TAP CHI SINH HQC, 2012, 34(4): 485-492 SANG L Q C VA NHAN DONG GEN MA HOA PECTATE LYASE
TU" Bacillus subtilis CO N G U 6 N G 6 C V I $ T NAM DS Thj Thu Hfing, V9 Hoii Bic*, Le V9n Trirdng Vi^n Cflng ngh§ sinh hpc, *[email protected]
T 6 M TAT: Pectate lyase (PL) Ii enzyme quan trpng trong b^nh thuc v§t do vi sinh v$t tiit ra PL phan huy polygalacturonate ciia thinh ti bao lh\rc vgt tgo ra cic oligogalacturonale. Trong bii bio niy chiing Idi cdng bo kit qui sing Ipc dupc 9 chiing Bacillus siibiilis ngu^n g6c Vift Nam cd khi n9ng sin xuat pectate lyase cao. Gii trj pH t6i uu phin ling plian hiiy popygalacluronate ciia cic enzyme niy tir 8,5-10, Gen mS hda pectate lyase (pel) ciia b6n chiing B. subiilis khic nhau di dupc cloning trong E. coli vi giai trinh t^
gen. Pectate lyase ciia 4 chimg vi kliuIn niy c6 420 amino acid (aa). Trinh t^ amino acid suy dien ciia cic enzyme nay c6 dd luong ddng tu 98,8-99,8% so vdi trinh tu amino acid ciia pectate lyase lir chiing B.
subtilis 168. Cd 10 vj tri amino acid bj thay d6i trong trinh t^r aa ciia 4 pectate lyase khi so sinh gitta chiing vdi nhau, Trinh tu aa ciia PL tit cic chiing phan i^p tir th\rc v^t bio thii hon cac chiing phin lap tit dit khi so sinh chiing vdi PL ciia B. subtilis 168.
Tu klioa: Bacillus subtilis. endopolygalacturonatc lyase, nhan ddng gen, pectate lyase, pectin acid.
MO DAU
Pectate lyase (PL) hay endopoly- galacturonatc lyase (EC 4.2.2.2) la enzyme quan trpng ttong benh Ui\rc vSt do vi sinh v§t gSy ra [2]. Enzyme nay phan cat nglu nhien lien kit a-1,4 glycosidic cua polygalacturonate ttong thanh tl bao th\rc vat thong qua phan img B-elimination tao ra cac oligogalacturonate co lien kit doi giita C4 va C5 a dSu ducmg khflng khir [2]. PL thucmg thay ttorig cac vi sinh vat gay benh thuc vgt nhu vi khuSn E. chrisanthemi [9], E. carotovo [10], B subiilis [14], nim mflc [8]. can day, PL cQng da duoc tim thiy trong yi khuin ira l?nh P. haloplanktis phan lap tir bien Nam cue [20]. Pectate lyase xiic tic phan img phan cdt CO chit pectin va pectin acid (polygalacturonate) a pH tfli uu ttong khoang 8- 10 [22]. PL CO ling dyng quan trpng trong cong nghifp det la logi bo pectin ttong vai bflng thfl, di thay thi chit kiim trong khau niu kiim (alkaline scouring), lam tSng chit lu?mg ciia vai bdng va giam thi€u fl nhilm moi ttuong do chat kiim gay ra [6.4, 15, 11,18].
Pectate lyase tir B. subtilis da dupc Nasser etaL(1990, 1993) [13, 14] nghien cim tir nhilng nam 90 cua thi ky tru6c, tuy nhiSn, hien nay chua CO cflng ttinh nao nghien cijru, giai ma trinh t\r gen cua gen ma hoa enzyme nSy tti cac chung B. subtilis phSn Up cr Vi?t Nam. Tron|
bai bSo nay, chung toi cong bfl nghien curu ve
sang Ipc cic chung B. subiilis sinh pectate lyase phSn ISp tir Vift Nam tir cac bp suu tSp chung gi6ng trong nude, kit qua nhan ddng va giai trinh tu cac gen n^y, dflng thcfi so sdnh trinh tir amino acid ciia chung voi cac gen ciing loai da bilt tten ngan hang gen de gop phin hilu bilt them vl tinh da dang cua enzyme nay.
