^ chi Cong nghp Sinh hpc 9(3): 391-396, 20[\
TINH S ^ C H P R O T E I N C O H O ^ T T I N H K H A N G N A M T l f C H U N G BACILLUS SVBTILISXL62
Do Thj Tuyfin, L& Dinh Quy^n, Q u y l n Dinh Thi, Nguy?n Ng^c Dung Vien Cong nghe sinh hpc
TOM T A T
Bacillus subtilis la vi khuan sinh tdng hop nhilu peptide khang ndm nhu iturin va bacillomycin Chiing cd oSu tnic peptidolipid msch vdng vfl cd hojit tfnh khdng nkm, tan huyet, Chiing XL62 da dupc phan l§p tir mlu dat va xic djnh chinh xdc loai B. subtilis dya trSn hinh thdi va phSn tich trinh tu 16S rDNA trong mgt nghien ciiru tmdc. Protein cd boat tinh khdng ndm dugc tinh s?ch tir djch nudi cdy ciia chiing B. subtilis XL62 qua 3 budc gdm tiia mudi ammonium sulfate, cgt sBc ky trao doi ion DEAE-cellulose va cot sSc ky loc gel Biogel PlOO Cdc phfin doan tinh sach cd ho(it tinh khdng nim tuang d6i in?inh va cd khoi lugng phfin tii khodng 21 kDa trfin di?n di do SDS-PAGE. Prolein khdng ndm ndy c6 hoai linh ire chl m?nh sir sinh tnrdng va phdt triln ciJa nfim Fusarium oxyspanim vd Rbizoctonia solani. Prolein tinh s^ch tit chung B. subtilis XL62 cd boat tinh khdng nfim mpnh doi vdi chiing nfim F oxysporum. Hoat tinh khdng nfim vin cdn duy tri khi u d 80''C, tham child lOCCtrong ISphut.
Tur khoa: Bacillus subtilis XL62. Fusarium oxysporum. prolein co hoat tinh khdng ndm. Rhizoctonia solani. tinh spch
MODAU
Cac protein va peptide co boat tinh khang nam deu duoc tach chifit phan Idn tir dong vat (lijima el al, 1993; Wang, Ng, 2002), thirc vat (Benhamou el al.
1993; Joshi e / a / , 1998; U r n efa/„ 2000), vi khu5n (Delcambe et al.. 1977; Kim, Chung 2004; Moyne el ai, 2004) va nkm (Lam, N§, 2001; Wang, Ng, 2004;
Theis el al.. 2005). Chit doi khang sinh truong nam
^^tifimgal compounds, AFC) la nhihig chat c6 boat tmh lie chl su sinh tnrong va phat trien ciia nam gay benh 6 cay trdng. Cac boat chSt khang nam nay deu CO ban chit la cac hoat chat thii cS,p, protein va .peptide. Dua tren cdu tnic hoac chiic nang, cac
protein va peptide co hoat tinh khang nam dugc phan loai thdnh nhilu lop khac nhau, ching han nhu chitinase va chitinase-like protein (Benhamou et al, 1993; Lam el al, 2000), ribonuclease (Liu et al.
2007), chdt uc chi protease (Joshi et al, 1998).
