BlEU HI^N

BlEU HI^N GENE MA H 6 A XYLANASE TCT N A M M 6 C ASPERGILLUS NIGER BH7 VAO E. COLI BL21

Tran Uen Ha, NguySn Thj Thiromg Thirang vd Nguyen VSn Cach

Vi$n Cdng ngh$ sinh hgc- Cong ngh§ tturc phim. Tnrang D^i hoc Bdch Khoa Ha Ngi T 6 M TAT

Xylanase (endo-p-1,4-xylanase, EC 3.2.1.8) l i enzyme quan trpng nhit trong so cac enzyme dui^C su dung dk thiiy phan hoan toan xylan; n6 xiic tic phin iing ±iiy phan cic lien k^t P-l,4-D-xylopyranosc trong xylan. Xylanase dirgtc fimg dvmg r^ng rai ttong cong ngh^ san xuat thuc in gia sAc, cdngn^^lhucphain,sanxuiigiay, sin xuit con nhi&ili^... Xylanase c6th^ dugc tim thiy o u h i ^ nguon khic nhau vi d|ic biet li tu vi sinh v§L Tuy nhien xylanase dirac sinh tfing h9p tii nim mSc la enzyme dugc mong dcri nhit vi chiing b ^ nhifit vi cd nhieu d$c tinh mdi khac. Chiing nim mflc Aspergillus niger BH7 dugc phSn lap tir sin phim Ifin men IniySn thong ciia Vi?t Nam. Gen ma h6a xylanase ciia A. niger BH7 c6 746 nuleodte. Vilng ma hoa gene xylanase (eds) ciia nim ni6c A.

niger BH7 dugc tach bing phin ihig overlap PCR vi dugc gin vao vector bilu hifin pRSET A. Vector tii t6 hgp RSET A-xylanase dugc bieu h i ^ trong £.co/i BL21. Xylanase dugc b i ^ hien ttiinh cdng oE. cofiBL21, enzyme tai t6 hgp co kich thuoc 35 kDa.

Hogl dO cia enzyme tii t6 hgp trong dich phi v6 ti bio (97,2 U/ml) cao hon ngoii mfli tnrong (3,17 U/ml).

THkhoa: xyianase, Aspergillus niger, gene xylanase, pRSET A vector, E. coli BL21.

MdroAU

Xylan Id polysaccharide chiSm h' 1^ Idrn thi> hai sau cellulose cr thanh t^ bao t h ^ v$t t6n tai a d^ng lien kdt ho^c khdng iiSn k^t vdi cellulose, lignin, pectin hay nhOng polysaccharide khdc dh duy tri c&u tnic cua thanh td tide. Xylan \d mOt hfin hp'p cd chi>a cdc nhom phg la cdc gfic acetyl, 4-0-meth^-D-glucuronosyl vd a-arabinofuranosyl llSn kfit vdi bp khung du-gc tgo bdi cac gfic xylopyranose. B$ khung ndy du-crc lien ket vd-i nhau theo kieu p-1.4-glyco2ide (Biely 1985; Juturu et al., 2012, Bissoon ef al.. 2002). Dfi thuy phdn hodn todn xylan c4n co sif kdt hgp cOa mot h$ thfing cdc enzyme.

Trong sfi dfi, enzyme quan h^ng nhfit Id endo-xylanase (endo-p-1,4-xylanase EC 3.2.1.8), xiic tdc phan Cmg thUy phdn cdc lifin kfit p-1,4-D-xylopyranose trong xyian (Bissoon et al.. 2002; Polizeli et al.. 2005). Enzyme xylanase du'gc irng dung rfing rdi ^ n g cfing ngh$ sdn xufit thi>c dn gia sue, cfing ngh$ thi/c phdm, sdn xudt gidy, sdn xudt cfin nhien li^u. ..

