Tap chi Cong nghe Sinh hpc 13(3): 965-971, 2015 TACH De:ONG VA BIEU HIEN GEN MA Holt ENDO-1,44-GLUCANASE B CiJA CLOSTRIDIUM THERMOCELLUM TRONG BACILLUS SUBTILIS Pham Luang Thing', Pham Thi My Tien', Trim Quie Twin', Nguyen Le TuAn Anh', Nguyen Mang NgQc Phtrong', Phan Thi Phirong Trang', Nguyen Thi Nguyen2, Nguyen Dinh Quan2, Phan Dinh Thin', TrAn Linh Nguyen Dire lining' i Trucing 2Trtrang Ogi hpc Khoa hpc to nhien Dai hpc Qu'dc gia Thanh phd Hd Chi Minh Dai hpc Bach khoa, Dai hpc Qudc gia Thank pito Hd Chi Minh Ngay nhan bai: 26.11.2014 Ngay nh4n dang: 12.9.2015 TOM TAT Viet Nam la met nhan quite gia doi dito v'e ngulin phe phttm ming nghiep yeti han 40 trieu tan ram m6i nrun De tan dung het Ton ca chat nay, cong nghiep san xuAt ethanol sinh hoc dei va mang Iai tiem nang to 16n, g6p phan giai quyet van killing hoing.nang Trong Q6, cac enzyme cellulase d6ng vai tr6 quan trong viec duly phan cellulose too ca chat cho qua trinh len men, san xuat ethanol Xuat phat lir thrc to Q6, chting toi ti8n hanh bieu hien Endo-1,4-8-glucanase B tai to hqp tit chung ky Clostridium thermocellum chung c6ng nghiep Bacillus subtilis Diu lien, chOng Ceti thu nhan gen ce/B ma h6a cho enzyme Endo-1,4-8-glucanase B ti} Clostridium thermocellum DSMI313 Sau de, churl* toi dOng h6a plasmid tai 18 hqp pHT43-ce/B va pHT1219-celB, kiem tra cac don mang plasmid tai to hop bang phtrcmg phap PCR khuan lac va giii trinh qt Cacplasmid tat to hqp duqc bier' nap vat) B subtilis 1012 va kiem tra kha nang bleu hien tren m8i twang long va ran Ket qua cho thay, nong d8 chat cam Ong IPTG la o,5 mM, chitty B subtilis tai to hqp cho wing phan giai nufai cay ten mei trtrang thach china ca chat CMC Dong thai, ket qua SDSPAGE va hoot tinh cho thity chung bieu hien c6 kha nang tiet CelB c6 hoot tinh khoang 80 U/ml nuai ay ten m6i trtgmg long a Tir khda: Bacillus subtilis Endo-1,4-6-glucanase B, Clostridium thermocellum, cellulose plasmid pHT MO DAU Qua trinh san xuAt ethanol sinh hoc thong thuang sO dung enzyme tin cac chting vi sinh vat de plan cat tinh 138t glucose rOi len men tao ethanol Tuy nhien, yin de thieu but luang thuc dang la not lo tan cAu nhu hien thi can phai thay dOi chin luqc, chuyen &I sang sir dung nguOn ca chAt doi clio han la cellulose nham thay the cho tinh bet M6t nhung nguOn cellulose doi dao, gid re, sin có tai Vi't Nam chinh rum Voi han 40 trieu tan ram mai nam, viec sir dung co chat san xuat ethanol sinh hoc se la mut the manh dm Viet Nam, d6ng thai g6p phan giam thieu nhiem moi tnr6ng, giam chi phi xir ly chit thai nong nghiep cho !tong dan (Dang Tuyet Phuany, 20101 Mac hien ce rat nhieu nguon vi khuan tai to hqp hay enzyme cellulase tai to hqp duqc sO dung de thity phAn cellulose nhung chua duqc nghien cOu situ rung Rieng Viet Nam, cac nghien ciru hien van b6 ngo NIA chua c6 nhieu thinh tuu d ang ke Xuat phat tO tinh hinh thur to neu tren, nh6m chung toi de xuAt huOng