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Tap chi Cong ngh.e Sinh hoc 13(1): 161-167, 2015 CiJA DUN POLY-HISTIDINE LEN KHA NANG BIEU HIEN TIET ANH ENDOGLUCANASE A ODA CLOSTRIDIUM THERMOCELLUM TRONG BACILLUS SUB77LIS WB800N Phom Luang Thing, Nguyin Huang Ngqc Pinning, Than Thj Phtning Trang, Tian Linh Thireic, NguyAn Dtrc Hoang Truerng Dgi hoc Khoa hoc it nhien, Dai hoc Quoc gia Thanh pha Ho Chi Minh Ngity nhan bai: 26.10.2014 Ngay nhan clang: 04.12.2014 TOM TAT Endoglucanase A (CeIA) t6 Clostridium thermocellum li enzyme chit, nhiet nem phirc he cellulosome c6 kha Wing phin cit cellulose met each hieu qui De c6 the a ding thu nhOn enzyme Ce1A, enzyme phii dtroc dung hqp vai duoi tinh the vi bieu hien met ching chit an toin, nuoi city vi c6 ruing tiet tot protein mai truing nuoi city Trong nghien dm duoi tinh the poly-Histidine vi chimg chit Bacillus subtilis dirge sir dung nhim khio sat sir anti huding cis duel tinh the len ning bieu hien vi hoot tinh dm enzyme CeIA Cic duoi tinh the poly-Histidine v6i so luqng amino acid Histidine khic (4xHis hoc 5xHis hoc 6xHis) duqc thiet ke dung hop tai ctiu C cua enzyme CeIA Mut de bleu hien vi hoot tinh cua cite protein di to hop ductc dinh gia thong qua kha ning gin cut Ni-NTA cue duoi poly-Histidine vi dual hoot tinh ph'in giai CMC to dtr6ng khir cua CeIA Ket qua phin tich thong ke ye anh htremg poly-Histidine len CeIA cho thiy, cite dieu kien nhiet de, nOng de chit cam (mg khic deu cho thiy duoi poly-Histidine dung hqp tAti diu C cis CeIA c6 inh hu6ng len sir bleu hien cfing nhu hoot tinh cua enzyme 0µ the li so lugng a.a His cing nhieu thi mire de bieu hien vi hoot tinh cua enzyme cing giim nhung fling gin dm protein dung hqp len cut Ni-NTA cing chit va hieu qui Tir klia: Bacillus subtilis, bleu hien tiet, CeIA, Endoglucanase A, poly-Histidine M6 DAU Cellulosome dm Clostridium thermocellum la mut nhiing phirc he enzyme cO kha fling phan cat Vet cellulose, dO Endoglucanase A (CeIA) la mOt nhang phin chit yeu cua phirc hg cellulosome (Demain et al., 2005), (Fontes va Gilbert, 2010), (Gold va Martin, 2007) De bleu hien va thu nhan CeIA tir chimg vi sinh vat Gram ducrng, ky va ua nhiet C thermocellum, chung chit bieu hien Bacillus subtilis la del tuonF tiem fling nhit vi: (i) Wang thing ve mat ciu true to bao vi ca hat cling la vi Ichuin Gram ducmg; (ii) da co he thOng plasmid ton tai dOc lap pHT43 (MoBiTec), cho phep bieu hien protein tai to hop ding tiet mai hieing (Phan et al., 2006), (Simonen va Palva, 1993); (iii) dieu kien nuoi city dun gian, chi phi thip nhung hieu qua tao sin phim va sinh khoi cao gip nhieu lin so vol viec nuoi cay va thu nhan CeIA tren C thermocellum (Schumann, 2007); (iv) la chung an toan GRAS (General Regarded As Safe) duqc cong nhan bai FDA (Foods and Drugs Administration), My (Schumann, 2007) Trong nghien ciru khfic, cluing toi da bieu hien c8ng CeIA tai to hop ding tiet ten B subtilis 1012 va B subtilis WB800N Trong nghien ciru nay, duoi tinh the poly-Histidine duqc dung hop h dau C dm protein CeIA nharn img,clung viec tinh the CeIA Tuy nhien, ban chat cua mang nguyen sinh chat, cfic dpi phan hi sinh hoc mang dien tich duong nhu du8i tinh che polyHistidine co the se bi han the di chuy'en qua ming qua trinh tiet Anh htreyng bai so lucmg His duoi poly-Histidine