Gan day, m§t so chung Bacillus subtilis da duoc ,lJng dyng d l kilm soat b?nh cSy trong (Hwang, Chakravarty, 1992; Asaka, Shoda, 1996; Wiilff el al; 2002; Okigbo 2005), do vi khuin nay ph6 biln trong dat, chju dirpc nhiet dg cao, sinh truang nhanh
•rang moi trudng Idng va sinh bao tur. Ngoai ra, B
«ibtilis thuong t6ng hgp cac loai peptide khing sinh . nho (khdi lirgng phan tur < 2.000 Da) khong phai tii
^.jibosome c6 ho^t tinh khang ndm, ching han nhu iturin (Delcambe et al. 1977; Stein 2005), surfactin
(Peypoux et al, 1999; Camllo et al, 2003), fengymycin, bacilysin (Loeffler el al. 1986), bacillomycin (Peypoux et al, 1980), mycosubtilin (Peypoux el al, 1986) va mycobacillin (Majumdar, Bose, 1960; Sengupta el al, 1971). B. subtilis hlu nhu Cling sinh tdng hgp ra rat nhieu loai protein (Moyne et al. 2004; Liu el al.. 2007). Tuy nhien, mot s6 it trong tdng sd cac loai protein tren da dugc cong bd la cd hoat tinh khang nSm
Trong nghien ciiu tmdc day, chiing tdi da phan lap va nghidn cuu hoat tinh khang ndm cua dich chilt ngoai bao tii B. subtilis XL62, uc chl manh cac ndm bdnh nhu Fusarium oxysporum va Rhizoctonia solani tren mdi tru(mg_ hot d^u tuong va thu dugc cac dilu kien len men tdi im d l sinh tdng hgp protein cd kha nang lie chl manh su sinh trudng va phat triln ciia hai loai nfim nay (D6 Thi Tuyen et al.
2010). Trong nghien ciiu nay, chiing tdi tach chiet, tinh sach protein c6 kha nang khang nam F.
oxysporum va R solani tu B subtilis XL62.
VAT LIEU VA PHl/ONG PHAP NguySn li^u
Chiing B. subtilis XL62 da dugc xac dinh loai bang phan tich trinh tir 16S rRNA, chiing nSm Fusarium oxysporum va Rhizoctonia solani dugc 391
phan lap tii cic mSu ddt do Phdng Vi sinh v$l ddt, Vi^n Cdng ngh| sinh hpc cung cdp.
M6i trirdiig nufii cdy
Mdi trudng PDA dCing dk thir ho^t tinh khdng nam: 20 g djch chiel khoai tSy, 0,2 g glucose, 0,2 g agar, M6i trudng NYD (g/1): 8 g cao thjL 5 g cao nam men, 10 g D-glucose,
Hda chdt
Sodium chloride, D-glucose tir Merck (Diic), ming Ipc Minisart (Biotech), agar (Vi§t Nam), DEAE-cellulose, Biogel PlOO, Trisbase, NaHP04, Na2HP04. Cac hda chdt thi nghiam khdc d^u d dang tinh khiil, M$t so nguydn li^u dCing thay thi nguSn carbon vd nita nhu tinh b$t khoai tdy, bgt d^u tuong dugc mua d ngodi thj trudng,
Thu nh^n djch Igc t i bfto
Djch len men cua chung B. subtilis XL62 sau 5 ngay nudi cdy a 30°C dugc ly tdm 15 phiit vdi 12.500 rpm. Thu djch n6i r^i Igc v6 triing qua mdng Ipc c6 kt'ch thudc lo 22 pm,
Xdc d)nh hoat tinh khdng ndm cua vi khuSn Bdo tir nam F. oxysporum vd R solani (Ix 10*
bdo tir/ml) dirge hda tan vdo I ml dung dich 0,2%
Triton X-100 va trdi deu IOO jil vdo dia petri chira moi tnrdng PDA. Due cac Id thach dudng kinh 10 mm, bd sung IOO |xl djch Igc ti bao ciia chung B.
subtilis XL62, giii d 4°C sau 2 - 3 h roi nuoi trong tii
am 30°C. Sau 2 - 3 ngdy nuoi cay, quan sat vdng tie che sinh trudng nam moc.
Doi vdl dich protein tinh s^ch, dia PDA dugc bd sung 0,1% ampicillin, d^t cdc khoanh gidy Igc thdm 15 }j.l djch protein tinh s^ch d cdc phdn do^n khac nhau, IJ 3 - 5 ngay d 30°C.
Tua ammonium sulfate vd tiikm tfch
Djch nudi cdy chiing B. subtilis XL62 trong moi trudng NYD sau 5 ngdy d 30°C dugc ly tdm 15 phiit vdi 12.500 rpm d 4°C. Djch ndi dirge tiia Idn 1 vdi 30% (w/v) ammonium sulfate. Sau ly tdm, djch ndi dugc bJa idn 2 vdi 70% (w/v) ammonium sulfate, khudy I gid d 30°C vd di qua dfim d 4°C. Djch tiia dugc ly tdm 20 phiit vdi 12000 rpm, Tiia dugc hoa vdo 4 ml d?m 20 mM phosphate pH 6,8. Dich protein dugc tham tich, ly tam vdi t6c d0 12.500 rpm trong 15 phut d 4°C vd lo?i bo phan kit tiia.