(Blely 1985; Jutum et al., 2012; Bissoon et ai.. 2002; Polizeli el ai. 2005 ; Kansoh, 2004). Xylanase cd thfi dirge tlm thay a nhifiu ngufin khac nhau vd ddc bi^t Id ti> vi sinh vft. Tuy nhien xylanase du'gc sinh tfing hgp ti> ndm mfic Id enzyme du'gc mong doi nhdt vl chiing ben nhifit vd cfi nhifiu dgc tlnh mij-l khdc. Chung nam moc A. niger BH7 duvc phan ldp tCr sdn phdm Ifin men truyfin thfing cua Vigt Nam. Gen md hfia xylanase ciia A. niger BH7 cfi 746 nuteotite (Lien Ha et al., 2013). N6i dung bdi bdo ndy sfi trlnh bdy ket qua tdch ddng, bidu hifn gene md hfia enzyme xylanase tCr ndm moc A. niger BH7 vdo E. coli BL21 nhdm nghien ci>u cdu tnic gene xylanase cung nhir khd ndng li'ng dgng xylanase trong thi,i'c tiin.

VAT Ligu VA PHlfONG P H A P NGHIEN COU Chung vi sinh vgt, tfi bao chu, vector SCF dung

ChOng ndm mfic A. niger 87 cfi hogt tlnh xylanase ti> bfi siru tdp gifing cua bfi mSn Vi sinh - Hfia sinh vd Sinh hpc phdn ti>, Vifin Cfing ngh$ sinh hpc - Cfing ngh$ thirc phdm, Tnrdmg Dgi hpc Bdch khoa Ha Nfii.

ChiJng Eschenchia coli BL 21.

Vector bidu hifin dirge chon Id vector pRSET A chCra promoter T7 idiiti dfing phien ma, gene chi thj khdng khang sinh ampicillin.

Hda chatvd thanh phdn moi t r u i n g nufii cdy Hda chat

Tris base, Ethylen diamin tetra acetic add (EDTA), CTAB, NaCI, p-mercaptoethanol. Sodium dodecyl sulphate (SDS), Natri axetat, ethanol 100%, clorofomn, isoamylalcohol, agarose, ethydlum bromide (EtBr), polyacrylamide, APS, TEMED , 4 logi dNTP, MgClz vd Taq DNA polymerase, primer dugc mua vd tfing hgp tir cfing ty Sigma (Iwly).

Birchwood xylan, Si04. Nai, isopropanol, proteinase K, RNase, (Merck, Dii-c); Wcol, Xhol, T4-//gase ( Fementas, Canada).

Thdnh phan mdi t r u i n g nudi cay

Mdi tar&ng sinh tdng hgp xylanase (A): NaNOj 3 g/l, MgS04.7H20 0.5 g/l, FeS04 0,01 g/l, KH2PO4 1 g/l, KCI 0,5 gfl, Cao ndm men 5 g/l, xylan 2 g/l. Agar 20 g/l.

Mdi tnrdng nudi ndm mfic Czapek (B): NaNOs 3 g/l, MgS04.7HzO 0,5 g/t, FeS04 0,01 g/l, KH2PO4 1 g/I, KCI 0,5 g/l, Cao ndm men 5 g/l, saccharose 30 g/i, Agar 20 g/l.

Mdi tnrdng nudi Luria-Bertanl (LB) : cao nam men 5 g/l, trypton 10 g/l. NaCI 5 g/l, agar 15g/l.

Tdt ca cdc mfii tnrdng dirge thanh tnjng ir 121 "C trong thd'i gian 15 phut

HOI NGHI KHOA HOC CONG N U H t oimn nvv>

Phirang phdp tach chiet DNA

Tdch chiet DNA.t&ng so '^

Ldy 0,2 - 0,3 g sinh khdi ndm mfic A niger BH7 sau 48 gid nudi cdy cho vdo fing eppendoftvd bfi sung 1 mM#m ly g ^ (100 mM Tns4«a pH 8,0.10 mM EDTA, 2% SDS, 1% p^necaptoethand 100 MQ Pr^^^^!!**L " i f i ^ K ^ f . ^ ' ^ . " * 65"C trong 1 gid ffii bfi s«ig Naa 5M dfi dat nfing dfi NaCI Id 0.7M va bfi sung CJAB 10% vd. thd tfch bdr^ 1/10 df^, Tidp Mc ii d 65"C troog 30 phuL Sau dfi, cho hdn hgp ctorofbrmnsoamyl alcohol (24:1) vao dich vdi ty lfi 1:1 Idc dfiu rAi ly tdm d 13000 vdng/ phut trong 10 phOt, d 4"C. Thu djch nfii vdo fing eppendoft mdi vdcho raopropanoi vdo ven ^ 1$ i:i.