V^T LI$U VA PHUONG PHAP NGHIEN ClTU Vgit lifu
Chimg vi sinh vgl, moi tnrang nudi cay Hai muoi chiing Bacillus subtilis nguon gdc Vift Nam tir bo suu tip chiing giflng cua ngan hang chiing giflng VTCC, D^i hpc Khoa hoc tu nhien, DHQG Ha Npi va Trung tam Cflng nghf sinh hpc Biolab dupc sii dung di sang Ipc enzyme pectinase. E. coli DH5a dupc sir dung lam chiing nhan dong gen. Moi ttucmg LB (1%
ttyptone, 0,5% yeast extract, 1% NaCI) dugc sir dyng lam mfli ttuong nuoi cay B. subtilis va E.
coli Khang sinh ampicilin dupc bfl sung vao moi trucmg nflng dfl 100 ng/ml klii cin thilt, Moi ttucmg LB 0,2% pectin ho$c moi trudng khoang chat Belistky [19] dupc sir dyng dl nuoi ciy B. sutilis cho muc dich thu PL, Primer, vector
Primer sir dyng trong thi nghifm c6 ttinh tu nhu sau; BSpelF: atcg aag cttatg aaa aaa gtg atg tta gc; BSpelR: atcg aag ctt tac tgc tga ctg tt.
485
Do Thi Thu Hang, Vo Hoai Bac. Le Van Truong Plasmid pJETl.2 sir dyng trong thf nghifm
nhfin dflng dupc mua tir hSng Fermcnias, Phuvng phAp
Sang IQC cdc chimg B. subtilis co hogt linh enzyme pectinase tren moi tnrdng thgch dia
Cac chung B. subtilis trong bO suu tjp chiing gi6ng dupc tt^ hda tren mfli iruimg LB th^cli dTa qua dem sau dd ciy vgch sang mfli truimg LB th^ch dia b6 sung co chit pectin 0,2%, nufli qua dem ttong tu Im 3 7 ^ . Cac dTa nufli ciy sau dfl dupc nhu$m v6i dung djch 0,5% Hecxadecyl ttimethyl amtnonium bromide (HTAB) (Sigma) de phdl hifn vflng thiiy phSn pectin nhu md td ciia Truong et al. (2001) [20]. Sau khi nhu^m vdi HTAB trong 1 h, c&c khuin l^c ciia cic chung sinh pectinase s€ xuit hifn v6ng thiiy phSn vdi CO chit pectin ttfn dia th^ch,
Thu nhgn enzyme pectinase ngagi bao Cic chimg B. subtilis dupc tre hda tren mfli ttudng LB long d 37''C, 200 vdng/phut qua dem, sau dd ciy chuyin 1% sang moi ttudng LB long ho$c mfli trudng khoing chat Belistky cd bo sung 0,2% co chit pectin di kich thich s^r san sinh pectinase. Sau 24h nuoi ciy d cimg dieu kifn, djch enzyme ngoai bao dupc thu bang ly tam 10.000 vdng/phut trong 10 phiit d 4''C df loai te bao, djch noi chira enzyme ngo^i bao dupc thu nh^n va bao quan d -20°C cho cic thi nghifm tiep theo.
Xac dinh hogl linh pectinase bdng phircmg phap dtf&ng khir
Ho^t tinh pectinase dupc x^c djnh bang phucmg phap dudng khir nhu mo la ciJa Bemfeld (1955) [1]. 50 fil djch enzyme ngo^i bao dupc bfl sung vao 450 ^I dung djch phan img chiia 0,2% pecdn tir citms (Sigma) trong dfm Tris 50 mM, NaCI 20 mM va CaClj 0,1 mM pH 10.
Phan ling dupc th\rc hifn d 42''C ttong 1 gid.