nghien cfru too hen hqp enzyme cellulase tai to hqp c6 kha nang thity phan cellulose Trong do, sir dung (long thai cac enzyme thuOc nham (i) endo-1,4-13D-glucanase phAn cat sqi cellulose tin ben too cac sqi cellulose nh6 ham, (ii) exoglucanase phan cat cac sqi nhe cellulose tin ben ngoai tao san phim IA cellobiose va (iii) f3-glucosidase too glucose tin cellobiose Qua da, g6p phan dua mgt giai phap kha thi cho yin de nang luqng tai sinh va xir phe phu pham nong nghiep Clostridium thermocellum la cluing vi sinh vat ky khi, ua nhiet, da duqc biet den tin rat lau bai kha nang san xuAt enzyme cellulase ben nhiet, ce hoot tinh rieng cao tren co chit cellulose (Demain et al 2005) Tuy nhien, viec nuoi cAy chimg vi sinh vat tren quy mit c8ng nghiep de san xuAt cellulase la nit kh6 khan, khi, tiem nang san xuAt loi chua c6 the kiem chOng duqc Do do, viec san xuAt cellulase tai to hqp tO C thermocellum se la giai phap hoin hao cho nen san xuAt enzyme c8ng nghiep nuOc 965 Pharr Lucmg Thing et al Hien nay, ben canh Escherichia coli thu6ng duqc sir dung sin xuat protein di to hqp, vi khuin Bacillus subtilis ding Bang duqc quan tam rat nhieu nhor sr bieu hien tiet hieu qua cac protein muc tieu cong nghiep va d Ong Op ding lee nong nghiep, y duqc va cong nghe thuc pham (Schumann, 2007) Endo-1,413-D-glucanase B hay Endoglucanase B (Ce1B) la met nhcmg enzyme duqc nghien ciru tir rat lau C thermocellum va c6 tient rang bieu hien cao i ca cap de phial ma va dich ma (Riederer et al., 2011) Nghien dm nham khfio sat sr bleu hien dm celB B subtilis sir dung he thong bieu hien pHT,,dtra tren promoter Pgrac Ce1B vai kha rang phan cat lien ket nei phan tir f3-1,4-glycosidic ben sqi cellulose, se la met nhitng thinh phan endo-1,4-13-D-glucanase dau tien, d6ng vai tre quan tong phirc he cellulase dung cho cac phan img thity phan sinh khoi ram VAT LIEU VA PHUONG PHAP Vet lieu Chimg vi sinh vat ky C thermocellum DSM 1313 tir ngan hang chimg DSMZ, CHLB Dirc duqc sir dung lam nguon gen de thu nhan celB Chung E coli OmniMAX {F' [proAB+lacrlacZAM15 Tn10(TetR) L(ccdAB)] mcrA A(mrr-hsdRMSmcrBC) 980(/acZ)AM15 A( lacZY A-argF) U169 endAl recAl supE44 thi-1 gyrA96 relAl tonA panD} duqc sir dung lam cluing chi de dOng h6a va luu gift cac plasmid Chimg B subtilis 1012 (leuA8 metB5 trpC2 hsdRMI) (Saito et al., 1979) duqc dimg lam chimg chit khao sat kha rang bieu, hien protein muc tieu tiet ngoai moi truing nuoi cay Cfic plasmid cua he thOng pHT nhu pHT43 va pHT1219 chira promoter Pgrac (Nguyen et al., 2007), day la cac plasmid thoi cho phep to (long, nhan ban E coli vii kieu hinh ch9n 19c la kiting ampicillin va c6 the duqc tao dOng vao B subtilis vii kieu hinh chon 19c kiting chloramphenicol Ca hai plasmid deu mang promoter nhan too Pgrac, c6 trinh at tin hieu tiet dm a-amylase SamyQ gifip tiet protein ngoai lai mei truing nuoi cay Khfic chinh cua hai plasmid la codon cua trinh at tin hieu tiet