L & dfiu rfii ly tdm d 13000 vdng/phiit trong 30 phiit, d 4''C. Rda pellet thu dtrge bang ethanol 70 %. Ly tdm d 13000 vdng/phut trong 10 phiit, d 4°C. Ldm khd, hda tan trong 50 pi TE (10 mM Tris. 1 mM EDTA)

Tdch chiet DNA plasmid

Plasmid dugc tdch theo phuomg phap Sambook et al., 2001 Phirang phap PCR

K h i ^ h dgi exon 1 vd exon 2

Cdc cdp mfii sd dyng dd khuech dgi exon 1 vd exon 2 mang trinh tg nhdn bidt Xhol, Ncoi (chd dirge ggdi chdn) ed trlnh tg nhir sau:

Exon 1: XynF1: 5"- GCeiCGAGATGCTCACCAAGAACCT- 3-; XynRI: 5 - CATTGTCCTGCGCACTTCCGGGGT- 3' Exon 2: XynF2: S -TTCATGGTGCGCAGGACATCACCTA- 3'; XynR2. 5'- GCCCATGGTTACTGAACAGTGATGG- 3'

Chu binh nhi$t phdn img PCR; Nhidt dd bidn tinh 94*'C trong 5 phut chgy 30 chu ky vdi nhifit dfi bidn tinh 94''C frong 30 gidy, nhifit dfi 58°C bdt d p 45 gidy vd nhi$t dfi keo ddi 72''C trong 45 gidy. Sau dfi nhifit dp kfio ddi 72''C trong 8 phiit Khudch dgi khung dpc md ciia gen xylanase

Cdp mfii su dung dd Mwdch dgi gen xyn ma hda xylanse mang hinh tg nh^n bidt Xhoi, Ncoi (chO' dirge gach chdn) cfi trinh tg nhu sau'

XynFI: 5"- GCCTCGAGATGCTCACCAAGAACCT- S'; XynR2: 5- GCCCATGGTTACTGAACAGTGATGG- 3'

Chu trlnh nhifit phdn iing PCR; Nhi#t dd bidn tlnh 94°C trong 4 phtit, chgy 30 ehu kJ vdi nhidt dfi bidn tfnh 94''C trong 30 gidy, nhidt dfi 50°C bdt c ^ 30 gidy vd nhi$t dfi kdo ddi 72''C trong 30 gidy. Sau dd nhigt dp keo ddi 72''C trong 8 phiH Phvtmg phdp dIf n dl tren gel agarose

Ldy 5 pi mdu DNA (100 ng/pl) dugc trfin vdi 2 pi thufic nhupm va tra vdo cdc gtdng tren gel 1% aganase. Sau khi tia mdu, gel duvc chgy di$n di b 100V vd 90mA. Sau dd, bdn gel di/gc nhufim trong dung djch EtBr 0,5 pg/ml trong 5-10 phut, n>a Igi bdng nude trudc khi sol gel.

Phuomg phdp tinh sgch DNA bdng b$ kit MINELUTE (QUIAGEN) Phirang phdp bidn ngp vdo E. coll bdng sfic nhi$t PlasmkJ dugc tdch theo phuong phdp Sambook efa/., 2001.