Phan irng enzyme dupc kit thiic bing vifc bd sung 500 pi 3,5 dinitrosalincilic acid (DNSA), dun soi 10 phiit, sau do dung djch phan img dupc lam ngupi din nhift dp phdng. Li tam 10.000 vdng/phut ttong 5 phiit ttudc khi do d budc sdng 530 nm tren may do quang phd. MOt don vj hoat tinh enzyme (unit) dupc dirih nghla la lupng enzyme pectinase phan cit ca chit pectin t^o ra 1 pM glucose trong 1 phiit.
Xdc djnh hogt tinh pectate lyase bdng phirang phdp phdl hi^n liin kit doi
Ho9t tfnh pectate lyase dupc xic djnh thong qua vifc phit hifn lifn kit dfli dupc t^o thanh sau phdn Cmg ciia enzyme vdi ca chit pectin acid. Phucmg phip dupc cai tiin tir phucmg phip cua Collmer (1988) [5]: 50 \i.\ djch enzyme ngo^i bao ISn men tir mfli trudng khoing dupc b6 sung vio 450 |il dung djch phan img chtia 0,2%
pectin acid 98% demethylation (Sigma) ttong dfm Tris 50 mM, NaCI 20 mM va CaCI: 0,1 mM pH 10. Phan img dupc thirc hifn d 42"C trong I gid. Phan img dupc kit thiic bang vifc bfl sung 500 ^I HCl 200 mM, Ii tSm 10.000 vdng/phut ttong 5 phut trudc khi do trfn may quang phfl d budc sdng 232 nm. D6\ chimg am dupc sir dyng bang nirdc cit thay cho djch enzyme.
Cdc phirang phap su d{ing trong thao tac vdi DNA
Phuang phip biln n^p plasmid vao E. coli, tach chilt plasmid tir te bio E. coll bing Ii giai vdi NaOH va SDS, tach chilt DNA genome tir Bacillus vi phucmg phap difn di DNA tten agarose theo md ta ciia Sambrook (1989) [16].
Phircmg phdp nhdn gen bdng PCR Gen pel dupc nhan len bing PCR sir d^ng Taq Polymerase ciia hing Fermentas. Chu ttinh phan img: Bien ti'rdi: 94°C trong 2 phut, bien tinh: 94''C ttong 30 giSy, gin mfli: 55°C ttong 30 giSy, keo dai: 72°C ttong 1,5 phiit, l?lp l?i 30 chu ky tir budc 2, keo dai: 72"'C ttong 10 phut, kit thiic: 4''C.
KtJ QUA VA THAO LU-jiN
sang Ipc cic chung B. subtilis san sinh pectinase
D I chpn dirpc cSc chimg B. subtilis mang gen pectinase, cac vi khuin B. subtilis dirpc mua tir bO suu t^p chiing gidng cua Trung t&ia Cong nghf sinh hpc Biolab va Bao tang gidng chuin Vift Nam (VTCC) dupc su dyng. Thdng tin cac chung Bacillus nay thi hifn trong bang 1. Cic chung vi khuin dupc tre hda sau dd cdy V9ch ttSn moi trudng th^ch dia 0,2% pectin nhu md ta ttong phin phirong phip. Sau khi nhu$m vdi HTAB, 17 chiing di xuat hifn vong thuy phan pectin tren dia th^ch ttong tdng sd 20
TAP CHI SINH HQC, 2012.34(4): 485-492 chimg vi khuan dupc su dyng, chi 3 chiing li
khflng cd vdng thiiy phSn (bang 1 vi hinh I).
Dilu nay chimg td hiu hit cic chiing B. subiilis ttong bO suu t§p nay diu cd kha nang sinh enzyme pectinase. Ket qua nay hoan toin phii
hpp vdi d$c diim cua loai vi khuin nay va phii hpp vdi cic nghien cim trudc diy vl vi khuin thuOc loii B. subtilis ciia Nasser et al. (1990), Nasser et al. (1993), Soriano et al. (2006) [13,14,17].
