TAP CHI PHAT BHA AWN, TAP 17, SO 14-2014 Bieu hien endoglucanase A dm Clostridium thermocellum vi khugn Bacillus subtilis • • • • Pham Luang Thing Phan Thi Phuvng Trang Trin Linh ThLrevc Nguyin DCrc Hoang Trireng Dai hoc Khoa hoc To, nhien, DHQG-HCM Bai &Win 24 thOng 09 nom 2014, nikin clang ngthy 26 thling 01 nom 2015) TOM TAT PCR va cher) vao plasmid pHT43, sau Endoglucanase A (CeIA) Le phan trinh ty, tin hieu tiet samyQ Plasmid pHT43quan trying phar he enzyme cellulase celA too se &Foy bien nap vac) cheing chiu nhiet (cellulosome) tir chang vi sinh vat bleu hien B subtilis 1012, c6 bleu hien ky Clostridium thermocellum Trong phCrc protease ngopi bao va B subtilis WB800N, he nay, CeIA cho kha nang phan cat cao d5 gay dat bien tren protease ngoai bao nhet cac lien ket (3-1,4 glucoside ter ben CeIA ter cac chOng B subtilis cluvr cam Cmg cac phan ta, cellulose va mOt so lopi bieu hien bang IPTG a cac wing khac polysaccharide to cac oligosec Tien hanh thu mau dich tiet protein charide Bacillus subtilis vi khuan Gram ngopi bao mOi twang nu6i PA), ter throng, hieu khi, phat trien nhanh, thuying den 24 gia sau cam Crng Kha nang bleu hien dOng san xuAt enzyme tong nghiep cOa chung clugc danh gia bang SDS-PAGE chang vi khuan co the &roc thing de Day /à va hoot tinh Endo-13-1,4-glucanase Nghien thay the cho chOng vi khuan ky khi, phat cdu chirng to co the bleu hien CeIA Men cham, C thermocellum de bleu hien B subtilis va ti6rn nang phat trie'n CeIA De too chOng B subtilis co Ma nang chOng co khth nang bieu hien CeIA cho cac tiet CeIA tai to hop, gen celA fir C Cmg dung they phan cellulose sau thermocellum clu'or thu nh en bang phan (mg Tip khda: Bacillus subtilis, Plasmid pHT43, Pgrac, CeIA, IPTG, Endo-13-1,4-glucanase D AU C thermocellum la vi khuan Gram dacmg, ky tuyet d6i duoc biet den vai he cellulase cho kha nang phan giai cellulose hieu qua nhat Chung vi sinh vat co kha nang tao phirc he enzyme chiu nhiet va dugc goi la cellulosome ngoai bao Phirc he bao g6m: endoglucanase, c6 kha nang phan cat lien ket p-1,4-glycosidic phan tti cellulose; exoglucanase hay On goi la cellobiohydrolases, c6 vai tre phan cat cellulose mach dai, tao san pham cellobiose don gian hon va protein 161, vita c6 vai Ire ket dinh cac enzyme cellulase vai vim tang caerng kha nang gan cilia enzyme len co chat, thong qua cac domain luc cao vai cellulose tren be mat Qua do, tang cuerng hoat tinh cilia enzyme cellulase len nhieu lan [14] Trong phirc he cellulosome, endoglucanase A (Ce1A) la enzyme dAu tien dime thu nhan va tinh the [8] Cau tao Trang 73 Science & Technology Development, Vol 17, No.T4-2014 CeIA rat don gian, chi bao gem I domain Dockerin (domain gin Va0 protein loci cita cellulosome) lien ket vai 18i enzyme thuijc nhom GH8 (Glycosyl hydrolase 8) [1] Ngoai ra, cac nghien cUu ve phirc he cellulosome cho thAy ring CeIA la dai dien nhat cim nh6m GH8 tren C thermocellum [3] va &roc bieu hien cao ca cap dO phien ma [10] va dich ma [13] Bacillus la nhom vi sinh vat gift vai tro chti dao nen ding nghiep sin xuat enzyme veri hon 60 % enzyme cong nghiep &rot sin xuat tir nh6m vi sinh vat [12] Trong d6, B subtilis da dirge irng dung cong nghiep tir rat lau tai Nhat Ban sin xuat the