Biểu hiện endoglucanase a của clostridium thermocellum trong vi khuẩn bacillus subtilis

TAP CHI PHAT BHA AWN, TAP 17, SO 14-2014

Bieu hien endoglucanase A dm
Clostridium thermocellum trong vi
khugn Bacillus subtilis
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•
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Pham Luang Thing
Phan Thi Phuvng Trang
Trin Linh ThLrevc
Nguyin DCrc Hoang
Trireng Dai hoc Khoa hoc To, nhien, DHQG-HCM
Bai &Win ngay 24 thOng 09 nom 2014, nikin clang ngthy 26 thling 01 nom 2015)

TOM TAT
PCR va cher) vao plasmid pHT43, ngay sau
Endoglucanase A (CeIA) Le thanh phan
trinh ty, tin hieu tiet samyQ. Plasmid pHT43quan trying trong phar he enzyme cellulase
celA too thanh se &Foy bien nap vac) cheing
chiu nhiet (cellulosome) tir chang vi sinh vat
bleu hien B. subtilis 1012, c6 bleu hien
ky khi 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 8 protease ngoai bao.
nhet cac lien ket (3-1,4 glucoside ter ben
CeIA ter cac chOng B. subtilis cluvr cam Cmg

trong cac phan ta, cellulose va mOt so lopi
bieu
hien bang IPTG a cac wing do khac
polysaccharide to thanh cac oligosec
nhau. Tien hanh thu mau dich tiet protein
charide. Bacillus subtilis là vi khuan Gram
ngopi. bao trong mOi twang nu6i PA), ter 2
throng, hieu khi, phat trien nhanh, thuying
den 24 gia sau cam Crng. Kha nang bleu hien
dOng trong 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 nay chirng to co the bleu hien CeIA
Men cham, C. thermocellum de bleu hien
trong 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 nay.
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
khi tuyet d6i duoc biet den vai he cellulase cho
kha nang phan giai cellulose hieu qua nhat.
Chung vi sinh vat nay co kha nang tao phirc he
enzyme chiu nhiet va dugc goi la cellulosome
ngoai bao. Phirc he nay bao g6m: endoglucanase,
c6 kha nang phan cat lien ket p-1,4-glycosidic
trong 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 nhau vim tang caerng
kha nang gan cilia enzyme len co chat, thong qua
cac domain ai 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
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Science & Technology Development, Vol 17, No.T4-2014

CeIA rat don gian, chi bao gem I domain
Dockerin 1 (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 duy nhat cim nh6m GH8 tren C.
thermocellum [3] va &roc bieu hien cao 6 ca 2
cap dO phien ma [10] va dich ma [13].
Bacillus la nhom vi sinh vat gift vai tro chti
dao trong nen ding nghiep sin xuat enzyme veri
hon 60 % enzyme cong nghiep &rot sin xuat tir
nh6m vi sinh vat nay [12]. Trong d6, B. subtilis
da dirge irng dung trong cong nghiep tir rat lau tai
Nhat Ban trong 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 ra 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 con co the Ong dung trong nuoi trong
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 khi 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

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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 do 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
trong binh nuoi ky, khi bang m8i throng 122, theo
huang dan cua ngan hang cluing DSMZ, a 60 °C
trong 4 ngay. Thu sinh kh& va tach chiet DNA
be gen theo quy trinh phenol/chloroform [5], do
neng dq va kiem tra de) tinh sach bing may
NanoDrop 1000.
Plasmid pHT43 (Hinh 1A) la meit plasmid
con thoi, co kha nang ton tai va sao chep dec lap
Men ca 2 chimg chi E. colt va B. subtilis. Plasmid
nay 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
ngay sau Pgrac promoter, cho phep tiet protein
tai to hop ra 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, 5 '-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 1. 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 khi giai trinh to duoc bien nap
vio B. subtilis theo quy trinh huang clan dm
MoBiTec. Khan lac B. subtilis moc duqc sau
khi 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
nhau tir 0,01 mM, 0,1 mM, 0,5 mM den 1 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 trong mei
truong long 100 niL LB-Cm 10 pg/mL ciing-Cs
cac ding de IPTG twang to nhu khi khdo sit tren

died diem khao sit, thu nhan 2 mL
dia.

dich nuoi cay da loai to bao bang ly tam a 10.000
vong trong 5 phut. De chuen bi mkt cho phan
tich SDS-PAGE, 1 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 1 % CMC, u 50 trong
30 phut. Sau do, dinh Wong diking khir tao thanh
bang thuec thO DNS [4]. Vat 1 IU hog tinh
enzyme la luting enzyme can thiet de tao ra
hrong throng khir Wang duong vat 1 pmol
glucose (0,18 mg glucose) trong 1 phin.
KET QUA VA THAO LUAN
Thu nhan gen to plasmid tai to hop pHT43ceL4
Ket qua do NanoDrop DNA be gen dm C.
thermocelhtm cho thay neng de DNA nam trong
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 trong 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
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Science & Technology Development, Vol 17, FIo.T4-2014

tren nein hang gen NCBI cling la 100 % khi 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 1 mM. Chi CO sir
khac biet re) ret ve kha nang phan giai CMC a nOng
dO 0,01 mM so voi 3 Ming de IPTG con lai. Nhu
vay, cac chimg B. subtilis 1012 va B. subtilis
WB800N mang plasmid tai CO hop pHT43-ce/A tao
thanh 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 6 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 khi do,
khan lac clOi chimg, khong xuAt hien vach
sang nao ten ban gel agarose 1 %. Dieu nay cho
thAy chimg t8i da tao thanh 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 1
% [2]. Ket qua thu nhan duoc trong Hinh 3 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 4 nOng
do IPTG 0,01 mM, 0,1 mM, 0,5 mM va 1 mM. Ket
qua phan tich ban kinh Wing phan giai con cho thAy
sir anh InrOng cua IPTG khong nhieu a cac ming dO
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0.1

0.5

I

onfoA !PT('

0.01

0.1

0.5

1

mM IPTG

pHT43-cr/A
pHT43
B. subilthsW13800N

lllnh 2. Ket qua dien di san phArn PCR kiem tra sir
hien dien cua tai to hop pHT43- celA 6 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 1 %.

