Tách dòng và biểu hiện gen mã hóa endo 1,4 β glucanase b của clostridium thermocellum trong bacillus subtilis

Tap chi Cong nghe Sinh hpc 13(3): 965-971, 2015

TACH De:ONG VA BIEU HIEN GEN MA Holt ENDO-1,44-GLUCANASE B CiJA
CLOSTRIDIUM THERMOCELLUM TRONG BACILLUS SUBTILIS
Pham Luang Thing', Pham Thi My Tien', Trim Quie Twin', Nguyen Le TuAn Anh', Nguyen Mang
NgQc Phtrong', Phan Thi Phirong Trang', Nguyen Thi Nguyen2, Nguyen Dinh Quan2, Phan Dinh Thin',
TrAn Linh
Nguyen Dire lining'
i Trucing
2Trtrang

Ogi hpc Khoa hpc to nhien. Dai hpc Qu'dc gia Thanh phd Hd Chi Minh
Dai hpc Bach khoa, Dai hpc Qudc gia Thank pito Hd Chi Minh

Ngay nhan bai: 26.11.2014
Ngay nh4n dang: 12.9.2015

TOM TAT
Viet Nam la met trong nhan quite gia doi dito v'e ngulin phe phttm ming nghiep yeti han 40 trieu tan ram
ra m6i nrun. De tan dung het Ton ca chat nay, cong nghiep san xuAt ethanol sinh hoc ra dei va mang Iai tiem
nang to 16n, g6p phan giai quyet van
killing hoing.nang
Trong Q6, cac enzyme cellulase d6ng vai tr6
quan trong trong viec duly phan cellulose too ca chat cho qua trinh len men, san xuat ethanol. Xuat phat lir
thrc to Q6, chting toi ti8n hanh bieu hien Endo-1,4-8-glucanase B tai to hqp tit chung ky khi Clostridium
thermocellum trong chung c6ng nghiep Bacillus subtilis. Diu lien, chOng Ceti thu nhan gen ce/B ma h6a cho
enzyme Endo-1,4-8-glucanase B ti} Clostridium thermocellum DSMI313. Sau de, churl* toi dOng h6a plasmid
tai 18 hqp pHT43-ce/B va pHT1219-celB, kiem tra cac don mang plasmid tai to hop bang phtrcmg phap PCR
khuan lac va giii trinh qt. Cacplasmid tat to hqp duqc bier' nap vat) B. subtilis 1012 va kiem tra kha nang bleu
hien tren m8i twang long va ran. Ket qua cho thay, 6 nong d8 chat cam Ong IPTG la o,5 mM, chitty B. subtilis
tai to hqp cho wing phan giai khi nufai cay ten mei trtrang thach china ca chat CMC. Dong thai, ket qua SDSPAGE va do hoot tinh cho thity chung bieu hien c6 kha nang tiet CelB c6 hoot tinh khoang 80 U/ml khi nuai

ay ten m6i trtgmg long.

a

Tir khda: Bacillus subtilis. Endo-1,4-6-glucanase B, Clostridium thermocellum, cellulose. plasmid pHT

MO DAU
Qua trinh san xuAt ethanol sinh hoc thong
thuang sO dung enzyme tin cac chting vi sinh vat de
plan cat tinh 138t thanh glucose rOi len men tao
ethanol. Tuy nhien, khi yin de thieu but luang thuc
dang la not lo tan cAu nhu. hien nay thi can phai
thay dOi chin luqc, chuyen &I sang sir dung
nguOn ca chAt doi clio han la cellulose nham thay
the cho tinh bet. M6t trong nhung nguOn cellulose
doi dao, gid re, sin có tai Vi't Nam chinh là rum ra.
Voi han 40 trieu tan ram ra mai nam, viec sir dung
co chat nay trong san xuat ethanol sinh hoc se la
mut the manh dm Viet Nam, d6ng thai g6p phan
giam thieu 0 nhiem moi tnr6ng, giam chi phi xir ly
chit thai nong nghiep cho !tong dan (Dang Tuyet
Phuany, 20101. Mac do hien nay ce rat nhieu nguon
vi khuan tai to hqp hay enzyme cellulase tai to hqp
duqc sO dung de thity phAn cellulose nhung chua
duqc nghien cOu situ rung. Rieng 6 Viet Nam, cac
nghien ciru nay hien nay van con b6 ngo NIA chua c6
nhieu thinh tuu d ang ke. Xuat phat tO tinh hinh

thur to neu tren, nh6m chung toi de xuAt huOng
nghien cfru too hen hqp enzyme cellulase tai to hqp