Bang 1 STT
1 2 3 4 5 6 7 8 9 10
. Ket qua sang loc ciic Ten chimg
VBSl VBS2 VBS3 VBS4 VBS5 VBS6 VBS7 VBS8 VBS9 VBSIO
NKUOII D9t 0,1t Dat B5C
Dac
DSt D.^t R 6 C 3 Y
D^(
Dit
chung B. subtilis cd kha nang phan huy pectin tren V6ns thCly phSn
+ + + + + + + + + + + + + +
STT II 12 13 14 15 16 17 18 19 20
Ten chiinE VBSll VBSl 2 VBSl 3 VBSl 4 VBSl 5 VBSl 6 VBS17 VBS18 VBS19 VBS20
Njtufin DSt DA(
Ronji Slln Dat Dit DSt Dat Dai Bk Dat
dTa th^ch V6nB thiiy phan
+ + + + + + + + + +
-
+ + T90 vdng thiiy phan vdi co chit pectin d mire dp binh thudng (+), miirc d<i manh (++) va khflng tj vong thuy phan (-).
Hinh I. Hinh anh mflt so chiing B subtilis tao vdng thiiy phan pectin tten dia thach LB 0,2% pectin. Thir tu cac Chung tir 1-10: VBS6 - VBSI5 Anh huorng cua pH den kha nSng ph3n huy pectin ciia pectinase tir cac chiing nghien cihi
Pectate lyase thuOc nhom enzyme pectinase phan huy pectin d pH tdi uu tir 8-10 [22], vi v(ty, cac enzyme pectinase hoat d^ng tdi uu d pH kiim nhilu kha nang sg la pectate lyase.
Trong thi nghiem nay, 9 ttong 17 chung vi khuin cd ho^t tinh m?uih vdi pectin tren dia th?ch dupc chpn dl nghiSn ciiu (bang 2).
cac vi khuin tren dupc nudi ciy tten mfli tnrdng LB Idng cd bfl sung 0,2% pectin dl kich thich sir tilt pectinase ngoai bio. Sau khi nudi ciy 24 gid d 37''C, enzyme ngo^i bao dupc thu
Ning d ; glucose (mM)
Hinh 2. Dfl thj dudng chuan glucose nh|n va sii dyng de lam phan irng vdi co chit pectin. Qk djnh lupng hoat tinh pectinase, phuang phap dudng khu dupc su dyng. Gia tri OD 530 nm thu duoc sau phan ling enzyme dupc chuyin ddi sang don vi unit dua theo dfl thi chuin glucose vdi phucmg trinh hfli qui y = 0,7267x - 0,0654, va r = 0,9981 (hinh 2).
Kit qua tten hinh 3 va bang 2 cho thiy, hai chiing VBS7 va VBSIO cd hoat tinh pectinase tfli uu lln lupt la pH 9,5-10 va pH 8,5-10 va 7 chung cdn l?i cd ho?it tinh pectinase tfli uu d pH 10. Nhu v§y, tit ca 9 chimg B. subtilis dupc lira chpn nuoi ciy trf n mdi trudng LB long bfl sung
Do Thi Thu Hang, Vo Hoai Bac. Le Van Truong 0.2% pectin diu cd kha nang sinh enzyme
pectinase kiim.
So sanh ho^t linh pectinase ciia cdc chiing nghifn ciiu d pH tdi uu cho thiy, hopt tfnh pectinase dupc xic djnli thip nhit la chiing VBS15 (17,5 unit), cao nhit la chung VBSll (38,9 unit) (hinh 3). Dilu nay cd thi giii llilch
ring, cic chiing B. subiilis tuy cimg loai nhung phin 19p tir cdc ngudn khac nhau cho nfn sS c6 s^
sai khic nhit djnh vl gen dSn din di lyc vdi pectin cCia cic enzyme nay sS khic nhau. M$t khic, khd nang bai tilt enzyme ciia cic chiing khic nhau thudng khdng gi6ng nhau, do dd, ho^t tinh pectinase tir djch ngo^i bao m^inh ylu khac nhau.