pHT1219 duqc thay doi nham lam giam sit bieu hien E coli, trinh to tin hieu tiet i pHT43 91 gen amyQ cita Bacillus amyloliquefaciens Ngoii ra, pHT1219 chira trinh at ma h6a cho Strep-tag sau vOng MCS Hai plasmid duqc sir dung lam 966 khung su6n d e tao plasmid pHT43-celB va pHT1219-ce/B Tao plasmid tai to hop pHT43-ce/B va pHT1219celB Dau tien, chimg toi tien hanh thu nhan gen celB (1) bang phucmg phap PCR vii cap m6i chuyen biet 5`ON917, AGCATCAGCAGGATCCGAAGGGTCATATGCT celB AatII R, 5'GATTTGG-3' va CTCA GGCCATGACGTCTTATTTATkGGEAA CTTATGC-3' vii kith think du doin 1639 by nham dung hqp vii trinh to tin hieu tiet c6 vi tri cat TSAVGS plasmid pHT43 Gen celB (2) cling duq‘c thu nhan bang phucmg phip PCR vii cap moi ON918, 5`va ON917 GGATGGCTCCAAGCGCTTTTATACGGCAACT CACTTATGCTACGC-3`, kith think gen 1641 by nham dung h9p vii trinh to tin hieu tiet co vi tri cat ASA-GS pHT1219 Mach khuon cho cac OIL img PCR la DNA be gen dm C thermocellum San pham PCR duqc tinh such, sau de duqc cat va not vao cac plasmid pHT43 va pHT1219 tai cac vi tri BamHI NIA Awn nham tao cac plasmid pHT43celB va pHT1219-celB Cac sin phim not duqc Wen nap vao chimg E coli OmniMAX Cac clang khuan lac c6 mang plasmid pHT43-ce/B va pHT1219-celB duac digit bang PCR khan lac vii cap moi tucmg img ON1209, 5'GGCCTAGATCTATGATTCAAAAAGCGAAAGC GG -3' va ON918 (sin pham khuech dai 1738 bp, doi vii plasmid pHT43-ce/B) va ON707, 5`AAAGGAGGAAGGATCAATGAGAGGAAGCA-3' va ON918 (sin pham Ichuech dai 1743 bp, dOi vii plasmid pHT1219-ce/B) Cac plasmid du tuyen duqc tach bang QIAprep spin miniprep kit (QIAGEN) va kiem tra bang phuang phap giii trinh tit Tao thing B subtilis vi o sit sy bieu hien aim CeIB teen dia Plasmid sau giii trinh to dtrqc bitn nap vao B subtilis 1012 bang phucmg phi') di en bien nap Khan lac B subtilis m9c duqc sau bien nap se duqc khao sat kha !tang bieu hien CeIB tren dia thach LB-Cm 10 (Luria Bertani - Chloramphenicol 10 pg/m1) ce chira 0,5% carboxymethylcellulose (CMC) va chit cam (mg IPTG Sau 24 gia nuoi cay, kha 'tang bieu hien cua chimg se duqc danh gia th8ng qua ban kinh veing phan giai CMC Dinh gii tang ban hien tiet CeIB dm B subtilis 1012 Top chi Cong nghe Sinh hoc 13(3): 965-971, 2015 Chung toi tien hanh bleu hien moi tru6ng long 100 ml LB-Cm 10 pg/ml ding a cfic nOng de IPTG tucmg tu nhu khdo sat tren dia moi thai diem khao sat, thu nhan ml djch nuoi cay di loai to bao bang ly tam 10.000 yang phut e chuan bj mau cho phan tich SDS-PAGE, ml djch enzyme duqc tua vai TCA (Trichloroacetic acid) a ruing de cuiii la 5% (Rajalingam et al., 2009) Song song do, chimg toi xfic d jnh hoot tinh CMCase ctia enzyme Ce1B theo quy trinh sau: 250 pl djch enzyme Arai 250 pl Natri Acetat buffer, pH 5,5 co chua 0,5% CMC, u 55°C 30 phut Sau d6, d jnh luring du 6ng khir tao bing thuOc thir DNS (Eveleigh et al., 2009) Val U hoot tinh enzyme la luqng enzyme can thiet de tao luqng du6ng khir tuang