c6 kha nang thity phan cellulose. Trong do, sir dung
(long thai cac enzyme thuOc nham (i) endo-1,4-13D-glucanase phAn cat sqi cellulose tin ben trong too
cac sqi cellulose nh6 ham, (ii) exoglucanase phan
cat cac sqi nhe cellulose tin ben ngoai tao san phim
IA cellobiose va (iii) f3-glucosidase too glucose tin
cellobiose. Qua da, g6p phan dua ra mgt giai phap
kha thi cho yin de nang luqng tai sinh va xir phe
phu pham nong nghiep.
Clostridium thermocellum la cluing vi sinh vat ky
khi, ua nhiet, da duqc biet den tin rat lau bai kha nang
san xuAt enzyme cellulase ben nhiet, ce hoot tinh rieng
cao tren co chit cellulose (Demain et al.. 2005). Tuy
nhien, viec nuoi cAy chimg vi sinh vat nay tren quy mit
c8ng nghiep de san xuAt cellulase la nit kh6 khan, trong
khi, tiem nang san xuAt loi chua c6 the kiem chOng
duqc. Do do, viec san xuAt cellulase tai to hqp tO C.
thermocellum se la giai phap hoin hao cho nen san xuAt
enzyme c8ng nghiep trong nuOc.
965


Pharr Lucmg Thing et al.
Hien nay, ben canh Escherichia coli thu6ng
duqc sir dung trong sin xuat protein di to hqp, vi
khuin Bacillus subtilis ding Bang duqc quan tam rat
nhieu nhor sr bieu hien tiet hieu qua cac protein muc
tieu trong cong nghiep va d Ong Op ding lee trong
nong nghiep, y duqc va cong nghe thuc pham
(Schumann, 2007). Endo-1,413-D-glucanase B hay
Endoglucanase B (Ce1B) la met trong nhcmg enzyme

duqc nghien ciru tir rat lau 6 C. thermocellum va c6
tient rang bieu hien cao i ca cap de phial ma va
dich ma (Riederer et al., 2011). Nghien dm nay
nham khfio sat sr bleu hien dm celB trong B. subtilis
sir dung he thong bieu hien pHT,,dtra tren promoter
Pgrac. Ce1B vai kha rang phan cat lien ket nei phan
tir f3-1,4-glycosidic ben trong sqi cellulose, se la met
trong nhitng thinh phan endo-1,4-13-D-glucanase dau
tien, d6ng vai tre quan tong trong phirc he cellulase
dung cho cac phan img thity phan sinh khoi ram ra.
VAT LIEU VA PHUONG PHAP
Vet lieu
Chimg vi sinh vat ky. khi C. thermocellum DSM
1313 tir ngan hang chimg DSMZ, CHLB Dirc duqc
sir dung lam nguon gen de thu nhan celB. Chung E.
coli OmniMAX {F' [proAB+lacrlacZAM15
Tn10(TetR) L(ccdAB)] mcrA A(mrr-hsdRMSmcrBC) 980(/acZ)AM15 A( lacZY A-argF) U169
endAl recAl supE44 thi-1 gyrA96 relAl tonA
panD} duqc sir dung lam cluing chi de dOng h6a va
luu gift cac plasmid. Chimg B. subtilis 1012 (leuA8
metB5 trpC2 hsdRMI) (Saito et al., 1979) duqc dimg
lam chimg chit khao sat kha rang bieu, hien protein
muc tieu tiet ra ngoai moi truing nuoi cay.
Cfic plasmid cua he thOng pHT nhu pHT43 va
pHT1219 chira promoter Pgrac (Nguyen et al.,
2007), day la cac plasmid con thoi cho phep to
(long, nhan ban trong E. coli vii kieu hinh ch9n 19c
la kiting ampicillin va c6 the duqc tao dOng vao B.
subtilis vii kieu hinh chon 19c kiting
chloramphenicol. Ca hai plasmid nay deu mang

promoter nhan too Pgrac, c6 trinh at tin hieu tiet dm
a-amylase là SamyQ gifip tiet protein ngoai lai ra
mei truing nuoi cay. Khfic nhau chinh cua hai
plasmid nay la codon cua trinh at tin hieu tiet trong
pHT1219 duqc thay doi nham lam giam sit bieu hien
trong E. coli, trong khi trinh to tin hieu tiet i pHT43
91 gen amyQ cita Bacillus amyloliquefaciens. Ngoii
ra, pHT1219 con chira trinh at ma h6a cho Strep-tag
sau vOng MCS. Hai plasmid nay duqc sir dung lam
966

khung su6n d e tao plasmid pHT43-celB va
pHT1219-ce/B.
Tao plasmid tai to hop pHT43-ce/B va pHT1219celB
Dau tien, chimg toi tien hanh thu nhan gen celB
(1) bang phucmg phap PCR vii cap m6i chuyen biet
5`ON917,
AGCATCAGCAGGATCCGAAGGGTCATATGCT
celB AatII R,
5'GATTTGG-3'
va
CTCA
GGCCATGACGTCTTATTTATkGGEAA
CTTATGC-3' vii kith think du doin 1639 by nham
dung hqp vii trinh to tin hieu tiet c6 vi tri cat TSAVGS trong plasmid pHT43. Gen celB (2) cling duq‘c
thu nhan bang phucmg phip PCR vii cap moi
ON918,
5`va
ON917
GGATGGCTCCAAGCGCTTTTATACGGCAACT