Hinh 3. Dfl thj anh hudng ciia pH tdi ho^t tinh pectinase ngo^i bio ciia cic chiing VBS6-VBS9 (a) va cac chimg VBS10-VBS15 (b) Bdng 2. Hoat tinh pectinase cua cac chiing nghien cim d pH tdi uu
sir 1 2 3 4 5 6 7 8 9
Ten chung VBS6 VBS7 VBS8 VBS9 VBSIO VBSll VBSl 3 VBSI4 VBSl 5
pH t6i uu 10 9,5 - 10
10 10 8,5- 10
10 10 10 10
Ho9t tinh peclmae (U) 24,7 22,3 22,5 36,5 25,5 38,9 30.1 20,1 17,5 Hoat tinh pectate lyase cua cac enzyme thu dupc
Pectate lyase phan cit ca chit pectin acid theo ca che chuyin lien kit B-elimination, san phim tao thanh mOt lien kit doi trong phan tir dudng galachironate. LiSn kit doi nay dl ding dupc phat hifn bang may do quang phfl d budc sdng 232 nm nhu mo ta ciia Macmillian et al.
(1966) [12]. D I khang djnh cac chimg Bacillus tren cd sinh PL hay khong, dich enzyme ngo^i bao
ciia cic chiing B. subtilis dupc nuoi ciy tren mfli tnrdng khoang chit Belisky diipc sir dyng de lam phan img enzyme vdi ca chit pectin acid 90%
demethylation (Sigma), la ca chSt dac trung cho nhdm enzyme PL. Sau khi kit thuc phan tog, ho^t tinh enzyme dugc dinh gid bang do tryc ti&) trSn may do quang phd d birdc sdng 232 run. Ket qua cho thiy, tat ca cdc chimg nghi&i ciru diu cho gii ttj OD 232 nm tii 0^4-0,71, khi cao so vdi nau
TAP CHI SINH HQC, 2012, 34(4): 485-492 ddi chtog am (0,005), Dilu nay chtog tfl enzyme
pectinase ciia cac chimg nay diu cd kha nang phan cat pectin acid t^o ra lien ket dfli trong san phim phan tog (bang 3), do dd, cd the kit l u ^ ring cac enzyme cua 9 chtog nghien ciru nay deu diu^c nhdm pectate lyase. Bdn chtog VBS6, VBS8, VBSll va VBSl3 cho ho^t tinli cao nhit (gid ttj ODtir0,51-0,7I)(bang3).
Bang 3. Hogt tinh pectate lyase ciia cac enzyme thu dupc
STT 1 2 3 4 5 6 7 8 9 10
Ten chting VBS6 VBS7 VBS8 VBS9 VBSIO VBSll VBS13 VBS14 VBSl 5 DC am
OD 232 nm 0,514 0,322 0,710 0,296 0,412 0,670 0,503 0,24 0,411 0,005
Hinh 4 San pham nhan gen pel tir cac chiing B. subtilis 1-4: VBS6, VBS8, VBSll \a VBS13; M. DNA marker. Bang DNA 1,5 kb dugrc chi bang mui ten, thang DNA chuan dH canh anh dien di.
Nhan ddng gen mi hda pectate lyase Nhdn gen pel lir cdc chiing B. subiilis bang PCR
Bdn chtog VBS6, VBS8, VBSll va VBS13 dupc lya chpn dl nhSn ddng cac genpel vao E. coii. DNA tdng sd cua cac chtog nay
dupc tich chilt va sir dyng lam khuon mlu cho phan tog nhSn gen, vdi c$p mdi d^c hifu BspelF vd BSpelR dupc thilt kl dya theo ttinh type/ ciia chung B. subtilis BS168 [14]. Sau khi kit thiic phan frng nhan gen, san phim PCR dupc kiem tra tren gel agarose 0,8%, trong dfm TAE IX. Kit qua tren difn di dfl cho thiy, ca 4 mlu DNA dupc sir dyng lam khufln mlu deu da khulch dgi m$t dogn gen khoang 1,5 kb diing nhu kich thudc ciia gen pectate lyase da dupc tinh toin tit trudc (hinh 4).