duang yeti nmol glucose (0,18 pg glucose) phut Xfc nh$n gr ban hien CeIB bing lai Western blot Djch nu& cay B subtilis 1012 mang plasmid pHT43-ce/B va pHT1219-ce/B a thai diem 14 gib sau cam ling yeti 0.5 mM IPTG duqc thu nhan va kiem tra Western blot vai khang the khang CelB dac hieu de pha Lang 1/10.000 Khang the thir cap khang IgG the dung hqp horseradish duqc sir citing, vach lai duce phit mau ban bang g ca chat 3,3',5,5'-Tetramethylbenzidine KET QUA VA THAO LUAN Thu nh4n gen celB tir DNA 136 gen cila C thermocellum A DNA be gen dm C thermocellum tach chiet duqc co nOng de 480 ng/pl, vai di) tinh sach duqc xac d jnh th8ng qua gia trj A260/A280 la 1,85 Nhu v#3,, co the kit luan rang DNA be gen hoan toan d am bao cho viec thu nhan gen trine tieu celB Gen celB, duqc khuech d oi tir be gen C thermocellum bang phan ung PCR bang cap moi dac hieu ON917 va vai kich thuerc gen la 1639 bp Sin pham PCR dirge phan tich tren gel agarose 1% (Hinh IA) cho thay co vach DNA vai kich um& nam khoang 1.500 by den 2.000 by dm thang chuin DNA San pham PCR sau tinh sach duqc xir ly vai enzyme cat giai han la BamHI va Aatll va tinh sach !An hal bang PCR purification kit (Qiagen, D ire) Trong met chien thuat khac, vai plasmid gtic la pHT1219 va chien luqc ding h6a In-fusion (Clontech, Mg), gen celB duqc thu nhan bang cap moi ON917 va ON918 Ket qua dien di agarose (Hinh I B) cho thay sin pham PCR cho vach nhat cao han vj tri 1.500 by ciia thang chuan DNA, tuang ung vai kich thuerc 1g thuyet la 1.641 bp Nhu vay, chimg toi da thu nhan ding gen celB tit DNA be gen dm C thermocellum, san sing cho viec tao plasmid pHT43-ce/B va plasmid pHT1219-ce1B Mon gen celB thu nhan duce chi chits phan ma h6a cho lii hoot tinh va dockerin domain: Phan gen ma hoa cho an trinh ter tin hieu tiet, bao g6m 27 a.a d Au tien MKKFLVLL1ALIMIATLLVVPGVQTSA dtrqc loci 136 vi chung toi mut% sir ding phlin trinh ti1r tiet ciia plasmid khung soon pHT43 va pHT1219 B 7000 4000 2500 1500 1000 700 400 by M I 7000 4000 2500 1500 1000 700 400 Hinh Sin pham thu gen celB bang phan Ong PCR tit DNA tx) gen C thennocellum (A) thu gen bang cap m6i ON917 vi celB_AatIl_R (sin pham 1.639 bp) cho &mg boa plasmid pHT43-celB (B) thu gen bang cap moi ON917 vi ON918 (sin pham 1.641 bp) cho ding hie plasmid pHT1219-celB Sin pham PCR duvc chay tren gel agarose 1%, Gieng 1: sin pham PCR thu gen celB tir DNA b8 gen C thermocellum, M: thang DNA ZipRuler Express 2, Thermo Scientific, M9) Kam tra plasmic! tai t4 tiny tao Cac plasmid khung soon pHT43 vi pHT1219 sau duqc xir bang enzyme cat gieri han BamHI va Aatll duce tinh sach vi ch thAn bj cho cac phim,img tao plasmid tai to hqp Ching toi tao plasmid tai to hqp th8ng qua phucmg phip not bang DNA T4 Ligase vi In-fusion vai chien luqc dirge trinh bay bang Tien hanh sang 967 Pham Luang ThAng et al loc clang bien nap bang phucrng phap PCR khuAn lac, san pham dirge phan tich tren gel