Tgp chi Cong nghe Smh hpc 11(1): 139-144,2013
BILL H I E N G E N CS\ \ L \ H O A C H I T O S \ N \ S E CL \ BACILLUS CEREIS H N 9 0 T R O N G PICHIA PASTORIS
Nguyen Thi Nggt Lien, \ u \ an Lgi, Pham Thanh Huyin, Nghiem Ngoc Minh, PhiQuydtTiSn I 'ien Cong nghe sinh hoc, I 'ten Hdn ldm Khoa hpc vd Cong nghe I 'iel Nam
TOM TAT
Trong nghien CLTU nay. gen < sn - gom 1362 nucleotide ma hoa eho chitosanase (CSN) cua Bacillus cereus HN90 - duoc khuach dai bang PCR. gan vao veclor tach dong pJETI 2 \a bi4n nap vao i^ bao Eschencbia coh XLI-blue Sau khi xu ly plasmid Ihu diroc tir E co/; vdi hai enzyme gidi han fcoRI iw A7wl. gen « « duoc nan vao veclor pPICZaA tao veclor bieu hien pPICZaA csn Ti^p do, veclor bieu hien duoc ni^cr vong lai vim;
AOXl bangSi/cl, chuyen vao Pichia pastoris X33 bang xung dien va sang loc tren mdi irucjng'iPDS bdsung zeocine ndng do tir 200 - l50()mgL"', Ket qua kiem tra bo gen nlm men bang PCR su dung cap mdi dac hieu 5' AOXl va 3' AOXl cho Ihay gen csn da duoc tich hop vao he gen n^m men Trong sd hon 100 dong nam men tai td hgp dugc sang loc, da lua chon dugc 8 ddng nam men lai to hop cd kha nang phal inen tren mdi trudng YPDS chua zeocine IOOO mg/ml va cd kha nang smh tong hgp C SN ngoai bao cao Dua tren kha nang bieu hi?n chitosanase. da lira chon diroc dong P pastons L6 sinh chitosanase cao nhat. dai 322.4 U ml sau 72 gid nudi cay Irong moi tnrdng BMMY duoc cam img boi 1,5% (v/v) methanol
Tir khda Bacillus • I, hieu hici chilosi, 6 liop, Puliia pa\it
MO DAU
Chitosanase (EC 3,2.1.132) - con duac gpi la chitosan N-acclyl glucosamino hydrolase - la enzyme ngoai bao thuy phan lien ket [J-1,4 gifra eac D- glucosamine frong noi phan Ur chitosan dii bi de acetyl hoa mpt phan. Chitosanase (CSN) diroc I'mg dung pho bien trong qua trinh lao cac dan xual co phan tir lupng Ihap cua chitosan nhu D-glucosamine va chiiooligosaccharides (COSs). COSs la san pham cua qua irinh thuy phan chitosan duai sir xiic tac cua chitosanase va eo Item nang img dung Itni irong cong nghiep thire pham, nong nghiep va cong nghiep dupc pham. Theo phuong phap truyen thong, COSs dupc san \uat bSng phuong phap hoa hoc Qu;i trinh nay lao ra ciic chuoi ngan oligosaccharides, nang sual ihap, chl phi tach chiet eao va khong than thien \ a i moi trirong. Trong nh&ng nam gan dav, su dung chitosanase Irong qua trinh thuy phan tren tro nen pho bien hon bm chi phi san xuat thap. it tae dpng den moi trirong va co kha nang lai su dung (Lee et al., 2006: Su et al. 2006).
Vm myc dich ihu dirpc luong Ion chitosanase.
ral nhieu nhom nghien cuu iren the gicn da lao ra cac chiing vi sinh vai tai to hop co kha nang bieu hi|n gen c,%n mS hoa cho chitosanase. Cho den nay, nhieu gen isn CO nguon goc tii eac vi sinh vai khac nhau nhu. Bacillus sp, CK4. Bacillus sp KCTC 0377 BP.
Bacillus sp, MET 1299. Bacillus sp, KI7.
Aspergillus sp. CJ22-326 (Yoon cl al. 2000.