Cdc bang DNA 1,5 kb nay dupc cit ra, lam s^ich, gin vio vector pJETl .2 bing T4 ligase sau dd bien ng.p vao E coli DH5a. Cac khuin lac mpc dupc ttfn mfli trudng chgn Ipc dupc tdch chilt plasmid vl phin tich bing Xhol va Xbal dl kiim tta gen clien trong vector. Ket qua difn di san phim cit plasmid tten gel agarose cho thiy, hSu hit cdc mdu plasmid deu mang dogn gen chen khoang 1,5 kb (kit qua khong trinh bay) Dilu nay chtog tfl cac gen pel da dupc gin vao vector va nhan len ttong E. coli.
Giai trinh t^ gen pW
Bfln ddng plasmid mang fragment kich thudc 1,5 kb ciia 4 gen pel khac nhau tren dupc sir dung dl giai trinh ty gen tren may giai ttinh ty gen tu dpng. Kit qua thu dupc 4 ttmh tu nucleotide co dp dai nhu nhau, mS hda cho mpt protein 420 amino acid. Trinh tu nucleotide ciia 4 gen pel tir chtog VBS6, VBS8, VBSll va VBSl3 da dupc dang Uen Genbank vdi ma s6 theo thir ty la JX083068, JX083069, JX083070 va JX08307I. So sanh trinh tu amino acid (aa) suy difn ciia 4 gen trfn bang phan min so sdnh BLAST tten NCBI cho thiy chtog cd dO tuang ddng vdi cdc trinh tu aa ciia PL tir B subtilis 168 lln lupt la 98,8; 99,7; 99,0 va 99,5%. Dilu nay chtog tfl ring 4 gen pel dupe nhan ddng tir 4 chtog vi khuin VBS6, VBS8, VBSll va VBS13 diu la gen ma hda cho pectate lyase.
Sy da dging ciia cdc pectate lyase phan lap tu B. subtilis nguon goc Vift Nam
Vdi myc dich ddnh gid sy da dang ciia cac pectate lyase phan lap tir Vift Nam, trinh ty aa ciia PL tir B. subiilis 168 dupc sir dyng dl so sanh theo hang ciing vdi 4 PL nay. Phin mim so sanh cym ClustalW dupc sir dyng. Kfit qua cho thiy, cd 9 vj tti aa khac nhau khi so sanh 489
Do Thi Thu Hang. Vo Hoai Bac. Le Van Truong gifta 5 trinh ty aa ndy vdi nhau (hinh 5). Khi so
sdnh rieng rO trinh ty aa ciia 4 PL phfin l(lp vdi PL tir B. subtilis 168 cho thiy cdc PL ndy c6 sy klidc nhau nhdl djnh, cy ihi?' PL ciia VBS6 c6 5 aa (L-I62, 11-181. A-248, M-249, Q-294), VBSScfl 1 aa (Q-294), VBSll cd4aa(K-118, T-140, Q-294. P-356) vd VBS13 cd 2 aa (D-69, A-376) sai klidc so vdi PL ciia B. subtilis 168 (hinh 5). Dilu thii vj Id PL tir VBS6 vd VBSl 1 phdn l$p tir dit cd s6 lugng aa khdc bift (5 vd
4 aa) nhilu hon PL tir VBS8 (1 aa) phSn l§p tir rl cfly vd VBS 13 (2 aa) phfin l§p tir rong syn.
Nhu v$y, dudng nhu cdc chtog phdn l^p tir thyc v$t cd trinh ty aa ciia PL it thay dfli hon so vdi cdc chiing phdn l^p tir dit. B, subiilis 168 cung Id chCing c6 nguon g6c tir chtog B. subiilis ATCC 6051 dupc phdn l§p tir m^t logi cd vdo nam 1875 [21]. B. subtilis 168 Ik chtog dupc gdy dOt biln d| dudng tir chtog B. subiilis ATCC 6051 tryptophan [3, 7].