Xdc <^nh hogt tinh xylanase si> dyng DNS

^ S ? S S *<,S^S?^^57*"'' ""= "^ *^'^ """•''"" '*"''"' * =°°= '™='""" - " - - *"=

K £ T Q U A V A THAO LUAN

Khufch d^l tlo,r, exoni v6i c»p m i l XynFI-XynR! v4 dofn exon2 vein c|p n i l XynF2-XynR2

d u ™ ^ a A i ^ u hB^vS i S S l i ? ^ , •• ° ? l ' " f ' « ' ' ^ ^'1'"^ ^ " " ^ (Srivastava ol al.. 2001). Khi sO

^^E. A^ZZ^IT^'X fnffZ?. "•• ™"- "°"= "^ ^ "^ """^ '« '" ""

si 347 v« * i n S I g^nl^natf B*4|^; ^'"' '^ *^ '"^ ^^ 9=" 1* »l « nucleotide si 278 din nuileoU*

^ i ^ ' ^ ^ ^ ' ^ V ^ T j ^ f ^ i ^ ' ^ ^ " ° " "*" ^" ^ •^' "* XynF2-X,nR2 6u^ tl,lit k4 dS nucteotide diu 5' cua doan e>», 2 ^ dSan e » „ * ? 5 S " i ! f " " '^" S ^ "^ « ' » ' B°?n exon m i l (dSu 3') mang 12

ooan exon tli„ 2 (diu 5) mang 12 nuclceollde dau 3' ciia doan exon tlii> ntlSt

)C CONG NGHE SINH HQC T O A N Q U O C 2013

Dogn 12 nucleotide ndy giup hai exon ndi vdi nhau vd day Id khu vgc dau tifin sfi dugc gan kdt Igi tgo eds cOa gen xylanase nhd phdn ung overlap PCR.

Hlnh 1: Dl^n di dfi sdn phSm phdn i>ng PCR khuech dgi exon ciia gen xylanase. 1: DNAmart(er1kb,2:M5uam tinh,3:Oogn exon 2; 4: Oogn exon 1

Hlnh 2. Dl$n di dfi sdn phdm phan dng PCR khuich dgi eds cua gen xylanase. 1; DMA marker Ikb; 2: M3U dm tinh; 3: Gen xylanase cdt t>d intron (eds xylanase); 4' Gen xylanase.

Kdt qud difin di trfin hinh 1 cho thdy vgch khufich dgi dogn ewxi 1 co kich thudc khoang 300 bp va doan exon 2 cfi kidi thude khodng 400 bp. Nhu' vdy, phan dng PCR da khudch dgi thanh c&ig hai dogn exon cua gen xylanase. Sdn phdm phdn irng PCR khufich dgi hai dogn exon dirac Idm sgdi bang bfi kit MINELUTE.

Khuech dgi eds cGa gen xylanase

Sau khi khufich dgi tfidnh cfing hai dogn axon mfit vd exon hai, dfi thu eds hodn chinh cOa gen xylanase, hai dogn exon ndy can dugc gan ket Igi vdi nhau vd dirge khudch dai Ifin bdng phdn dng PCR de &iu khoi lugng Idn edc eds cQa gen xylanase. Sau phdn dng overlap PCR, sdn pham dugc difn di trdn gel agarose 1%.

Dd hai dogn exon rifing lfi cd thfi gan kdt Igi dirge vin nhau thi nhigt dp bdt cgp moi trong phdn dng vdi cdp mdi XynFI- XynR2 phdi du^c hg thdp. Khi hg thdp nhifit dd bdt cgp mfii, dogn exon l$p se cd thfi dfi ddng gdn kdt Igi vdi nhau dd tgo thdnh eds hoan chinh. Tidp theo, mfii XynFI vd mfii XynR2 sd bdm trdn sgi DNA vd khuech dgi todn bfi eds cda gen xylanase.Trfin difin di dfi Hinh 2, sdn pham cfi kfch thudc ldn hgn 500 bp vd nhd hgn 750 bp. Chinh vi vfiy, cd thfi nhdn dinti rang phan PCR da gdn kdt thdnh cfing hai dogn exon va khufich dgt thanh cfing eds cua gene md hda xylanase.

Do ha thdp nhifit dfi bat c$p mol nfin trong qua trlnh tfing hgp sgi DNA cfi the xdy ra l5i tfing hgp sai nudeotide Chlnh vi vdy sau khi tach dong cdn gidi trlnh tg dogn gen gan vdo vector de dam bdo rdng dd tdch ddng thanh efing hay khfing thdnh cdng gen x^anase. Ket qud cho thdy san pham over lap PCR cd kich thudc 678 bp va dogn intron kich thudc 68 bp da bj logi bo.Vdy ed thd kfit ludn gene xylanase cda A. niger BH7 cd kfch thudc Id 746bp, trong dfi eds cd kfch thudc 678 bp vd dogn intron cfi kfch thudc 68 bp.