Yatsunami ci at.. 2002: Choi cl al , 2004, Pyoung ct cd, 2004, Ll el cd. 2008) da diroc tach dong \;i bieu luen trong Escherichia coli Chitosanase tir chung Bacillus sp. CK4 duac bieu hien trong E coli co hoat tinh CSX dat 0.5 U'ml dich nuoi cay lYoon el al, 2000) Doi vol chitosanase tir chung Aspeigiltiis sp, CJ22-326. khi bieu hien trong E coli. hoat tinh enzyme thap htm nhieu so vin CSN tai to hop bicu hien irong / ' pasinns (Li el al. 2008) Nam 200H.
Lec va cong su da bicu hien ihJnh cong gen csn tir chung .Aspergillus jiigiinaliis JXSD-97 trung / ' pastons GSI 15 Hoai tinh CSN sau qua tnnh loi iru thanh phan moi trucmg dat cao nhat IOO U/m! Ben canh do. da co nhieu no Iuc lap trung vao sang loc chung bang dot bien va toi iru hoa eac qua tnnh nham thu CSN voi ham luong va hoat tinh cao Tuy nhien. luong chitosanase san xuat ra chua dap img diroc trong san xuat cong nghiep va thuong mai (Lee el at.200H)
Hien nav: trong cac he ihong bieu hien thi nam men P. pastons dang duoc su dung rong rai irong san xuat protem lai to hop lir cac nguon khac nhau do chiing c6 cac dac tinh nhtr nuoi cay don gian trong moi Iruimg khoang re lien, co the dal smh khoi cao trong nuii iruong lot thieu va de dang nang cap quv mo san xual Dac biet, chung chu nav bieu hien gen ngoai lai a mirc cao duoi sir kiem soat chat che cua promolor AOXl (Yang el at. 20t()| (i ViOt l ^ >
Nguyen Thi Ngpc Lien el ai Nam. cho d^n nay chua c6 cong trinh nghien cim
nao cong b6 kgt qua bieu hien chitosanase tir 6.
cereus trong he thong bieu hien P. pastoris Trong nghien ciju nay. de nang cao boat tinh tong sd ciia chitosanase, chiing toi nghien ciru bieu hien gen csn co nguon goc tit chimg B cereus HN90 (Ma s6 truy cap cua gen ma hoa I6S rRKA tren GenBank: HQ438588) trong nam men P pastons X33 nhim thu enzyme ngoai bao hoat tinh cao va s a n l u o n g lon
VAT LIEU VA PHU'ONG PHAP
Chiing giong vi sinh vat, plasmid \ a vat lieu khac Chiing £• c(j/f XLI-blue [A(mcrA)l83 &(mcrCB- h.sclSMR-mir)l73 endAl siipE44 thi-l gyr.496 ret Al lac] (Stralagene, My) dugc su dung de nhan dong gen Chung nim men P. pastons X33 duoc su dung lam chung chu bieu hien CSN, chiing co kha nang sinh truong t6t fren moi Iruang YPD va moi truong khoang, rat thuan tien cho viec nuoi cay, chpn Ioc tren moi truong co bo sung zeocin
Plasmid pCR"2 I csn co chua doan gen csn (Ma s6 truy cap tren GenBank FJ68239I) ma hoa chitosanase ciia chung B subtilis HN9n dupc sir dung lam khuon cho phan img PCR Bp kil tach dong ClonJET'^'{Fermentas, \1v) dupc su dung de tach dong gen. Vector pPICZaA (Invitrogen, My) duoc sir dung de thiet ke vector bieu hien Nhan dong gen c^/; trong vector p J I i T l . 2
Dua Iren trinh tu va cac \ i tri giai han cua gen csn trong plasmid pCR2 l''::c-sn, su dung phan mem Clone Manager (Sci-Ed Software, Ver. 8 0, NC.