VBS13 Bs-iea VBSll
VBS13 BS-16a VBSll VBse VESG
VBSll VBS8 VBS6
NKKVMUVTALPlXlLTPAaAHAADLOHQTLQSNIXJWOAYSTOTTaOSKASSSNVYTVaNRN 6 0 MKKVMLATALFLOLTPAQANAADLOHQTLGSHDOWGAYSTOTTGOSKASSSNVYTVSNRN 60 NKKVMLATALFLaLTPAaANAADIXJHQTIX3SNIX3WOAY3TaTTOOSKASSSNVYTVSNRN 60 MKKVMLATALPl/)LTPAaANAADIX)HQTLaSNIX)W0AY3T0TT0aSKASSSNVYTVSNRN 60 MKKVMLATALFLOLTPA0ANAADLOHQTLG3NDOWOAY3TGTTQG3KASSSNVYTVSNRN 60
I 1 IK 1lYIKQTtDMNVDDNLKPtXJLNDYKDPEYDLDKYbKAYDPSTV ~ I 1 IK IIYIKGTlDMNVnDNLKPLOLNOYKDPBYDLDKYLXAYDPST»
JTPKIIYIKGTIDMNVDDNLKPLGLHDYKDPEYDLDKYLKAYDPSTI ITPKJIYlKQTIDMNVDDNLKPLGLNDYKDPEYDLDKYl.KAYDPS'n L.,V5A:,GI'flirn ri'KlIYIKOTIDMNVDDNLKPLGLNDYKDPEYDLDKYLKAYDPSTW]
KEPSGTOEEARARSQKNQKHRVMVDIPAMTTIVGSGTNAKVfcONFQriCSDMVriRNIi KEPSGTOEEARARSOKNOKBRVMVDIPANTTIVGSGTNAKVKGNPQlKSDHVriRl KEPSGTQEEARARSOKNQKnRVMVDIPAHTTrVGSGTNAKvfcGNFQrKSDKVIIRNIl KEPSGTQEEARARSOKNOKBRVMVDIPANTTrVGSGTNAKvfcGNFQIKSDNVlIRHI!
KEPSGTQEEARARS0KNQKJHVMVD:PAMTTIVGSGTNAKV§3GNFQIKSDKVIIRNIi VB313 DAYDYFPQWDPTDGSSGNWNSQYDNlI
B S - 1 6 8 DAYDYFPQWDPTDGSSGNWNSQYDNlB V B S i : DAYDYFPQWDPTDGSSGNWNSQYDNlI VBSa DAYDYFPQWQPTDGSSGNWNSQYDNIi VBSe DAYDYFPQWDPTDGSSGNWNSQYDNlI
biGGTHIWIDHCTFNDGSRPDSTSPKYYGRKY 2 4 0 WGGTHIWIDHCTPNDGSRPDSTSPKYYGHKY 2 4 0 pJGGTHIWIDHCTFNDGSRPDSTSPKYYGRICY 2 4 0 pjGGTHIWrDHCTFNDGSRPDSTSPKYYGRKY 2 4 0 J H G G T H I W I D H C T F N D G S R P D S T S P K Y Y G R K Y 2 4 0
VBS13 QHHDGQTDASNGANYITMSYNYYHDHDKSSIFGSSDSKTSDDCKLKITLHHNRYfclVQR 300 B S - 1 6 8 QHHDGQTDASNGANYITMSyNVYHDHDKSSIFGSSDSKTSDDGKLKITLHHNRYWlVQR 300 V B S l l QHHDGQTDASNGANylTHSyNYyHDHDKSSIFGSSDSKTSDDGKLKITLHHNRYmrVQR 3 0 0 VBSB QHHDGQTDASNGANyiTMSYNYYHDHDKSSIFGSSDSKTSDDGKLKITLHHNRYWIVQR 3 0 0 VBS6 QHKDGQTDASNGAI^ITHSyNyYHDHDK53IFG5SD3KTSDDGKLKITLHKNRYpiIV0R 3 0 0 VBS13 APRVRFGQVHVVN«YYEGSTS3SSYPFSYAWGrGKSSKIYAQNMVIDVPGLSAAlJlSVF 3 6 0 B S - 1 6 e APRVRFGQVHVYNNYYEGSTS3SSYPFSYAWGIGXSSKIYAQNNVIDVPGLSAAIMISVF 3 6 0 V B S l l APRVRFG0VHVYNNYYEGSTS3SSYPFSYAWGrGKSSKIYAQmJVIDVPGLSAAIMISVF 3 6 0
vase APRVRFGQVHVYNNYYEGSTSSSSYPFSYAWGIGKSSKIYAQKNVIDVPGLSAAKBISVF 360
VBS6 APRVRFGQVHVYNNYYEGSTSSSSYPFSyAWGrCKSSKIYAQMNVIDVPGLSAAKlISVF 3 6 0 VBSl 3 SGGTALYDSGTLLNGI
Bs-iee SGGTALYDSGTLLNGI VBSl 1 SGGTALYDSGTLLNGI VBS8 SGGTALYDSGTLLNGI VBS6 SGGTALYDSGTLLNGI
}1NA5AAN01.S53VGWTP5LKQSIDASANVKSNVINQAGAGKLK 420 } 1 N A S A A N G L S S S V G W T P S L H G S I D A S A N V K S N V I N Q A G A G K L N 420 JINASAANGLSSSVGWTPSLHGSIDASANVKSNVINQAGAGKLN 420 JINASAANGLSSSVGWTPSLHGSIDASANVKSNVINQAGAGKLN 420 JINASAANGLSSSVGWTPSLHGSIDASANVKSNVINQAGAGKLN 420