pliant tir dau nanhNatto [6] Do do, viec sir dung B subtilis de bieu hien, sin xuat he enzyme cellulase cila C thermocellum se co (kw nhung uu diem so vai cac chimg bleu hien truyen th6ng khac nlur Escherichia colt hay Saccharomyces cerevisiae: (i) Ttrang deng ve mat eau trim to bio vi ca hai cUng la vi khan Gram duong; (ii) He tilting plasmid ton tai dee lap pHT43 (MoBiTec), cho phep bieu hien protein tai to hop clang tiet moi truerng; (iii) Dieu kien nuoi cay don gian, chi phi thap nhung hieu qua tao san pham, tao sinh Idt6i cao gap nhieu lAn C thermocellum; (iv) La chimg an town theo chuin GRAS (General Regarded As Safe) cluot cong nhan bed ca quan Quin ly Thu& vi Thuc pliant Hoa Ky (FDA-Foods and Drugs Administration, My) Ngoai ra, sinh khei B subtilis co the Ong dung nuoi thity sin, phan b6n sinh hoc, cho pile]) tan dung het tot da sin pham cua quy trinh sin xuat enzyme VAT LIEU VA PHUCING PHAP Vet lieu Cluing vi sinh vat Icy C thermocellum DSM 1313 tir ngan hang chting DSMZ, CHLB Dirc Plasmid bieu hien tiet pHT43 va cac chiing bleu hien B subtilis 1012, B subtilis WB800N Trang 74 dime cung cap bed ding ty MoBiTec (dai dien la Trung tam Khoa hoc vi Cong nghe Sinh hoc, Truang Dai hoc Khoa hoc Tu nhien, DHQGHCM) Cac enzyme, hoa chat can thiet cho dong hem, bieu hien protein va hoat tinh enzyme tai to hop Nuoi cay, thu nhen DNA be gen dm C thermocellum va tao plasmid tai to hop pHT43-ce/A C thermocellum DSM 1313 duqc nuoi cay binh nuoi ky, bang m8i throng 122, theo huang dan cua ngan hang cluing DSMZ, a 60 °C Thu sinh kh& va tach chiet DNA be gen theo quy trinh phenol/chloroform [5], neng dq va kiem tra de) tinh sach bing may NanoDrop 1000 Plasmid pHT43 (Hinh 1A) la meit plasmid thoi, co kha nang ton tai va chep dec lap Men ca chimg chi E colt va B subtilis Plasmid chiu su kiem sok bed promoter Pgrac, bao gem promoter groES ket hap vOi lacO operator va trinh to Shine-Dangamo cua gsiB [7] Ben canh c16, trinh to tin hieu tiet Samy0 dtroc gin sau Pgrac promoter, cho phep tiet protein tai to hop ngoai mei twang nuoi cay Gen ma hoa endoglucanase A (Ce1A) dtroc khuech dai tir DNA l) gen cila C thermocellum bing cap mOi Ce1A_F, '-GGC CATAGATCTGCAAACAC T GTGTCAGCGGCA-3' va CelA_R, 5'GGCCATGACGTCTTAATAAGGTAGGTGGG GTATGCTC-3'.San pham PCR ding vai plasmid pHT43 &rot xir ly bang BglIl Ara AatII la nguyen lieu cho phan Ong nei tao plasmid tai to hop pHT43-c,e/A Tiep theo, plasmid pHT43celA (Hinh 1B) dtroc bien nap vao chung E coli OmniMAX Tien hanh PCR khuan lac de sang loc khuari lac tai to hop, tach plasmid bang QIAprep spin miniprep kit (QIAGEN) vi kiem tra ket qui tao ding bang phuong phap giai trinh tu TAP CHI PHAT TRIEN HHACN, TAP 17, SO T4-2014 A B pH T43-ceIA 9404 bps Ifmh Ban de gen cll.