Sflc k^- trao a6i ion trgn c^t DEAE-ceUulose Dya trSn co sd ciia phdn ling trao dfli anion giiia protein trong hSn hgp vd cdc nhdm ion dimethylaminoethyl tich di$n duong ciia chdt mang trfin c^t DEAE-cellulose (dimethylaminoethyl- cellulose), khi h6n hpp protein qua cgt cdc protein tich di?n dm gdn vdo c$t vd sau do dirge ddy khoi cgt bang dung djch muoi chira ion c6 di lire manh han d6i vdi nhdm trao d^i ion ciia chdt mang tren cgt,
Ba ml dung djch protein sau khi tham tich dugc dua 16n c^t DEAE-cellulose (2,6 x 60 cm) dd cdn bang vdi d^ni 20 mM phosphate pH 6,8. Protein tren cOt dugc day ra bdng d§m 20 mM phosphate pH 6,8 chiia 1 M NaCl vdi tdc dd ddng chay 25 ml/h (Hinh 1). Thu thi tich moi phdn do^n 1,5 tnl. Sau khi phan doan trdn c0t, 3 dinh cd nong dp protein cao it phdn do^n 7 (dinh I), phan doan 17 (dinh 2) va phan doan 22 (dinh 3) dugc thu l^i. Cdc phan doan cd hoat tinh khdng nam m^nh tiep tyc dirge dua len cgt Biogel PlOO.
sac k^ IQC gel Biogel PlOO
Ld cpt phan tdch hdn hgp protein theo nguyen tdc Ipc gel, Biogel PlOO la mgt polyacrylamide trong dd phdn tli cua chimg lien ket vdi nhau bdi cdc lien ket ngang tao thanh sang phdn tii bdi he thong 16 lirdi xdp. Dya vdo sy khdc nhau ve kich thirdc vd phan tli lirgng ciia cdc protein co trong hon hgp de phdn tdch.
Cdc phan doan protein (the tich 6 ml) sau khi qua cgt DEAE-cellulose cd ho^t tinh khang nam cao, dirge dira len c$t sdc ky Ipc gel Biogel PlOO (2,6 x 60 cm) dd cdn bdng vdi dgm 20 mM phosphate pH 6,8. Protein dupc ddy ra bdng dpm 20 mM phosphate pH 6,8 vdi t6c dg ddng chay 25 ml/h. Thu 10 phdn d o ^ the tich moi phan do?n 1,5 ml. Djch protem tinh S9ch dugc dimg di ddnh gid hoat tinh khdng ndm F. oxysporum va R. solani.
Di^n di SDS-PAGE
Khoi lirpng phan tu tirong ddi vd dp sach ciia protein tinh s^ch dirpc ddnh gid bdng didn di tren gel polyacrylamide (Laemmli 1970).
Xdc djnh hdm lupng protein
Hdm lugng protein dugc xac dinh theo Bradford
(1976).
|cA( Cong nghe Sinh hpc 9(3): 391-396, 2011
': I Dich thA L y t a m 10 p h u t
vo-i 12.500 rpm
LQC q u a m i i n g Ipc m i n i p o r e
- > I Thi> hojit linh khdng ndm [
Phan d o ^ n t r £ n ci
TiJa vd-i a m m o n i u m sulfate 30% (v/w)
TOa vd>i a m m o n i u m sulfate 70% (v/w)
Phan d o ^ n tren c $ t
^ >i
nh 1. Quy trinh tinti sgch pnatein c6 hoat tinh khang n i m Hoat chat t i n h s ^ c h
Ly tfim 15 philt vo-i 12.500 rpm
L y t f i m 15 phiit vd-i 12.500 rpm, Tiia t h u du'O'c hoa vdo 4 ml d^m, loai
mudi bang thdm tich.