Hinh 5. So sdnh theo hang ttinh ty amino acid cua PL lir 4 chtog 5. jHi/i7« phdn l§pd ViftNam vdiPLtilr5. 5u6r//w 168.
Cdc amino acid khdng tuong dflng dupc ddnh diu bdng bdi den.
KET LU^N Da nhdn ddng vd gidi ma trinh ty gen nm hoa Da sang Ipc dupc 9 chtog B. subtilis sinh ^ " ^ ^ Pf,^*^*^ l^^^f, ''f" '^'T^A^v^^i pectmase ua kifm ngufln gdc Vift Nam. Cdc
chtog nay diu cd ho?t tinh enzyme pectate lyase.
VBS8, VBSll vd VBS13 cd ngufln gdc Vift Nam. Trinh ty aa ciia PL tir cdc chtog phSn I ^
490
TAP CHI SINH Hgc. 2012, 34(4): 485-492 tir thyc v$t bdo thii han cdc chtog phdn l$p tir ddt
khi so sdnh chtog vdi PL cua B. subiilis 168.
L&i cdm ffn: Cflng trinh dupc tdi trp kinh phi cua dl tdi nghifn ciiu khoa hpc cdng nghf tiem nang thuOc chuong ttinh "Nghien ciiu phdt triln va tog dyng cflng nghf sinh hpc" ciia B$ Klioa hpc vd Cflng nghf. Cdc thi nghifm dupc tiln hanh c6 sii dyng ttang thilt bj ciia phdng Thi nghifm ttpng diem ve cflng nghf gen, Vifn Cflng nghe sinh hpc.
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SCREENING AND CLONING OF PECTATE LYASE GENES FROM Bacillus subtilis ISOLATED IN VIETNAM
Do Thi Thu Hang, Vo Hoai Bac, Le Van Truong Institute of Biotechnology, VAST SUMMARY
Pectate lyase (PL) is an important enzyme in plant pathogenesis, PL is secreted by microorganisms. PL degrades polygalacturonate of plant cell wall product oligogalacturonates. In this rcpon, we described the screening of nine strains of 5. subtilis. which isolated from Vietnam with ability to produce pectate lyase. The optimal pH for enzymatic degradation of polygalacturonate was from 8.5 to 10. The genes encoding pectate lyase from 4 different strains were cloned in E. coli and sequenced, Pectate lyase of these 4 strams contains 420 amino acid (aa). The deduced amino acid sequence of these PLs showed 98,8-99.8% identity to PL from B. subtilis 168. There are 9 aa positions were changed in the amino acid sequencing of 4 trains in comparison between them and B. subtilis 168. The amino acid sequences of PLs of strains from plant were more conserve than those of Pl-s of strains from soil in comparison with PL from fi. subtilis 168.
Keywords: Bacillus subtilis, cloning, endopolygalacturonatc lyase, peetate lyase, pectin acid.
Ngay nhdn bdi: 10-4-2012