USA) de Ihiel ke cap mot co gan them trinh ty cat gioi han EcoRl va Xbal: ppF ( 5 ' - CGAATTCGCTGACAACATAATGAATGG-3') va (5"-CICTi6>iTCCTTTCTTAAGTAACCATTCTC -3') Gen csn duac khuech dai bang phan img PCR theo chll trinh nhiet 94"C" trong 5 phut, 30 chu trinh (94"C trong 90 giay, 52"C trong 90 giay, 72°C trong 90 giay), 72"C trong 10 phut roi giu mau a 4"C San pham ctia phan tmg PCR duoc kiem fra bang dien di tren gel agarose 1%. iinh sach bang kit PureLink^^' - DNA Purification (Invitrogen, My). Do gen csn dupc khuech dai bang enzyme Taq DNA polymerase nen dau 3" cua gen c6 them nucleotide adenine Vi vay, iruoc khi dua vao vector pJETI 2 de tach dong, gen csn duoc tao dau bang llieo lurang dan su dung cua
nha san xuat.
Phan ling ghep n6i gen csn vao vector pJETl.2 dupc tidn hanh n h a enzyme T4 DNA ligase Thanh phSn phan img g6m: DNA tinh sach, 3 nl (50 - 100 ng), vector da m o vong: I |il (50 ng), T4 ligase buffer (lOX), I jil, T4 DNA ligase (5Vt\il): 1 pi, nuac cat khir ion vo triing. 4 pi Hon hap phan img duac frpn deu va u a 22"C frong 1 gia, Sau do hen hanh bi^n nap DNA plasmid tai to hpp vao te bao E coll XLI -'blue bang phuang phap soc nhiei theo phuong phap da dirac mo ta truoc day (Phi Quyet Tiin e r a / . 2011).
Cac t^ bao E coli XLI - blue mang plasmid lai to hpp pJETI 2:.csn dugc chon loc tren moi frucmg LB CO hd sung IOO pg/ml ampicillin va plasmid dupc tach khoi E coli bang kit PureLink™ - Plasmid Extraction (Invitrogen, My). Kiem tra sirco mat cua gen csn va vector pJET! 2 trang te bao E coll XLI - blue thong qua ket qua dien di kiem tra plasmid sau khi da xu ly vol hai enzyme gioi han EcoRl vil Xbai.
Thiet ke vector bicu hien gen csn trong P. pastoris Xu ly vector pJET1.2:ci77 va pPICZaA dong thoi vai hai enzyme giai han EcoR\ va Xbal. Tmh sach gen csn, vector pPICZaA va thuc hien phan irng ghep n6i DNA bang enzyme T4 ligase (Invilrogen, My) a 22"C frong thai gian I gia Bien nap toan bo san pham nhan dugc sau phan img ghep n6i DNA vao E coli XLl-blue, Tach plasmid pPICZaAT.i(7 fri E coll, xu ly ddng thai voi hai enzyme gicri han f'coRI va Xba\ de kiem Ira kich thuac cua gen csn dugc chen trong vector bieu hien pPICZaA. M a vong plasmid pPICZaA':c5n tai viing AOXl bing enzyme gioi han Sad va bien nap vao nam men P pastons X33 bang xung dien theo phuang phap da dupc mo ta truac day {Do Thi Huy6n el al, 2005) Cac dong P. pasioris X33/pPICZaA''Ci-/7 tai t6 hgp duac lua chgn tren dia thach YPDS (!% cao n i m men, 2% pepton, 2°/o dextrose, sorbitol IM) co bo sung zeocin co nong do khac nhau, tir IOO - 2000 pg/ml de sang Igc cac dong Ie bao CO tich hpp nhieu ban sao ciia ket cau bieu hien (expression cassette) vao genome ciia nam men.