% plasmid thuong mai pHT43 va plasmid pHT43-celA (A) pHT43 (MoBiTec) voi trinh tu khang ampicillin cho E coli va trinh to khang chloramphenicol cho B subtilis (B) pHT43-ce/A chira promoter Pgrac va gen celA sau trinh Ur tin hien tiet SamyQ To chill B subtilis tai to hop vi san lec cac ding dB to hop bieu high CeIA slang tiet Plasmid sau giai trinh to duoc bien nap vio B subtilis theo quy trinh huang clan dm MoBiTec Khan lac B subtilis moc duqc sau bien nap se &roc kiem tra stir hien dien cfut plasmid pHT43-ce/A Tien hanh PCR khan lac cac chimg tat to hqp B subtilis 1012 va B subtilis WB800N bang cap mei CeIA_F vi ON632 (TAGGCGGGCTGCCCCGGGGACG) San pham PCR co kich thubc du doan la 1392 bp Sir dung gel agarose 1% a phan tich ket qua Tir cac khuan lac cho phan Ong PCR duang tinh, khao sat khit nang bieu hien CeIA tren dia thach LB-Cm 10 (Luria Bertani-Chloramphenicol 10 ig/m1) co chira 0,5 % carboxymethylcellulose (CMC) va 'tiling de chit cam img IPTG khac tir 0,01 mM, 0,1 mM, 0,5 mM den mM Sau 24 gib nuOi cay, kha nang bieu hien dm chimg se duoc &nit gia thong qua bitn kinh vOng phan giai CMC Dsinh gia kha Ming ban hien tit CeIA dm B subtilis 1012 va B subtilis WB800N Chung tot tien hanh bieu hien mei truong long 100 niL LB-Cm 10 pg/mL ciing-Cs cac ding de IPTG twang to nhu khdo sit tren died diem khao sit, thu nhan mL dia dich nuoi cay da loai to bao bang ly tam a 10.000 vong phut De chuen bi mkt cho phan tich SDS-PAGE, ml dich enzyme ducic tita vai TCA (trichloroacetic acid) ir neng de cuei la 10 % Song song d6, chOng toi xac dinh hoot tinh CMCase dm enzyme CeIA theo quy trinh sau: 250 pi dich enzyme a yen 250 gLnatriacetat buffer, pH 5,5 c6 chira % CMC, u 50 30 phut Sau do, dinh Wong diking khir tao bang thuec thO DNS [4] Vat IU hog tinh enzyme la luting enzyme can thiet de tao hrong throng khir Wang duong vat pmol glucose (0,18 mg glucose) phin KET QUA VA THAO LUAN Thu nhan gen to plasmid tai to hop pHT43ceL4 Ket qua NanoDrop DNA be gen dm C thermocelhtm cho thay neng de DNA nam khoang 391 ng/pL, vai de tinh sach the hien qua OD260/0D280 la 1,92 Tien hanh tao ti plasmid tai to hqp pHT43-celA nhu phlin vat lieu va phtrcmg phap nghien ciru Ket qua giai trinh tu cho thay stir tucmg &Ong cita trinh tU muc tieu so vOi thiet ke la 100 % Nth ket qua BLAST Trang 75 Science & Technology Development, Vol 17, FIo.T4-2014 tren nein hang gen NCBI cling la 100 % so sanh v6i trinh tir gen ceL4 tren cluing C thermocelltun ttr nhien Tao chiing B subtilis 1012 vi B subtilis WB800N mang plasmid tai to hop B subtths 1012 bp B subtilis WB800N 0HT43-ceM pHT43-csIA KL1 KU KL3 KL1 KL2 KL3 duct: khao sat 0,1 mM, 0,5 mM va mM Chi CO sir khac biet re) ret ve kha nang phan giai CMC a nOng dO 0,01 mM so voi Ming de IPTG lai Nhu vay, cac chimg B subtilis 1012 va B subtilis WB800N mang plasmid tai CO hop pHT43-ce/A tao CO kha nang ti6t CeIA co hog tinh cao tren moi twang chin ca chat CMC B subtills 1012 5000 2000 1200 1392 by 500 Ket qua PCR khan lac de kiem chimg plasmid tai to hop pHT43-celA cac chiing B subtilis 1012 va B subtilis WB800N (Hinh 2) cho thay cac dong vi khan B subtilis tai to hop deu cho vach san pham PCR khan lac co kich that ly thuyet 1392 by nim gia vj tri 1200 by va 2000 by dm thang chuAn DNA Trong do, khan lac clOi chimg, khong xuAt hien vach sang nao ten ban gel agarose % Dieu cho thAy chimg t8i da tao ding cac chUng tai tO hop B subtilis 1012/pHT43-ceL4 va B subtilis WB800N/pHT43-ceL4 Khan sat kha nang hien hien CeIA teen mai truing Viec danh gia kha nang bleu hien CeIA dang tiet ciia chting bang &a thach LB-Cm 10 chira 0,5 % CMC de dang cho ket qua chi sau 24 gi6 nuoi cAy a 37 °C, va 15 phut nhuiam bang Congo Red % [2] Ket qua thu nhan duoc Hinh cho thAy yang phan giai tir cac chlIng B subtilis mang plasmid pHT43-ceL4 Lan han rat nhieu so van cac mau chirrig am mang plasmid pHT43 a ca nOng IPTG 0,01 mM, 0,1 mM, 0,5 mM va mM Ket qua phan tich ban kinh Wing phan giai cho thAy sir anh InrOng cua IPTG khong nhieu a cac ming dO Trang 76 0.1 0.5 I onfoA !PT(' 0.01 0.1 0.5 mM IPTG pHT43-cr/A pHT43 B subilthsW13800N lllnh Ket qua dien di san phArn PCR kiem tra sir hien dien cua tai to hop pHT43- celA cac chang B subtilis tai to hop Dei chimg am la san pham PCR chimg B subtilis 1012 mang plasmid pHT43 Ket qua dien di duoc phiu'tich tren gel agarose % 0.01 pHT43-celA pHT43 cm Ilnh Kha nang phan giai CMC cua chung bieu hien B subtilis 1012 va B subtilis WB800N mang plasmid tai to hop pHT43-celA cac ming de IPTG la 0,01 mM, 0,1 mM, 0,5 mM va mM Dei chimg am la thong B subtilis mang plasmid pHT43 Thanh mau trang the hien ban lcinh vong phan giai co chat CMC Thanh mau den the hien thuerc co kich thu6c la cm Khan sat kha nang bieu hien CeIA tren mai truing long Qua trinh khdo sat nuoi cAy lie a 24 gi6 sau cam ling cho phep danh gia chinh xac han cac yeu to anh hang den sir bleu hien CeIA c6 hoat tinh nhu Ming de chAt cam Ong IPTG, theri gian nuoi cAy, qua do, c6 the xay dung tien de cho vi'ec tOi tru h6a quy trinh va ap dung tren quy mo 16n han (1 L hoac L) d day, chimg tor sir dung phuong phap de danh gia kha nang bieu hien la SDS-PAGE va hoat tinh dich nuoi city Xac nhein su bieu hien CelA tilt to B subtilis 1012 va WB800N Tnrac lien hanh ldiao sat kha nang bieu hien, chi:mg toi titn hanh lai Western Blot de xac nhan chinh xac sir bieu hien CelA djch nuoi cay B subtilis tai thin diem gi6 sau cam ling bing 0,1 mM MTG TAP Cal HAT TRIER AWN, TAP 17, SO 14-2014 M lllnh ket qua icac dinh sv bieu hien CeIA B subtilis (A)KM qua lai Western Blot dich tiet B subtilis veri Ichang the the> da dong Ichang Ce1A; Gieng 1: dich tiet B subtilis 1012 mang plasmid pHT43-celA; Gieng 2: dich tiet B subtilis 1012 mang plasmid pHT43 (dei chfmg am); M: thang chuan protein; Gieng 3: dich tiet B subtilis WB800 mang plasmid pHT43Q.Lteichirng am); Gieng 4: dich tiet B subtilis WB800 mang plasmid pHT43 (B)Cau true protein Ce1A tai to hop then ngan hang dft lieu Uniprot CeIA gem domain chinh la loi hoat tinh (kich thuerc khoang 43 lcDa) va Dockerin nhom I (kich dm& khoang kDa) Kich thus c: 42.275 Da pl: 5,53 VI tri: as 41 - as 393 Kich thuetc: 7.479 Da p1: 8,65 Vitthaa 411 -aa477 \ Lai hoar tinh Dockerion: I OMNI 4111M • 1.•■••■ MINN •MIP • *".• ( cit \ Kich think: 451 am (A) Ket qua lai Hinh 4A cho they CeIA tao B subtilis 1012 co kich thtroo khoang 43 kDa dich tiet B subtilis WB800N, CeIA van gift dirge vach kich thuac la 50 kDa va 43 kDa Ngoai ra, a mAu ded chimg la dich tiet B subtilis mang plasmid pHT43, khong c6 vach lai nao &roc phat hien Dieu cho they ket qua lai tot, khang the CeIA la Bac hieu va khOng tao san phAm Iduing dac hieu nao vii protein ngoai bao mia B subtilis 1012 va B subtilis WB800N Enzyme Ce1A co kich think 49754 Da (Hinh 4B) du trim CeIA bao g6m domain Dockerin loai (kich thy& khoang kDa) lien ket v&i loi hoat tinh enzyme (43 kDa) Tuy nhien, phan lai gap cuen rieng re va doc