agarosc 1% va so sanh kich thuerc veri thang chuan DNA (Hinh 2) Cac plasmid tiem nang dirge tach chiet va tie!) tic sir dung phucmg phip giii trinh tit de xac nhan Ket qua dat duce la sirtucrng (long 100% giira trinh tu IS1 thuyet veri trinh to nhan du qc tir cone Macrogen (Han Quoc) phan tich bang phan mem Clone Manager 9.0 Bang Chian Ittqc dong hda tao plasmid pHT43-celB via pHT1219-ce/B Ten gen celB (1) celB (2) Kfch thteerc Plasmid khung su*n Chien thuat to &mg Plasmid san M61 PCR phAm khuen loc celB_Aall I_R 1639 by pHT43 T4 DNA Ligase pHT43-ce/B ON917 ON918 1641 by pHT1219 In-fusion M61 PCR thu gen ON917 pHT1219- celB Kich thutoc ON1209 ON918 1738 by ON707 ON918 1743 by 7110 ww 1743 — ID IMP ins 7911 A B Hinh Kat qua PCR khan lac cac plasmid pHT43-ce/A (Hinh A) va pHT1219-ce/B (Hinh B) Cac gieng 1, 2, 3, va la cac khan lac dy, tuygn, M la thang DNA ZipRuler Express 2, Thermo Scientific, M9) Khao sat kha nang bieu hien ctia celB mai trtrirng ran Khuan lac dun B subtilis mang plasmid pHT43celB va pHT1219-ce/B sau sang 16c duce cham sang dia LB-Agar-Cm, b6 sung 0,5% CMC va IPTG veti n6ng dO 0,5 mM u 24 gi6 a 37°C Kha nang bleu hien cira CeIB dirge danh gia thong qua ban kinh wing phan giai co, chat mai tnremg Ket qua khao sat dirge trinh bay hinh Quan sat ket qui kha nang bleu hien cellulase B (Hinh 3) to thay ca hai chimg B subtilis mans plasmid pHT43-ce/B va pHT1219-celB deu xuat hien vong phan giai va Ian hun so veri mau khung dirge cam irng bang IPTG Dieu chirng to hai chimg B subtilis mang plasmid pHT43-ce/B va pHT1219-celB deu c6 kha nang Neu hien tiet EndoI ,4-B-glucanase B mei tnremg nuoi ch IPTG pHT43-ce/B pHT1219-celB Hinh Kha nang phan giai CMC cua hai chting B subtilis mang plasmid pHT43-celB va pHT1219-celB ten dia thach 968 Tap chi Cong nghe Sinh hoc 13(3): 965-971, 2015 Mem tra kha 'tang tiet Endo-1,4-ll-glucanase B bang phwang phip SDS-PAGE De khao sat kha nang tiet protein tai to hqp m8i titan long, hai chring B subtilis chfra plasmid pHT43-ce/B va pHT1219-ce1B duqc nudi cAy moi trureng LB vai khang sinh Chloramphenicol 37°C va tee to lic 250 vong/phiri Thu matt gi6 OD600 dot 0,8 va bit diu cam ung vai IPTG a nOng (le 0,5 mM, tiep ttic thu mau sau 2, 4, 6, 8, 10, 12 va 14 gia nuoi cAy Thi nghiem &Km tien hanh tuang tir voi mau chimg am 111 B subtilis 1012 mang plasmid pHT43 duqc cam ung IPTG va thu mau sau 14 gia nu6i city Ket qua thi nghiem Quqc trinh bay a hinh Ket qua (lien di SDS-PAGE cho ditty so vai mau d8i chirng am, cac voch tai to hqp xuat hien thei diem 10 gib va 12 gia sau cam Ong cac chung B subtilis 1012/pHT43-ce/B va B subtilis 1012/pHT1219-celB Cac voch tuang ung veri kich thtrac 61 kDa va 53 kDa so yeti thang chttin protein Trong de, vach 61 kDa tucmg ung vai kich thtrac ly thuyet dm protein Ce1B sau loci b6 trinh tv tin hieu tiet SamyQ Cau trite dm enzyme CelB bao gem domain chinh la loi hoot tinh (kich thtrac 53 kDa) ca