la 4, NIA a.a duqc khao sat nghien ciru VAT LIEU VA PHIJONG PHAP Ttio cac plasmid tai t6 hqp Cac plasmid pHT1454 (CeIA-4xHis), pHT1455 (Ce1A-5xHis) va pHT 1456 (Ce1A-6xHis) duqc tao tir plasmid khung stRm IA pHT1202 mang trinh to tin hieu tiet cua gene amyQ tir Bacillus amyloliquefaciens va promoter manh Pgrac212 (Phan et al., 2013) Phucmg phi!) dong hOa duqc sir 161 Pham Lucmg Thing et al ding la In-fusion yeti cac cep mei thu gene d#c hieu cho celA dung hqp duel 4xHis (ON915/0N916E), 5xHis (ON915/0N916D) va 6xHis (ON915/0N916C) (phg lgc Si) Ket qua deng hea duqc kiem tra bang phuong phip giai trinh tlr vai Op mei ON314 va ON1207 To cluing B subtilis tai to hip Cac plasmid tai to hop pHT duqc Wen nap vio B subtilis WB800N (Nguyen et aL, 2011) bang phucmg phap bien nap to nhien theo quy trinh Wang din cua MoBiTec (MoBiTec GmbH Company, 2012) Khang sinh ch9n 19c hi chloramphenicol neng dO 10 µg/ml Sau 24 gib, 6 37°C, cac khan lac dv tuyen duoc sang 19c lai bang PCR khuan lac vii c(ip moi duqc sir ding la ON707 va ON916, sin phim to CO kich thus* la 1479 bp Khoo sit kha fling bieu hi§n tat CeIA (ra tren vbng phan giii Cac chung tiem nang duqc nuoi cay tren dia thach LB-Cm10 (10 pg/m1 chloramphenicol) chira 0,5% CMC va chit cam ung IPTG v&i cac ming de khic 0; 0,01; 0,05; 0,1; 0,5 va mM U 24 gib 37°C, nhuem thuec thir de Congo 1% phut, Ilia bang NaCI M Kha nang phin giii CMC cang cao thi yang phan giai cang len (Yoon et al., 2007) Xic nh#n kha nang bieu hi§n flit CeIA djch nuoi ay B subtilis Te boo B subtilis WB800N mang plasmid tai to hip duqc nuoi cay moi trutmg LB long 37°C Khi m#t de to boo did gia trj ODA khoang 0,8 — 1,2, cam ling vol IPTG ming de la 0,1 mM Sau cam ung ducrc6 gia, tien.hanh thu ml mAu dick net, tira TCA non de cuoi la 5% (Rajalingam et al., 2009) lei tien hanh rhan tich SDS-PAGE va Western Blot vei khang the the da deng khang CeIA Killing the thu dip dung hop enzyme, Horseradish peroxidase (HRP) duqc phat hien bang co chat 3,3',5,5'-tetramethylbenzidine (TMB) Xic nh.n sv din t31 cua dubi tinh the 4xHis, 5:His vi 6xHis Sic ky hrc ky thu#t tinh the dva tren tic ac hieu cua ion Nit' yen a.a His (Uppsala, 2002) Trong de, phan tir imidazole to se earth tranh veri His viec bam len cot Ni-NTA Ming de imidazole can thiet cho viec dung ly cling cao, tuong tic dm duoi tinh the veri pha tinh Ni-NTA cang Ian Cac duOi dung hip 4xHis, SxHis va 6xHis se duqc kiem tra sir hien dien thong qua danh gia kha 162 nang gin Ni-NTA bang sic ky Neng dO imidazole c3nh tranh tang clan theo se Wong a.a His du8i dung hop 4xHis, SxHis va 6xHis Dinh gii anh huteng se Wong a.a His duoi dung hop len khg nang flit CeIA cilia B subtilis Chung B subtilis WB800N duqc xic nh#n bieu hien tiet CeIA vol eau trac day de dm 4, va His du8i dung hqp Tien hinh nuoi cay va cam ling bleu hien CelA dun hqp cac ming de chat cam ung IPTG tir 0,1 den mM Djch nuoi cay t6 gib den 24 gia sau cam trig duqc thu nh#n de danh gia hoot tinh theo quy trinh cal bien cua IUPAC (International Union of Pure and Applied Chemistry) (Ghose, 1987) Phucrng phap th8ng ke sir dung cho danh giit sv khgc bit ye kha nang bieu hien la ANOVA met chieu ket hop plan tich Tukey's significant difference, sal so chip nhOn la 5% KET QUA Tao chfing B subtilis c6 mang plasmid tai to hop Cac khuan lac tuyin B subtilis duqc nuoi cay tren moi trutmg chen 19c LB-Cm10 sau 24 gib se duqc kiem tra sv hien dien cua plasmid tai to hop bang cep moi ON707 va ON916 veri san phim PCR co chira gene celA dv kien 1479 bp San phi.= PCR duqc kiem tra tren gel agarose 1% (Hinh 1) cho thay cac khuin lac duqc hra chip deu cho sin pham PCR dung %raj kich thuOc dg doin Day se la cac khuin lac duqc sir ding viec sang 19c kha nang phan gal CMC buOc tiep theo Kha nang phan giai CMC cua die chiing B subtilis tai to hop Vangphan giii CMC duqc hinh thinh CelA t6 cac khuan lac duqc flit moi trueng xung quanh phan cat cAu tire CMC, yang moi trubng thiu co chit se khong bit mini veri dung djch Congo red 1% Ket qui Hinh cho they dueng kinh Wong phan giii tirog can theo ding de chit cam ung IPTG Tuy nhien, ket qua Wong phan giai cho they kha nang ti6t CelA auk chimg B subtilis WB800N kh6ng hoin toin tuyen tinh vai ruing de chit cam ung IPTG, hieu qua t3o enzyme Qat t6i da ming •10 chat cam ung la 0,1 mM Tap chi Gong ngh" Sinh hac 13(1): 161-167, 2015 Bacillus subtilis WB800N by pHT1456 pHT1455 pHT1454 M KL I KL2 KL I KL2 KL1 KL2 5000 2000 1479 by 1200 800 500 Hinh San phArn PCR khuech dal vOng gene celA tip khuen lac B subtilis WB800N mang plasmid tai to hop pHT1454, pHT1455 va pHT1456 Bacillus subtilis WB800N 0,00 0,01 0,05 0,10 0,50 1,00 (mM IPTG) • • • • • • • • • •• • • • • 0 O • 0 10 Hinh yang phan giai CMC cua cac chi:mg tai to hop B subtilis WB800N mang cac plasmid pHT1454, pHT1455 va pHT1456 b cac ming dO chat cam 0,ng IPTG va dieu Van nhiOt dO nu6i cay Chung vi khuen mang plasmid pHT43 (MoBiTec GmbH Company, 2012) khong mang gene ma h6a cho CeIA duvc sir dyng lam doi chCrng am phan CeIA tai to hgp clich, nuoi cay vi sinh vet Dieu dirge the hien qua ket qua SDS-PAGE Hinh vai vach protein tai to hop (vi tri ki hieu bang B subtilis WB800N la claing chu cid duac dot cham mau trang) a kich thuac khoang 50 kDa, tucmg bien loai protease ngo4i bao, nhien, cac ang vai kich thuac 1Y thuyet ciia protein CeIA (49,14 protease lai ciia no van co kha nang phan cat met kDa) cho tai vach 40 kDa, twang drig vai domain lei Phan tich SDS-PAGE ve Western Blotting eau &tic CeIA dung hop 163 Plum Luang Thing et al hoot tinh cua protein Ce1A (39,4 kDa) Ket qui duqc xac djnh loi bang phial tich Western blot v6i khang the dac hieu cho protein CeIA V6i cac voch dim, ro vi chi xuAt hien phan (loon tiet cac chung B subtilis mang plasmid pHT1454, pHT1455 vi pHT1456, kit qui lai cho thiy twang tac protein — khang the (lac hieu, kheng c6 bit kk tin hien miu nio too thinh djch nuoi ciy chung doi chimg B subtilis mang plasmid pHT43 Ben conh Q6, cac gieng tuung ring v6i cac chimg mang plasmid tai to hqp, voch cau trac cua CeIA too thinh la tuong tIng yeti kit qua (lien di SDSPAGE li domain loi hoot tinh vi CeIA Cray dti kh6ng bj phan cit Trong Q6, tin hieu lai cua Ce1A Ichong bi phan cat (GH8 — Doc 1) cao ham Ilan so v6i loi hoot tinh (GH8) Kit qua phan tich cho thiy CeIA tai to hop bieu hien tren B subtilis WB800N bi phan cit nhung phin lan protein dung hop van gill duqc cau truc nguyen vgn v6i duel poly-Histidine diu C vi c6 the tinh the duvc bang sac kji luc voi Ni-NTA Bacillus subtilis WB800N A M s'2) 414:c•b`55 iz4 4.41 "SI° i
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