu 0,25; ldn 0,6, 0,75; 1,0 vd 1,5 g/l (Hlnh 6 a). Td v^n tfie phdn dng enzym vdi ca chit D-alanine d cdc nfing dO khdc nhau, dyng diUij Lineaweaver-Burk qua dd xdc cHnh dupe vgn tfie eye dgi (Vm) Id 0,15 tiM/s vd K™ = 0,468 g/l (tuang duang 5,25 mM) {Hinh 6 b). Tuong ty nhu vdy, qua sy thay dli nfing dfi H2OZ theo Uidi gian phdn dng vdi nfing dd ca chit DL-alanlne dya vdo dfi thj Uneaweaver-Burtt, Km vd V™ cda DAAO dupC xde djnh vdl ea chit DL-alanine tuang dng Id: Kn,= 0.682 g/l {7,66 mM) vd Vmw Id 0,13 pmol/s (Hlnh 6 e, d). Tuang ty nhu v|y. dc ttidng sfi Km vd Vmax xde djnh dupe ttidng qua dfi ttij Lineaweaver vd Burii vdi D vd DL-Methionine (ndng dd ca ehit thay dfii ti> 0,025 ^ 0,8 gfl) lln lugt Id 0,023 gfl {0.15 mM). 0,10 pmol/s vd 0,041 g/l (0,27 mM), 0,12 U/ml.s (Hlnh 7).
Hlnh 6. Biln dfii v|n tfie phdn dng theo nfing d? D-alanine (a) vd DL-alanine (c), dfi thj Lineaweaver -Burk cda DAAO vdi c<
chdt D-alanine (b) co- chat DL-aiantne (d).
•4<Bia»DUM|vq
T
., . - 3
Hlnh 7. B i l n dfii v|ln toe phan d n g theo nfing d $ D-methlonine (1) vd phuomg trlnh Uneaweaver-Burk cua DAAO tdi tfi hffpvM CC c h i t D-methlonlne (2) vd DL-methlonlne (4).
Bang 4. Cac thdng sfi dpng hoc cda DAAO tdi to h p p t r o n g cac phan i r n g x u c tdc v d i D-amino acid khdc nhau.
Co c h i t Km (mM) Vm„(umol/s) D- alanine
DL - alanine D - methionine DL - methionine
0,15 0,13 0.10 0,12
N h u Vdy, h i n g sfi Miehealis M e n t e n Km ttilp n h l t v d i D-methionine, t i l p d d d i n DL-methionine, DL-alanine vi 0- alanine. V§n toe e y e dgi Vmax thay dfii t u a n g d n g t u 0,1 * 0,15 pmol/s. t r o n g d d D-alanine eho Vmax 16^ n h l t O^^'^- pmol/s. D-methionine cho gia tii n h d n h l t (0,1 pmol/s) { B d n g 4 ) . Trong lifin c a c h i t d u p e nghidn c d u , D-methionlne W 1 hidn tinh dgc hieu cao nhdt v d i D / \ A O . Cac hfin h p p d o n g phdn D L c d di I y c v d i e n z y m e n h f i h a n c d c dfing phSnP .1
tuang img, vl trong hfin tiyp hai dfing phdn, duy chi co dfing phan D ttiam gia phdn dng chuyen hda, trong khi dd dfing P^ j - Widng phdi Id ea chit phdn dng ciia enzyme ndy. Guriie vd dfing tdc gid (2007) cho ring sy, ed mdt eda dfing A '-r'"^*'^'°"''^6 t™"g *i*n hpp phdn dng giy uc c h l mdt phin hogt tinh DAAO. Nhifiu nghidn cdu cho ttiay DAAO CO ngudn gfic tu T. variabilis ttil hidn di lye cao vdi edc D-amino aeid cd mgch ben kfch thudc ldn vd ky nude, frong d6 eao nhdt Id D-methionine, D-phen^alanine va D-tryptophane (Pollegtoni et ai, 2008). Cdc gia tii Km xdc dinh eho DAAO ti> T. vanabilis cung ttiay dfii nhieu ttieo ca chit sd dung: 0,2 mM (D-phenylalanine); 0,29 mM (D-Met); cephalosporin C (0,83 va 2,4 mM) vd 6.5 mM (D-alanine) (Schrader, Aridreesen, 1996).