kha nang plan cat ca chAt cellulose vo Qinh hinh va dockerin loci c6 vai tre lien ket CeIB yen protein gia d6 CipA qua trinh hinh thinh phut hg cellulosome CeIB sau &Km tiet ngoai mai truang nu6i cAy se bj protease ngoai bao dm B subtilis 1012 phan cat va dockerin loci 1, IA domain rieng biet yea loi hoot tinh so bj looi be khei cau trite cria Ce1B Tiep theo, de cO cai nhin re) ham ve voch cAu trim CeIB bieu hien tren B subtilis 1012, chung toi tien hanh phan tich Western blot yeti khang the dic hieu khang CeIB duqc nhin tir nhenn nghien ciru cria giro su Pierre Beguin, met nhiing nh6m elAu tien bieu hien cong CeIB tai to hqp (Petit et al., 1985) B subtilis 1012 pHT43-ce/B 10 12 kb M (-) B subtilis 1012 plIT1219-ce1B 10 12 (gib) 80 CelB 60 50 40 30 25 ■Wle CelB Hinh Kha nang tiet CeIB cua chUng B subtilis mang plasmid pHT43-ce/B v9 pHT1219-ceIB tren m6i trutmg long bang SDS-PAGE (-): dich nu6i cay B subtilis mang plasmid pHT43 sau 14 gid cam Ling IPTG 0.5 mM: 4, 10 12 gitr thu mau sau duvc cam Ong bang IPTG 0,5 mM Ket qua lai Western blot Ket qua lai yen khang the clic hieu khang CeIB duqc trinh bay hinh 5, d6, c6 the thAy duqc protease ngoai bao di phan cat enzyme CeIB tir kich thy& thuyet IA 61 kDa xueng kich thuac 58 va 53 kDa, twang Ong v6i domain lei hoot tinh Cac vach tin hieu lai d ec hieu via mau d of chimg khong c6 bit kj, tin hieu lai nao hinh Ben conh Q6, cAu true CeIB cung cap boi ngfin hang dir lieu Uniprot va Pfam cna EMBL-EBI (European Molecular Biology Laboratory - European Bioinformatics Institute) cho thay c6 domain rieng biet loi hoot tinh va Dockerin loci Trong Q6, Dockerin loci I to bai yang lip loi ciut motif gan Canci va lien ket vai 101 hoot tinh CelB bing met clop as c6 ciu ft-6c clan gian Dva vac) tin hien lai thu Qu qc teen ming nitrocellulose, c6 the thay Qtr qc CeIB v6i kich thu6c 61 kDa duce bieu hien ca chung B subtilis khao sat, bj protease phan cat vach kich thy& la 58 kDa va 53 kDa Trong de, voch 58 kDa lA CeIB bj phan cat phAn domain dockerin tool 1, Gal vi tri giila motif gan Canxi va voch 53 kDa cAu truc lei hoot tinh CeIB Qi lo0i bo holm toil) dokerin loci 1, vai cau trite dtr &An hinh 5B Deng that ket 969 Phom Lutrng Thing et al qua cho thay plasmid pHT43-celB cho hieu diem bit diu bleu hien tiet tot CeIB la gier sau qua tiet tot ham plasmid pHT1219-ce/B Va theri cam irng bang 0,5 mM IPTG B subtillt 1012 B subtills 1012 pHT43-ce/B pHT1219-ce/B M 10 12 10 12 (506) A It CelB - Dockerin Poll CelB - 1/2 Dockain CelB Glycoside hydrolase superfamily (53 kDa) Dockerin Iola I (8 kDa) CclB Dockerin full as CelB 1/2 Docker's' CelB Hinh Ket qua lai Western blot v6i khang the thd chuyen Wet cho CeIB Hinh A: ket qua lai v6i meu krang Ong 100 pl djch nuoi cey B subtilis mang plasmid pHT43-ce/B va pliT1219-ce/B ding v&i chung dei chi:mg mang plasmid pHT43 khOng mang gen (-) Hinh B: du true protein CeIB tren ngan hang di), lieu