Bifiu hign gen x^anase trong E.co// BL21

Sau khi gdn eds gen xylanase vdo vector pRSET A bang phdn dng gan kfit nhd sg hogt dfing cua enzym TA-ligase, vector tdi tfi hgp dugc bidn nap vdo E. co/fBL21 bang phirong phdp sdc nhifit. Djch tfi bao dugc dem edy trdi trdn mdi trudng LB dgc, dugc bd sung khdng sinh Ampicillin de chpn Ipc. Sau khi nudi d 37''c qua dem cfi khd nhilu khuan Igc mpc Ifin trfin mdi tnj^ng dgc hpp petri.

Hinh 3. Xdc djnh hogt dd enzym xylanase bing Hinh 4. Di#n dl do protein tong sfi ciia djch enzym th6 n$i bdo ciia te bao phuong phip due l6. A D|ch len men, B: Djch phd E. cof/tii to hgp. 1: Marker protein (KDa): 2: Djch enzym xylanase thS sau 8 v& td bao ^ •^"1 "^^9 IPTG; 3: Dich enzym xylanase tho sau 0 gicr cam t>ng IPTG Mdt khudn lgc E. coh BL21 nfing re dugc Idy va nudi thu enzyme xylanase tai tfi hgp trong mdi trudng LB long bfi sung ampicillin, cdm ung IPTG, Enzyme xyianase tai tfi hgp dugc tfing hgp trong £. coli BL21 dugc xdc djnh ir dang nfii bdo vd nqoai bdo bang phugng phdp khuech tdn due lo thach vdi nong dd xylan 0,4%.Te bdo dugc pha bdng phuong phap sifiu ani tifin hdnh 5 lan (sifiu am 45 gidy, nghi 45 gidy) trong dfim.Ket qud tren Hinh 3 cho thay hoat tinh i^anase trong

H O I N G H ! K H O A H O C C O N G N G H E S I N H H O C Tr'f^

d j d i Ifin m e n cua d i u n g tdi tfi h g p ( H i n h 3 A ) thdp h o n s o v d i hogt tinh ^ l ^ ^ f f , ^ " 9 ^ J ^ ^ ^ ^ ^ * { ^ ^ ° ^^^^^ ^J^' ^ ^ ndy h o d n toan p h u h g ? v d i ly thuyfit rdng trong d i u n g E. coli B L 2 1 , protein tdi to h g p t h u d n g n d m t r o n g t f i b d o . ft kh.

d u g c tidt ra mdi t n r d n g nudi cdy ( A h m e d et at., 2009).

Dfch p h d v d t d bdo thu d u g c b d n g p h u o n g phdp sifiu d m d u g c s u d y n g dd xde d j n h p r o t e i n t f i n g s 6 ttieo p h u g n g phdp didn d^ gel p d y a c r y l a n S ^ 10 % Difin di dfi trfin H i n h 4 eho thdy xylanase d d d u g c bidu hign v d h a m l u g n g protein tdng Ifin s a u 8h ttidi gian c d m u n g v d k i c h ttiuifrc ctia protein tdi to h g p 35 kDa.

D j d i phd v d td b d o v d djch Ifin m e n d f i u d u g c xdc djnh hogt t i n h . Xylanase d u g c bifiu h i f i n d £ coli p h f i n l d n d dgng nOi bdo, hogt d f i cua e n z y m e tdi tfi h g p npi bdold 97,2 U/ml. trong khi d d hogt d f i e n z y m e n g o d i m o i t n ' o ' n g 3,17 U/ml. Bd ttiu d u g e enzyme tdi tfi h g p efi hogt Igc c a o ttii vific tfii u u h o d cdc didu kien dnh h u d n g d f i n q u a t n n h b i f i u hifin vd qud trlnh Ifin m e n cdn d u g e fifip tuc nghifin c d u .