De kiem fra su co mat ctia gen csn fren bp gen nam men P pastons X33/pPICZaA,:c5«, sii dung c^p m6i 5 ' A O X l (5'-GACTGGTTCCAATTGAC AAGC-3') va 3'AOXl (5'-GCAAATGGCATTCTG ACATCC-3') da thuc hien phan img PCR Iheo huang dan ciia hang Invifrogen-My
140
Tgp chi Cong nghe Sinh hoc U(ly 139-144,2013
Bicu hien CSN trong nam men P. pastons De bieu hien CSN trong ti bao nim men, cac dong nam men lai to hgp P. pasioris X33/pPICZaA::c5n dupc nuoi ciy trong thai gian 72 giir tren moi Iruang BMGY ( 1 % cao nim men, 1%
pepton: 100 inM potassium phosphale, pH 6,0, 1,34% YNB: 4x10"-% biolin: 1% glycerol) a 30°C, ODtiiunm dat 2-6 don vi. Sau 66, chuyen 5-10% (v/v) giong sang moi truong BMMY (thanh phan giong inoi Iruong BMGY nhung thay glycerol bang 0,5%
(v/v) methanol). Trong qua trinh nuoi cay, cii sau 24 gio Ihi thuc hien cam iing bang each bo sung vao moi iruong nuoi cay 0,5% methanol (v/v). Chiing nam men P pastoris X33/pPICZaA nuoi cay tren hai moi truong BMMY va BMGY vai che do cam ling mong tu dugc su' dung Iam dot chiing am trong thi nghiem nay.
\ a c dinh hoat ti'nh chitosanase
Hoat tinh chitosanase xac dinh bang phuang phap quang pho hap phu, sa dung colloidal-chilosan {Sigma, Due) lam co chat Hon hop phan img bao gom 0,4 ml colloidal-chilosan 1,0% va 0,4 ml dung dich enzyme CSN, u o 37"C trong 30 phut Phan img duoc dirng lai bang 20 pi NaOH ION Tiep do, ly tam 6 12.000 vong/phul tiong 5 phut. Lugng duang kliir giai phong ra trong phan irng duoc xac dmh bang phucmg phap DNSA o buoc song 540nm (Miller, 1959). Mot dan vi boat tinh chitosanase dugc xac dinh la lugng enzyme can thiet de giai phong I pmol D - glucosamine trong thiri gian mot phiit a pll 6,0 (Pyoung el al, 2004)
KET QUA THAO LUAN
Khuech dai va tach dong gen ma hoa CSN Bang ky ihuat PCR, gen csn da dugc khuech dai lir khuon pCR2.I®:'c5n cho mot bang DNA duy nhal CO kich thud'e 1,4 kb tren gel agarose 1% (Hinh I A), Gen csn sir dung trong nghien ciai (duac dang ky tren GenBank (NCBI) duai ma so truy cap JF68239I) da dupc giai va phan lich Irinh tu, cho do dai 1362 kb, ma hoa cho protein gom 453 amino acid (Vu Van Lgi e / o / , 2011).
San phim phan img PCR khucch dai gen csn dupc linh sach va gan vao vector tach dong pJETI.2, Sau khi dupc bien nap vao te bao kha bien E. coli
XLI-blue va nuoi fren moi frudng LB co b6 sung 100 pg/ml ampicillin, plasmid pi£Tl.2-.:csn dupc tach va xu Iy ven hai enzyme gicri han EcoRl va Xbal. Ket qua dien di kiem fra plasmid pJETI 2-:csn sau khi xu ly vai enzyme gim ban cho ihiy hai bang DNA ro net c6 kich thuoc lin lugt ia 1,4 kb va 3,0 kb the hicn cho gen csn va veclor pJETI.2 tren gel agarose (Hinh IB) Tir nhung ket qua Iren cho diiy gen csn da dugc chen vao vector tach dong pJETI,2.