0.01

pHT43-celA

pHT43

cm
Ilnh 3. Kha nang phan giai CMC cua chung bieu
hien B. subtilis 1012 va B. subtilis WB800N mang
plasmid tai to hop pHT43-celA 6 cac ming de IPTG
la 0,01 mM, 0,1 mM, 0,5 mM va 1 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 do co
kich thu6c la 1 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 5 L). d day, chimg tor sir dung
2 phuong phap de danh gia kha nang bieu hien la
SDS-PAGE va do hoat tinh dich nuoi city.
Xac nhein su bieu hien CelA tilt to B. subtilis
1012 va WB800N
Tnrac khi 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 trong djch nuoi
cay B. subtilis tai thin diem 8 gi6 sau khi cam ling
bing 0,1 mM MTG.

TAP Cal HAT TRIER AWN, TAP 17, SO 14-2014

2 M 3

4
lllnh 4. ket qua icac dinh sv bieu hien CeIA 6 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 2 domain chinh la loi hoat tinh (kich thuerc khoang 43 lcDa) va Dockerin
nhom I (kich dm& khoang 7 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 trong Hinh 4A cho they CeIA tao
thanh 6 B. subtilis 1012 co kich thtroo khoang 43
kDa trong khi 6 dich tiet B. subtilis WB800N,
CeIA van gift dirge 2 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 nay 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 1 (kich thy& khoang 7 kDa) lien ket v&i loi
hoat tinh enzyme (43 kDa). Tuy nhien, 2 thanh
phan nay lai gap cuen rieng re va doc lap yeti
nhau [1]. Do 86, protease ngoai bao se cat phan
Dockerin loai I tnroc, trong khi lai enzyme \Teri
cau true phirc tap va chic chin hon se it bi anh
huemg b6i hoat tinh cila protease. KM qua nay
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 là bieu hien nhieu
loai,protease ngoai bao, do d6, san pham lai dich
tiet Ce1A chi cho 1 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 8 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 nay hoan town
phis hop vai ket qua lai veri 2 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 2 vach nhel co kich thuerc
khoang 50 kDa va 43 kDa 6 cac chimg B. subtilis
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Science & Technology Development, Vol 17, No.T4-2014

1012 mang plasmid pHT43-ceiA, trong 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 thanh tong tren
chung B. subtilis 1012. Mac du sin pi-tam tiet bj
protease phan cat met phAn 6 cac thai diem 4, 6,
8 va 10 gia sau cam ung, tuy nhien, a cac thai
diem 12, 18 va 24 gia, van co met luvng CeIA
mm tao thanh va chua bi phan cat boi protease
dm B. subtilis 1012. Ket qua nay hoan toan phis
hap vai ket qua lai Western Blot trirerc do — Hinh
4. DOng then, khi doi chieu ket qua do 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 trong khoing 120 den 140
mU/m1 djch nuoi cey. Dieu nay cho thay ring lei
boat tinh enzyme van con 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 do hoat tinh a thai diem
cam img IPTG (0 gib) cho thay lugng duang khir

tao thanh 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 5. 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 do.

-CelA

50

-CeIA

D
• 0.1 mM IPTG
■0.5tuM !PTO
■ I mM IPTG

6

Trang 78

12
8
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
thanh a kich thuOc la 50 kDa. Kich thirac nay
tuang Ong v&i vach xuat hien 6 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
2 den 24gia sau cam img a neng de IPTG 1 mM
(Hinh 6B). Trong khi do, 6 cac mau chimg am
(cherig mang plasmid pHT43 va thing mang
plasmid pHT43-cell kh8ng duoc cam /mg (0
mM — Hinh 6B) thl vach nay khong xuat
hien.Nhu viy, thing toi ket luan day la vach
protein tai to hop CelA bieu hien vuqt men do co
so cam img bad IPTG. Luang Ce1A tao thanh
tang dein qua cac gia nuoi cay va dat tei da trong
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 thanh 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 1 mM, Itiong Ce1A tao thanh la Wang
throng nhau trong khoing thai gin tir 8 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 0 gia cho ket qua tao
&Tang khir la rat thip, khong the phat hien &roc
khi dinh lueng kii bang thuec thir DNS. Nhu
viy, co the thay rang boat tinh phan giii CMC
cia dich tiet B. subtilis la do so bieu hien dm
vector tai to hop pHT43-ce/A.

B. subtilis WB800N
paw

▪

A

B

C

b 6. 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) 1 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 do
la mU/mL dich tiet moi
truang da loai to bit°, vai
1 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
0

• I niM IPTG

10 ,

1.2,

Thni gmn thu man quo)

18

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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 thanh 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 trong vice bieu
hien enzyme cellulase tai to hqp, phuc vu cho

viec thin' phan nguon co chat nhu ram ra it Viet
Nam.
1D - tat &roc thtrc hien nlurr str hj tro kith phi cna Sd

Khoo hoc ra 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 2 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.

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TAP CHI PHAT TRIER KWH, TAP 17, SO T4-2014

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