K£TLUAN

Od bifiu hign ttidnh cfing gene m a hda xylanase t d n d m m o c A. niger B H 7 v d o E. cc^i B L 2 1 . E n z y m e tdi t f i h g p cfi kh6l l u g n g 35kDa. Hogt tinh xylanase ttong djch chiet td bdo Id 97.2 U/ml v d trang djch idn m e n Id 3,17 U/ml.

T A I U ^ U T H A M K H A O

AhmedS, Riaz S. JamB A(2009). Molecular cloning of fungal xylanases an overview. >lpp'Wicrotttrf ao(ec/)no/ 84:19-35 Blely P(1985). MKTOfaial xylanolytic systems. Trends in Biotechnoi 3. 286-289

Bissoon S, Chfistove L, Singh S (2002). Bleach boosting effects of purified xylanase fnam Jhennornyces lanuginosusSSBP on bagasse pulp Pmcess Biochem 37: 567-572

Jun H, Bing Y, Keying Z, Xuemei D, Daiwen C (2009). Expresston of a Tnchodenna reesei p- xylanase gene in Escherichia cob'and acUvity of the enzyme on fiber-txxxid substrates. Protein Exp Pur 67 1-6

Kimura T, Suzuki H, FuruhasM T, Abuiatanl K.,MorimDto K, Sakka K, Ohmiya K (2002). Molecular cloning, characterization and expresston an^ysis of the xynF3 gene from Aspergillus oryzae. Biosci Biotechnoi Biochem 66' 285-292.

Lien Ha T, Thuong NTT, Cach NV (2013). Cloning xylanase gene from Aspergillus niger BH7 in Eschenchia cdi TOP 10. Journal ol Science md Technokigy Techier UntversiSBS 9 3 : 4 ( M 3 .

Polizsll ML, RizzatUAC, MonS R, Terenzi HF, Jorge JA, AmorimDS (2005). Xylanase from fijngi: properties and industrial application Appli Microbiol Biotechnoi G7 577-591 .

Sambnaok & Russel. (2001). Molecular ctoning - a laboratory manual (third edition ) Cold Spring Harbor Leboratory

Zhou C . Bai J. Deng J, Wang J. Zhub J. Wu M.Wang W (2008) Ctoning of a xylanase gene from Aspergillus usamii and its expression

\n Eschenchia cod. Biores Technol 99 831-838,

Ohta K. Moriyama S. Tanaka H. Shige T, Akimoto H (2001). Purification and charat^enzation of an acidophilic xylanase from Aureobaaklium paOulansvar. Melanigemim and sequence analysis of the encoding gene. J Biosci Bioeng 92:262- 27 Srivastava P, Mukherjse KJ (2001). Cloning, charecterlzation and expression of xylanase gene from Bacillus lyticus In Escherichia cdl and Baoflus subhlis. PreperBiochem Biotechnd 31(4) 389-400

EXPESSION XYLANASE GENE OF Aspergillus niger BH7 IN E. coli BL21

T r a n Lien H a ' , N g u y e n T h i T h u o n g T h u o n g , N g u y e n V a n C a c h

Sclmd o f Bioltchnology • Food Technology. Hanoi University o f Science and Technology

SUMMARY

E n ^ = . y l ™ . (=„do-M,4-xyl.n„e EC 3 2,1 8) i, ,he mo,t important enzyme for eompleled hydi<,lym xykn It digeste, xyta I M ?"v7 ' " ™ ' ° t r r - " ' " ' • " • " ™ " " " » "^ " ^ P«>«"»6, food technology/p.pJmakri p S g , l l . , ! . J!^ V ^ r ° ' *™»s>«l'ilily aid novel characteristics. Xylanase gene from Asperilllw, ni„er BH7 ttSormLl it^B E » ? B T I ^ n ™ ^i^^^^^^^^ " " T ™ '"SET A vector The recombinant pRSET A - x y I « , a s = S r Is

.bcb,or™dcSiei^.i^3.,?ur*;s^^^^^

* ; » m > r * , xytoase. A^r^llu, „ , g „ . gene xylanase, pRSET A vector, E. cell BL21 Aolhor ot comspooamco- Email: ha tranlien@hust.edu vn