Thiet ke vector bi^u hien pPICZa,A::cs/i Gen csn duac cat khoi veclor tach dong bang hai enzyme giai ban EcoRl va Xbal va gan vao vector bicu hien pPlCZaA tai hai vi trf cat tuong iing dc tao veclor bicu hien frong P pasioris
Ket qua fren hinh 2 cho thay, vector bieu hien pPICZaA: csn sau khi xii ly voi hai cnzjnie gioi han cho hai bang DNA co kich thuac lan luot la 1,4 kb va 3,6 kp tuang ung vai kich thuoc cua gen csn \ a vector goc pPlCZaA Do vay, vector bieu hien pPICZrrA csn da duoc thict ke thanh cong Tao chung P. pasioris tai to hop eo khii niing bieu hien CSN
Vector tai to hop pPICZaA r.wi sau khi mo vong bang enzyme giai han Sac I. dutrc bicn nap viio chung nam men P pastons X33 bfiiig xung dicn Sau khi lien hanh nuoi cay cac tc hao nam men ircn moi trudng YPDS co nong do zeocin lan luot la 500, 1000 \ a 2000 mg L"' chung loi da thu dugc 100 dong te bao nam men tai to hop va da sang Ioc duoc 8 dong co kha nang smh truang tot tren YPDS chiia IOOO mg.L''. Kiem Ira su co mat cua gen csn tren he gen ciia cac dong P pasioris X33/
pPICZaA..c5/i cho thay, cac dong nay deu lao ,san pham PCR dac hieu, khuech dai gen csn nam giiia viing dac hieu 5' AOXl va 3' AOXl, gom hai bang CO kich Ihudc lin lucrl la 2 0 kb va 2 2 kb (Hinh 3), Bang 2,0 kb the hien gen csn (~ 1,4 kb) va viing dem 5' AOXl va 3 ' AOXl 6 hai dau ciia gen csn cd kich thudc khoang 0,6 kb. Bang 2,2 kb the hien vimg gen oxy hda methanol (alcohol oxidase gene - AOXl) cua chung nam men gdc X33 Trong khi dd, san pham PCR thu duoc tir chung nam men P pastons X33 khong duac tich hop vdi ket cau bieu hien chi CO mdl bang duy nhat vdi kich thudc la 2,2 kb (ddi chiing am) Nhu va>, pPICZ«A csn da lich hop thanh cdng vao bp gen cua nam men P pasioris X33.
Nguydn Thi Nggc Lien et al
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Hinh 1, Oien di So san pham PCR khuech i3ai gen csn (A) va plasmid pJET1,2,csn dugc xi> ly boi h.ai enzyme giai ban EcoRl va Xjbai (B) tren gel agarose 1,0% M Ttiang DNA chuan, 1 gen csn, 2, Plasmid p J E T I 2 csn
Hinh 2. Dien di 66 san pham cat vector bleu hien pPICZaA::csn bSng enzyme gioi han.. Thang DNA chu§n, 1 pPiCZaA: csn du'oc cat boi EcoRl va Xiial,
Hinh 3. Kiem tra sg' co mat cua gen csn thung qua san pham PCP l^huech a^i Jung gen tu 5 ' A O X I ldi uung 3'A0X1 voi khuon DNA la bO gen cua n^m men tai to hop P pastons X33/ pPICZoA,.csn U. Thang DNA chLjan,1-8 San pham PCR SLf dung khuon DNA la bp gen ciia cac dong nSm men tai to hop L l , L2, L3, L4, L5, L6, L7, L8. 9 San pham PCR si> dung khuon DNA la genom cua n^m men P. pastons X33
Dong n.im men I.ii 16 bao
Hinh 4. Hoat tinh CSN cua cac dong nam men lai to hpp Iren moi truong bieu hien B M U Y theo tho'i gian nuoi cay
Bieu hien chitosanase trong nam men P. pastoris Cac ddng te bao nam men P pastoris X33/
pPICZaA:cjn(Ll, L2,_L3, L4, L5, L6, L7, L8) cd kha nang sinh trudng tdt Iren moi trucmg YPDS cd
chiia zeocin ndng do IOOO mg.L"' dugc nuoi cay
va kiem tra hoat tinh CSN tren hai moi tnrang
fai^u hien la BMMY va BMGY, cam iing hang
1,5% (v/v) methanol. Ket qua cho thiy tit ca cac
ddng nam men tai to hgp deu cd hoat tinh CSN va
142• '^'dP ^M Cdng nghe Smh hoc\Ul):\ 39-144. 2013
^°f. " " ' ^ ^"^ynie thu d u g c tang d i n theo thai gian nudi cay. Dac biet, khi nuoi c i y tren mdi frumig BMMY ddng L5 va L6 cd boat tinh CSN cao n h i i , dat Ian lugt 263,9 U/ml va 322.4 U/ml sau 72 gid len men (Hinh 4 ) . Trong khi dd, cac enzyme sinh tdng hop bai n i m men gdc P pasioris X33 tich hgp vector gdc p P I C Z a A h i u n h u khdng c6 phan iing vdi CO c h i t chitosan (Hinh 4)
Tir cac ket qua nghien ciru cho t h i y , chung tdi da tao thanh cdng chting nam men P. pastons tai td hgp cd kha nang smh tdng h o p CSN ngoai bao cao, frong do ddng L6 cd hoat tinh cao nhat sau 72 gid nudi cay. De nang eao hoat tinh CSN sinh tdng h g p bdi nam men tai td h g p P pastons, cac nghien ciiu tiep theo ve dieu kien len men, di^u kien tinh sach va dac tinh enzyme tai td hgp se d u a c iiep tuc nghien ciru.