Thi^ h o ^ t t i n h khang nam
ET QUA VA THAO LUAN
Sail khi xac dinh ho^t tinh khdng ndm cua dich loi cay chung B subtilis XL62 trong mdi trudng iT) sau 5 ngay d 30°C vd djch protein thd thu r?c sau khi tiia vdi muoi ammonium sulfate ndng ' 70% (w/v) da chiing td djch protein cd boat tinh ang nam F. oxysporum rat hieu qud (Hinh 2B).
lu nhu djch protein thd ndy da irc chi dugc sy sinh fdng vd phdt trien ciia nam trong vdng dudng kinh oang tii 20 - 30 mm (Hinh 2B).
Protein dugc phan tach thdnh 3 dinh ro rang tren
; ky dd trao ddi ion DEAE-cellulose (Hinh 2A).
c phan dogn nay dupc thir ho^t tinh khang nam F.
i>sporum, kit qud cho thdy dich protein tmh sach bO da CO hoat tfnh khdng nam vdi vong uc che sy h trudng vd phdt trien ciia ndm trong vdng dudng kinh khodng tir 5 - 10 mm (Hinh 2B).
Trong bai bao nay chung tdi di sau vdo nghien
• ^ va tinh sach cdc protein cd ho^t tinh khang ndm
tir dinh I (peak I). Cdn cdc protein cd hoat tinh khang nam tir dinh 2 va dinh 3 se dugc cdng bd d cac bao cao tiep theo.
Cac phan doan 5 d^n 11 xung quanh protein thu dugc tir dinh I duoc trdn l^i va dua len cgt Bioge!
PlOO. Sau sdc ky Biogel PlOO, thu dugc 2 dinh chinh (PI) vd dinh phu (P2) (Hinh 3A), Do sach va khdi lugng phdn tit ciia cac phan doan dugc ddnh gia so bg tren gel dien di 12,5% polyacrylamide nhugm bac sir dyng thang protein chuan (Fermentas).
Protein thu dugc kha sach d phdn doan cd bang - 2 1 kDa (Hinh 3B). Khdi lupng phdn tit cua protein khdng nam nay khac so vdi mpt so protein khang ndm dugc tinh sach tir B. subtilis nhu bacisubin (41,9 kDa) (Liu el al, 2007), YxjF (12,23 kDa), endo-l-4-P-glucanase (46,60 kDa), YnfF (45,39 kDa), BamD (44,94 kDa), putative sensor kinase (53,38 kDa), bacillomycin D synthetase C (309,04 kDa) (NCBI search) vd X98I1I (59,0 kDa ) (Xie el al, 1998).
t)o I hi rtyft el al
Pi'^ik 2 , Pc.ik 3
i i
n u, 20 C:i'. |)liMii(loan
Hinh 2 Sac ky do r;l(- |ih,-in do;:iii qua (.;()! sac ky irno clot ion OEAE-cellulobe (A), (B) Hoai linh khang F oxysporum cua cac ph.in doan prolnin sau khi qua col DEAE-cellulose (DC- dot chung am - dich moi Iruong NYD, DN dicfi ndi sau ly lam. DT ilicltlua P l , P 2 v a P : i cac phan tioan d dinh 1, 2 va 3)
kDa DN M 1 2 3
5 20
Hinh 3 Sac ky 66 (A) va dien di dd (B) cac phan doan linh sach qua cot Biogel P100 iDN dich protein iho 1 -~4 cac phan doan 1 -4 qua col Biogel PlOO)
Hinh 4. Hoat tmh khSng n i m ciia prolein dugc tSch ra tu B subtilis XL62 trSn hai chiing ndm F. oxysporum (A) va R solani {B),(DC{-) dem 20 mM phosphate pH 6,8, DC(+) djch ndi ti> chung B subtilis XL62; fd 2, fd 3, fd4, fd5 djch pnatein tmh sach sau khi qua cdt Biogel P100
I ^ chi Cong nghe Sinh hoc 9(3): 391-396, 2011
^•^ Cac phuong phap tdch chiet protein cd hoat tinh
^ n g nam tir B. subtilis XL62 diu gi6ng nhu cdc i ^ i r o n g phdp tach chiet protein khdng ndm khdc da , .i:6ng bd (Wang and Ng 2002; Wang and Ng 2004;
Ngai et dl, 2005), Cdc phuang phdp sdc k^ trao d6i ion DEAE-cellulose va sdc ky Igc gel Biogel PlOO da dupc sii dung de tach chiet cdc protein cd ho^t ' -itjnh:khdng ndm trong cdc nghian citu trudc ddy
(LameM/., 2000; Lm el al. 2007).