K £ T LUAN
Da ttiu dupe DAAO tinh sgch cd hogt Hnh dgc hi$u 215,6 U/mg protein, tang 5,7 lln so vdi djeh thd ban dau bang phuang phdp sdc k]? di Iyc tren eOt ProBond™ column c6 gin Ni^*
Ba xdc djnh dupe mOt sfi dgc tfnh eda DAAO b> chdng tdi tfi hgp: Nhigt dp tfii uu SOX; pH tfii uu 8.5; bin frong viing nhigt dp dudi 45 C vd t i i n frong dat pH ti> 7 - 9.
Enzym DAAO bi dc ehi bdi ion kim logi Cu"" vd Hg** va cdc chit GL-7-ACA, benzoat, H2O2 va pymvic acid.
Ba xdc djnh dupe cdc ttidng sfi dpng hpc cua DAAO vdi ede ea chit D, DL-alanine, D, DL-mettiionine, Km vd Vm ldn lupt nhu sau: Km: 5.25; 7,66; 0.15; 0,27 mM vd V^: 0,15; 0,13; 0,1 va 0,12 pmol/s.
LM c i m orn: Nghiin ciiu ndy dufrc tai Irfrbal Quy phdt tnen Khoa hpc va Cong ngh$ Quae gia (NAFOSTED) trong di tdi ma so 106.03-2011-07.
TAI Lieu THAM KHAO
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PURIFICATION AND PROPERTIES OF RECOMBINANT D-AMINO ACID OXIDASE FROM TRIGONOPSIS VARIABILIS KCTC 0864
Phan Thi Hong Thao\ Dang Thi Thuy Duong'', Phi Quyet Tien\ Vu Thi Hanh Nguyen'', Quan Le Ha^ Le Gia H / ' institute of Biotechnology, Vietnam Academy of Sdence and Technology
Institufe of Biotechnology and Food Technology, Hanoi University of Sdence and Technology
SUMMARY
D-amino acid oxidase (DAAO, EC 1.4.3.3) catalyzes oxidation of the ammo group of D-amino acid to form corresponding a-keto acid, ammonia and hydrogen peroxide. This enzyme can be obtamed from many different organisms, but DAAO originated from Trigonopsis variabilis is the most prefencd for bio-transfonnalion in industrial scales. In this Study, recombinant D-amino acid oxidase originated from E coli BL21(DE3)-rfaao was purified on NTA-Ni column with the recovery yield of 67,32%. The specific activity of purified enzymes on D-alanme as substrate gamed 215.6 ^mol/ mg protein, which was 5,7 fold higher than that of the cnide extract. D-amino acid oxidase has molecular mass of 39 kDa as analyzed on 12,5% SDS-PAGE gel. The enzyme was stable at temperatures up to 45''C and pH of 7.0 - 9.0, while its highest catalytic activity was observed at 50°C and pH 8.5. Severals of organic acids, such as pyruvic, glutaric and benzoic acid inhibited DAAO activity at the concentrations of 10, 20 and 30 mM, respectively.
Strong inhibition was observed with the presence of Cu'* and Hg^* ions at low concentrations, i.e 1 mM and 1 nM, respectively.
HQI NGHI KHOA H6L lUUU I^UI II, S^WWm
Both ions Mg^ and Zn'* increased DAAO acdvity up to 117^ 122% at the concentration of 1 mM, whereas Fe'', Co" and Mn'' showed insignificant effects m tbe enzyme activity, llie K., and 'V„„ values of the purified DAAO were calculated by Lioeawnvv and Budc equation, giving 5,25: 7,66; 0,15; 0,27 mM and 0,15; 0,13; 0,1 and 0,12 funat/s on D-alanine, DL-alanine, D-n ' ' and DL-meduonine as substrates, respectively.
KeytPonts.D-vmao add oxidase, Trigonopsis variabilis, inhibitor, recombinant enzyme. BL21(DE3)-<faao.
• Author for coTOSspoodence: Tel: +84-4-38363257; Email: [email protected].