lap yeti [1] Do 86, protease ngoai bao se cat phan Dockerin loai I tnroc, lai enzyme \Teri cau true phirc tap va chic chin hon se it bi anh huemg b6i hoat tinh cila protease KM qua tucmg tir mot nghien tiro kliac ve bieu hien Ce1A cling cho ket qua lai Western-Blot a kich Alm& Wang *rig khoang 43 kDa [9] B subtilis 1012 la Khoi hapg: 49.754 Da pl: 5.58 (B) chimg duo; nghien cuu tir lau [11] Tuy nhien, B subtilis 1012 co nhuqc diem bieu hien nhieu loai,protease ngoai bao, d6, san pham lai dich tiet Ce1A chi cho vach a kich thu6c 43 kDa tuang Ung veri lbi hoat tinh Nham khac phuc van de nay, chimg t6i sit dung chimg B subtilis WB800N de Neu hien CeIA B subtilis WB800N la chimg Neu hien da bi dot bien mat protease ngoai bao [9], chinh vi vay, ceu trim hoan chinh oda Ce1A, bao gom ca Dockerin loai I va loi hoat tinh se kheing bi pfitm cat Milieu ben protease, cho phep gift nguyen &roc kich thirac day dii cite enzyme CelA (49,754 kDa) Dieu hoan town phis hop vai ket qua lai veri vach protein la 50 kDa va 43 kDa Wang irng, v6i kich thuerc day du va 16i hoat tinh cue CeIA da bi phan cat Dockerin loai I Khci ming bleu hien CelA b cluing B subtilis 1012 Cac ket qua SDS-PAGE (Hinh 5A, 5B, 5C) cho they xuat hien vach nhel co kich thuerc khoang 50 kDa va 43 kDa cac chimg B subtilis Trang 77 Science & Technology Development, Vol 17, No.T4-2014 1012 mang plasmid pHT43-ceiA, khi, chiing del chi:mg B subtilis 1012/pHT43 thi khong xuAt hien Nhir vay,chUng toi co the ket luau ring CeIA dirge bieu hien tong tren chung B subtilis 1012 Mac du sin pi-tam tiet bj protease phan cat met phAn cac thai diem 4, 6, va 10 gia sau cam ung, nhien, a cac thai diem 12, 18 va 24 gia, van co met luvng CeIA mm tao va chua bi phan cat boi protease dm B subtilis 1012 Ket qua hoan toan phis hap vai ket qua lai Western Blot trirerc — Hinh DOng then, doi chieu ket qua hoat tinh B pHT43 cel A pHT43 prnm A kDa 200 djch nuoi ch (Hinh 5D) cho May enzyme van co hoot tinh cao, nam khoing 120 den 140 mU/m1 djch nuoi cey Dieu cho thay ring lei boat tinh enzyme van kha nang phan giai ca chAt CMC mac chi da bj loai be domain Dockerin loai I beri protease ngoai bao dm B subtilis 1012 Ben earth do, ket qua hoat tinh a thai diem cam img IPTG (0 gib) cho thay lugng duang khir tao rat thap, dual 0,1 mg/mL nam ngoai gia trj duang chuin glucose ce the phat hien &roc bing phuang phap djnh luang vai thuec thir DNS IA 0,1 mg/mL — 3,2 mg/mL subtilis 1012 pHT43-ce1A-0.1mM 8h 8h 4h 6h 8h 10h 12h 18h 24h 100 ITmh K61 qua phi!' tich icha nang bieu hi6n CeIA cua 60 CeIA elA B subtilis 1012 biing SDSPAGE va danh gia hoat tinh (A) N6ng d6 IPTG chmc khao sat la 0,1 mM, (B) 0,5 mM va (C) 1,0 mM (C) Luang matt tuong duang 200 [IL djch tiet mei tnrUng nuoi cey duqc sat dung cho phan tich SDS-PAGE (D) Ket qua chuth gia hoat tinh djch nuoi cay Luang dung djch thing cho danh gia hoat tinh enzyme la 50 uL/lan -CelA 50 -CeIA D • 0.1 mM IPTG ■0.5tuM !PTO ■ I mM IPTG Trang 78 12 10 71101 gum dm mite Outs) TAP CHI PHAT TRIER KHACN, TAP 17, Si 14-2014 Khii nang bieu hien CelA a chfing B subtilis WB800N Nhu (la trinh bay, Ce1A bieu hiOtt,$ Br subtilis WB800N bao gem protein hoan chinh va loi hoat tinh da loai bo domain Dockerin loai