KET LUAN
Da thiet ke thanh cong vector bieu hien pPICZaA"Ci77 m a n g gen csn ciia vi khuan B cereus liN90 Vector nay da d u a c tieh hgp ihanh cdng ket cau bieu hien (expression cassette) mang gen csn vao bo gen nam men P pastoris X33 Trong sd cac ddng nam men tai lo h o p d u a c chon Igc. ddng L6 cd kha nang khang zeocine ndng do 1000 mg/ml va sinh long hgp CSN boat tinh eao nhat, dat 322,4 U/ml sau 72 gid nudi cay trong mdi trudng bieu hien BMMY cdcam img bdi 1,5% (v/v) methanol,
Loi cam o'n; Cong Irinh ducrc thuc hicn nha kinh phi cuu Bt) Cong ihuang cdp clio de ldi md so DTOS 10/CNSHCB thuoc de dn ••Ptidl Inen vii ung dung cong nghe sinh hoc trcmg lmh vuc cdng nghiep che bien den ndm 2020" Xin chcin thanh cam an su bo tro ve Irang thiet hi cua Phong thi nghiem trong diem Cong nghe gen, Vien Cong nghe smh hoc. I 'ien Hdn Uhn Khoa lioc va Cong nghe Vicl Nam
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\tu rnhiol (>Hf) .1727-1714
Nguyen Thi Ngoc Lien elai
E X P R E S S I O N O F G E N E CS.\ E N C O D I N ( ; C H I T O S A V A S E O F BACILLUS CEREUS HN90 JN PICHIA PASTORIS
Nguyen Thi .Ngoc Lien, \ u \ a n Loi, P h a m Thanh Huyen, Nghiem Ngoc Minh, Phi Quyet Tien"
Institute of Bioiechnolog)-. Vieinam Academy of Science and Technology
SUMMARY
In the presenl research, gene csn which consists of 1362 nucleotides and encodes chitosanase (CSN) of Bacillus cereus HN90 was amplified hy PCR, ligated in vector pJETI 2 and transformed into Escherichia coh XLI-blue After treatment of extracted plasmid from E coh with both resinciion enzymes EcoRl va Xbal, gene csn was inserted into plasmid pPICZuA to generate expression veclor pPICZaA: csn The resuhing veclor was Ihen linearized at the AOXl locus hy Sad, transformed into Pichia pastoris X33 strain by eleclroporalion and resuhing Iransformanls were screened on agar plate YPDS supplemented with aniibiouc zeocine at various concenlraUons The examination of yeast chromosomal DNA by PCR using specific primers 5" AOXl va 3"
AOXl indicated that Ihe gene csn was integrated into genome of P pasioris X33 Among over 100 transfonnants tested, eight recombinant strains showed the both capabilities lo grow on YPDS medium containing lOOO-mg^ml zeocine and synthesize extracellular CSN Based on Ihe enzymatic assays, Ihe rccombinanl siiain P pastons L6 revealed the highest chitosanase activity, reaching up 322 4 U,L" after 72- hour cullivation m BMMY medium induced by 1.5% (v/v) methanol
Keywords: Bacillus cereus, csprcssion of enzvme, recoinhinani chilosanasc. Pichia pasions
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