- Dich protein sau tinh sach trfin cdt Biogel PlOO dugc xac dinh hoat tinh khdng ndm F. oxysporum vd R. solani tren dia PDA. Dich protein tinh s^ch cd boat tinh uc che m^nh su sinh trudng vd phdt triSn cua cd ndm F. oxysporum lln R. solani (Hinh 4).
Dinh chinh (PI) dupc thoi ra tii cgt Biogel PlOO cd ho^t tinh khang ndm vd tren di^n di SDS-PAGE cd khdi lupng phan tu khoang 21 kDa (Hinh 3B).
KET LUAN
Protein cd hoat tinh khang nam dugc tinh sach tir dich nudi cdy B. subtilis XL62 trong mdi trudng NYD, sau khi qua 3 budc tinh sach: tua mudi 30- 70% ammonium sulfate, sac ky trao d6i ion DEAE- cellulose vd sdc ky loc gel Biogel PlOO. Tren gel dien di 12,5% polyacrylamide, protein tinh s^ch cd khdi lupng phan tii - 2 1 kDa. Cac phan doan tinh sach cd boat tinh khdng ndm tuong ddi manh.
Protein khang nam nay cd khd nang itc ch£ m^nh su sinh trudng va phat trien ciia ndm F. oxysporum vd R. solani, dac biet Id ddi vdi chiing ndm R. solani.
Lei cam an: Cong trinh dupc hS trp td- Chtttrng Irinh trpng diem phdt trien vd ting dpng cong nghe smh hpc trong Imh vtrc nong nghiep vd phdt trien nong thdn den ndm 2020, de tdi: Nghien cwu cong nghe sdn xudt chi phdm BCF phong chong benh cdy trong do ndm Fusarium sp. va Rhizoctonia solani, 2009-2011.
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PURIFICATION OF AN ANTIFUNGAL PROTEIN FROM BACILLUS SUBTILIS STRAIN XL62
Do Thi Tuyen, Le Dinh Quyen, Nguyen Ngoc Dung, Quyen Dinh Thi*
Institute of Biotechnology
SUMMARY
Bacillus subtilis strains produce the antifiingal peptides such as iturin and bacillomycin. They have a cyclic structure of peptidolipid and potentially antifungal and hemolytic activity. The strain XL62 was isolated from a soil sample and identified as B. subtilis on the basis of its morphological, biochemical and physiological characteristics, and I6S rDNA analysis in a previous study. An antifungal protein was isolated from a culture of the strain B. subnhs XL62. The purification procedure compnsed of three steps, ammonium sulfate precipitation, ion exchange chromatography on diethylaminoethyl DEAE-cellulose and gel filtration chromatography on Biogel PlOO column. The purified protein showed a molecular mass of-21 kDa on SDS- PAGE and exhibited inhibitory activity on mycelial growth of Fusarium oxysporum and Rhizoctonia solani.
The purified antifiingal protein from B. subtilis XL62 exhibited inhibitory activity against both F oxysporum and R. Solani. The F. oxysporum strain showed the highest sensitivity to the antifimgal protein. Antifiingal activity was retained at temperatures up to SOX, even at 100°C, afrer 15 minutes incubation
Kqnvords: Antifiingal protein. Bacillus subtilis XL62. Fusarium oxysporum. purification. Rhizoctonia solani
' Author for correspondence: Tel: 84-4-37568260; Fax: 84-4-38363144; E-mail: quven<S),ibl.ac.vn