I Trong do, chi yeu la protein hoan chinh Ce1A tao a kich thuOc la 50 kDa Kich thirac tuang Ong v&i vach xuat hien cac gieng 6, 8, 10, 12 va 18gia sau cam Ong a cac nOng d0 0,1 mM va 0,5 mM IPTG (Hinh 6A) va cac giLg tir den 24gia sau cam img a neng de IPTG mM (Hinh 6B) Trong do, cac mau chimg am (cherig mang plasmid pHT43 va thing mang plasmid pHT43-cell kh8ng duoc cam /mg (0 mM — Hinh 6B) thl vach khong xuat hien.Nhu viy, thing toi ket luan day la vach protein tai to hop CelA bieu hien vuqt men co so cam img bad IPTG Luang Ce1A tao tang dein qua cac gia nuoi cay va dat tei da khoang 10 den 18gio sau cam /mg Ben canh do, ket qui danh gia hoat tinh (Hinh 6C) ming phan anh cbing ket qua dien di SDS-PAGE (Hinh 6A vi 6B) 'Trong do, lacing Ce1A tao dat ngirting tei da tai thai diem 8gia sau cam Ung Nhu vay, thing Col co the ket luan rang khoang theri gian Cat nhit dimg nuoi cay va thu nhin enzyme Ce1A la 10 den 12gia sau cam Ong Ming de IPTG cam Ong la 0,1 mM, 0,5 mM va mM, Itiong Ce1A tao la Wang throng khoing thai gin tir den 24gia sau cam Ong IPTG (Hindi 4), itch hoat tinh enzyme tai to hop thu throe khoang 70 mU/mL dich nuoi cay Cling twang to nhu chimg B subtilis 1012, mau thu dich tiet B subtilis WB800N tai thai diem gia cho ket qua tao &Tang khir la rat thip, khong the phat hien &roc dinh lueng kii bang thuec thir DNS Nhu viy, co the thay rang boat tinh phan giii CMC cia dich tiet B subtilis la so bieu hien dm vector tai to hop pHT43-ce/A B subtilis WB800N paw ▪ A B C b Phan tich kha nang biL hiL CelA cia subtilis B thing WB800N bang SDSPAGE va clanh gia boat tinh (A) Ming tins chid cam ung IPTG la 0,1 mM va 0,5 mM va (B) mlvl Doi veri phan tich SDSPAGE, luvng mau tuong throng 200 1.11, dich tiet duac nap vac, moi gieng 'tett gel polyacrylamide 12,5 % (C) Ket qua ithc dinh hoat tinh ducrc anti gia thong qua don vi la mU/mL dich tiet moi truang da loai to bit°, vai mU/mL = Una lim MITaicefA-0.1mM Olt eh 1011 1211 01, en 100 191 1110 On On tan tm ten 80 70 60 50 • 0.100■1 0'T13 40 O0.51•81 IPTG 30 20 RI B 10 • I niM IPTG 10 , 1.2, Thni gmn thu man quo) 18 Trang 79 Science & Technology Development, Vol 17, No.T4-2014 KET LUAN Chung toi da taothanh cong cluing B subtilis 1012 va B szihtilis WB800N mang plasmid pHT43-ceL-1 va bieu hien ding enzyme tai to hop CeIA tir C thermocellum co hog firth cao tren co chit CMC Ket qua ban dau cho that' tiem nang drig dung Ian ciia B subtilis vice bieu hien enzyme cellulase tai to hqp, phuc vu cho viec thin' phan nguon co chat nhu ram it Viet Nam 1D - tat &roc thtrc hien nlurr str hj tro kith phi cna Sd Khoo hoc Cong tighe Theinh pita 1fri Chi Alinh, nnm 2012-2013 1kt sd 178,2013 HD-SKI-KW Production of endoglucanase A of Clostridium thermocellum in Bacillus subtilis • • • • Pham Luong Thang Phan Thi Phuong Trang Tran Linh Thuoc Nguyen Duc Hoang University of Science, VNU-HCM SUMMARY inserted in the C-terminus of signal peptide Endoglucanase A (CeIA) is a component of amyQ (samyQ) in plasmid pHT43 The of thermostable complex - cellulosome which plasmid pHT43-celA was then transformed is produced by anaerobic bacterium into B subtilis 1012 and WB800N, an Clostridium thermocellum In this complex, extracellular