Uniprot va cac kieu ceu trot du, down la san phem cat cua cac protease ngoai bac) tit b6i B subtilis Trong de, c6 domain chInh la domain host tfnh (kfch thirOc 53 kDa) va Dockerin loci (8 kDa) 90 80 spHT43-ce/B • pHT I 219-celB § 6° -a Kt T :2 30 b '0 S 10 10 12 Thbi gian nuoi coy sau cam img IPTG 14 (gi6) Hinh Kat qua hoot tlnh cellulase B tiet mai taterng nuoi cey cua chung B subtilis mang plasmid pHT43-celB va B subtilis mang plasmid pHT1219-celB K4m tra host tinh ctia dich nuoi cav Tu ket qua kiem tra hoot tinh (Hinh 6), chung to djch nuoi coy cua ca hai cluing B subtilis 1012 deu c6 hoot tinh phan buy CMC, nhien, khong co sq khac Net Ian ve kha nang phan giai giiia chiing bleu hien B subtilis 1012 mang plasmid pHT43-ce/B va chung B subtilis 1012 mang plasmid pHT1219celB Float tinh cellulase cao nhat la 12 gi6 sau cam (mg IPTG, va ket qua phi' hqp vai ket qua phan tich SDS-PAGE ding nhu la Western Blot D Ong thai, ket qua ding cho thay rang, 101 hoot tinh CeIB van cho kha nang phan cat CMC, do, hoot tinh CeIB khong bi anh twang nhieu bai protease ngooi bio ctia B subtilis 1012 970 KET LUAN Tir cac IC& qua dot duqc a tren, chiing Ceti ket luan rang di tach dong va bleu hien tong gcn CelB tir C thermocellum DSM 1313 dual clang tiet B .subtilis va cho hoot tinh phan giai CMC dot 80 U/mL Ket qua g6p phan cho viec tim hieu sq bieu hien enzyme tai to hqp dam le huemg den viec MIA() sat sts bieu hien him hqp cellulosome CO hoot tinh B subtilis cac nghien cau tiep theo Liri cam cm: De tai Ave ho trot beri du an JICA-JST can Tricang Doi hoc Bach khoa Dgi hoc Quiic gin TP Ho Chi Minh ma so JICA-JST-2012-06 Top chi Cong nghe Sinh hpc 13(3): 965-971, 2015 TA! LIEU THAM KHAO Clostridium thermocellum Ann Inst Pasteur Microbiol 136B: 113-124 Ding Tuyet Phuang (2010) Sir dung ram ri Viet Nam de sin xuAt dAu sinh hnc (bio—oil) Arcing Luang Tai Tao Viet at: http://deviNam Available renewable.com/2011/04/29/su-dung-rom-ra-san-xuat-dausinh-hoc/ [Accessed August 30, 2012] Rajalingam D, Loftis C, Xu JJ, Kumar TKS (2009) Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate Protein Sci Publ Protein Soc 18: 980-993 Demain AL, Newcomb M, Wu JHD (2005) Cellulase, Clostridia, and Ethanol Microbio Mo! Biol Rev 69: 124-154 Eveleigh DE, Mandels M, Andreotti R, Roche C (2009) Measurement of saccharifying cellulase Biotechnol Biofuels 2: 21 Nguyen H, Phan T, Schumann W (2007) Expression Vectors for the Rapid Purification of Recombinant Proteins in Bacillus subtilis Curr Microbiol 55: 89-93 Pare D, Beguin P, Millet J, Aubert JP (1985) Heterologous hybridization of bacterial DNA to the endoglucanases A and B structural genes celA and celB of Riederer A, Takasuka TE, Makino S, Stevenson DM, Bukhman YV, Eisen NL, et al (2011) Global gene expression patterns in Clostridium thermocellum as determined by microarray analysis of chemostat cultures on cellulose or cellobiose Appl Environ Microbiol 77: 1243-1253 Saito H, Shibata T, Ando T (1979) Mapping of genes determining nonpermissiveness and host-specific restriction to bacteriophages in Bacillus subtilis Mol Gen Genet MGG 170: 117-122 Schumann W (2007) Production of recombinant proteins in Bacillus subtilis Adv Appl Microbiol 62: 137-189 CLONING AND EXPRESSING ENDO-1,4 GLUCANASE B FROM CLOSTRIDIUM THERMOCELL UM IN BACILLUS SUBTILIS Pham Luong Thang', Pham TM My Tien', Tran Quoc Tuan', Nguyen Le Tuan Anh', Nguyen Hoang Ngoc Phuong', Phan TM Phuong Trangl, Nguyen Thi Nguyen2, Nguyen Dinh Quan2, Phan Dinh Tuan z, Tran Linh Thuoc', Nguyen Duc Hoang1421 University of Science, Vietnam National University, Ho Chi Minh City of Technology, Vietnam National University, Ho Chi Minh City 2University SUMMARY Vietnam is one of countries having the most abundant agricultural waste with more than 40 milion tons of straw produced annually For complete reuse of this cellulose substrate, bioethanol industry is founded and able to solve the world energy crisis problem In a typical bioethanol producing process, the cellulose hydrolyzing ability of cellulase enzymes is a vital issue From that reason, we expressed recombinant Endo1,4-13-glucanase B (CelB) from anaerobic Clostridum thermocellum in potential host trains Bacillus subtilis Firstly, gene celB was isolated by PCR reaction from C thermocellum DSM1313 DNA chromosome The recombinant plasmid pHT43-celB was generated by T4 DNA ligase reaction while pHT1219-celB was created by In-fusion cloning technique The transformants were checked by colonies PCR and confirmed by sequencing Bacillus subtilis 1012 strains carrying plasmid pHT43-celB or pHT1219-celB were used to study the expression of CelB in both halo technique on agar plate and activity measurement by inoculating in liquid medium The results showed that in the 0.5 mM of IPTG inducer concentration, recombinant B subtilis cells could hydrolyze CMC substrate in the plate In addition, SDS-PAGE combined Western blotting and enzymatic activity assay results demonstrated that B subtilis can secrete active CelB, equal to 80 Unit/ml of cultured medium Keywords: Bacillus subtilis, Endoglucanase B, Clostridium thermocellum, cellulose, plasmid pIIT Author for correspondence: E-mail: ndhoang@hcmus.edu.vn 971 ... Production of recombinant proteins in Bacillus subtilis Adv Appl Microbiol 62: 137-189 CLONING AND EXPRESSING ENDO- 1,4 GLUCANASE B FROM CLOSTRIDIUM THERMOCELL UM IN BACILLUS SUBTILIS Pham Luong... plIT1219-ce 1B 10 12 (gib) 80 CelB 60 50 40 30 25 ■Wle CelB Hinh Kha nang tiet CeIB cua chUng B subtilis mang plasmid pHT43-ce /B v9 pHT1219-ceIB tren m6i trutmg long bang SDS-PAGE (-): dich nu6i cay B subtilis. .. CeIB teen dia Plasmid sau giii trinh to dtrqc bitn nap vao B subtilis 1012 bang phucmg phi') di en bien nap Khan lac B subtilis m9c duqc sau bien nap se duqc khao sat kha !tang bieu hien CeIB