protease deficiency strain Next, CelA has the highest catalytic activity and it CeIA production was induced by IPTG at can cleave randomly 8-1,4 glucosidic linkage different concentrations Cultural supernatant of cellulose to smaller oligosaccharides was collected from to 24 hours after Bacillus subtilis a Gram positive and aerobic addition of IPTG The expression levels were fast growth bacterium, is commonly used to evaluated by SDS-PAGE and endo-8-1,4produce industrial enzymes This bacterium glucanase activity This report demonstrates can be used to replace the anaerobic slow that CelA can be expressed in B subtilis and growth bacterium, C thermocellum, to its potentials for development of bacterial produce CeIA To create B subtilis strains strains which can produce CeIA to hydrolyze which can secret recombinant CelA enzyme, cellulose in the future celA gene was amplified using C thermocellum DNA genome by PCR and Keywords: Bacillus subtilis, Plasmid pHT43, Pgrac, CeIA, IPTG, Endo-f3-1,4-glucanase Trang 80 TAP CHI PHAT TRIER KWH, TAP 17, SO T4-2014 TAI LIEU THAM KHAO [1] P.M Alzari, H Souchon, R Dominguez, The crystal structure of endoglucanase CeIA, a family glycosyl hydrolase from Clostridium thermocellum, Structure, 4, 265-275 (1993) [2] P Cornet, I Millet, P Beguin, J.P Aubert, Characterization of two eel (Cellulose degradation) genes of Clostridium thertnocellum coding for endoglucanases, Nature Biotechnology, 1, 589-594 (1983) [3] A.L Demain, M Newcomb, J.H.D Wu, ethanol Cellulase, Clostridia, and Microbiology and Molecular Biology Reviews, 69, 124-154 (2005) [4] D.E Eveleigh, M Mandels, R Andreotti, C Roche, Measurement of saccharifying cellulase, Biotechnology for Biofitels, 2, 21 (2009) [5] M.R Green, J Sambrook, Molecular cloning: a laboratory manual Cold Spring Harbor Laboratory Press (3th, 2012) [6] T Hara, A Aumayr, Y Fujio, S Ueda, plasmid-linked of Elimination polyglutamate production by Bacillus subtilis (natto) with acridine orange, Applied and Environmental Microbiology, 44, 1456-1458 (1982) [7]• H.D Nguyen, T.T.P Phan, W Schumann, Expression vectors for the rapid purification of recombinant proteins in Bacillus subtilis, Current Microbiology, 55, 89-93 (2007) [8] J Petre, R Longin, J Millet, Purification and properties of an endo-beta-1,4glucanase from Clostridium thermocellum Biochimie, 63, 629-639 (1981) [9] T.T.P Phan H.D Nguyen, W Schumann, Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis, Protein Expression and Purification, 46, 189-195 (2006) A [10] Riederer, T.E Takasuka, S Makino, D.M Stevenson, Y.V Bukhman, N.L Elsen, B.G Fox, Global gene expression patterns in Clostridium 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mOi Ce 1A_ F, '-GGC CATAGATCTGCAAACAC T GTGTCAGCGGCA-3' va CelA_R, 5'GGCCATGACGTCTTAATAAGGTAGGTGGG... pi-tam tiet bj protease phan cat met phAn cac thai diem 4, 6, va 10 gia sau cam ung, nhien, a cac thai diem 12, 18 va 24 gia, van co met luvng CeIA mm tao va chua bi phan cat boi protease dm B subtilis. .. 451 am (A) Ket qua lai Hinh 4A cho they CeIA tao B subtilis 1012 co kich thtroo khoang 43 kDa dich tiet B subtilis WB800N, CeIA van gift dirge vach kich thuac la 50 kDa va 43 kDa Ngoai ra, a mAu