CACTTATGCTACGC-3`, kith think gen 1641 by
nham dung h9p vii trinh to tin hieu tiet co vi tri cat
ASA-GS trong pHT1219. Mach khuon cho cac OIL
img PCR la DNA be gen dm C. thermocellum. San
pham PCR duqc tinh such, sau de duqc cat va not
vao cac plasmid pHT43 va pHT1219 tai cac vi tri
BamHI NIA Awn nham tao thanh cac plasmid pHT43celB va pHT1219-celB.
Cac sin phim not duqc Wen nap vao chimg E. coli
OmniMAX. Cac clang khuan lac c6 mang plasmid
pHT43-ce/B va pHT1219-celB duac digit bang PCR
khan lac vii cap moi tucmg img là ON1209, 5'GGCCTAGATCTATGATTCAAAAAGCGAAAGC
GG -3' va ON918 (sin pham khuech dai là 1738 bp,
doi vii plasmid pHT43-ce/B) va ON707, 5`AAAGGAGGAAGGATCAATGAGAGGAAGCA-3'
va ON918 (sin pham Ichuech dai là 1743 bp, dOi vii
plasmid pHT1219-ce/B). Cac plasmid du tuyen duqc
tach bang QIAprep spin miniprep kit (QIAGEN) va
kiem tra bang phuang phap giii trinh tit.
Tao thing B. subtilis vi khi o sit sy bieu hien aim
CeIB teen dia
Plasmid sau khi giii trinh to dtrqc bitn nap vao
B. subtilis 1012 bang phucmg phi') di en bien nap.
Khan lac B. subtilis m9c duqc sau khi bien nap se
duqc khao sat kha !tang bieu hien CeIB tren dia
thach LB-Cm 10 (Luria Bertani - Chloramphenicol
10 pg/m1) ce chira 0,5% carboxymethylcellulose
(CMC) va chit cam (mg IPTG. Sau 24 gia nuoi cay,
kha 'tang bieu hien cua chimg se duqc danh gia
th8ng qua ban kinh veing phan giai CMC.
Dinh gii khi tang ban hien tiet CeIB dm B.
subtilis 1012

Top chi Cong nghe Sinh hoc 13(3): 965-971, 2015
Chung toi tien hanh bleu hien trong moi tru6ng
long 100 ml LB-Cm 10 pg/ml ding a cfic nOng de
IPTG tucmg tu nhu khi khdo sat tren dia. 0 moi thai
diem khao sat, thu nhan 2 ml djch nuoi cay di loai to
bao . bang ly tam 6 10.000 yang trong 5 phut. e
chuan bj mau cho phan tich SDS-PAGE, 1 ml djch
enzyme duqc tua vai TCA (Trichloroacetic acid) a
ruing de cuiii la 5% (Rajalingam et al., 2009). Song
song do, chimg toi xfic d jnh hoot tinh CMCase ctia
enzyme Ce1B theo quy trinh sau: 250 pl djch enzyme
Arai 250 pl Natri Acetat buffer, pH 5,5 co chua
0,5% CMC, u 55°C trong 30 phut. Sau d6, d jnh
luring du 6ng khir tao thanh bing thuOc thir DNS
(Eveleigh et al., 2009). Val 1 U hoot tinh enzyme la
luqng enzyme can thiet de tao ra luqng du6ng khir
tuang duang yeti 1 nmol glucose (0,18 pg glucose)
trong 1 phut.
Xfc nh$n gr ban hien CeIB bing lai Western blot
Djch nu& cay B. subtilis 1012 mang plasmid
pHT43-ce/B va pHT1219-ce/B a thai diem 14 gib
sau cam ling yeti 0.5 mM IPTG duqc thu nhan va
kiem tra Western blot vai khang the khang CelB dac
hieu 6 de pha Lang 1/10.000. Khang the thir cap
khang IgG the dung hqp horseradish
duqc sir citing, vach lai duce phit mau ban
bang
g ca chat

3,3',5,5'-Tetramethylbenzidine.
KET QUA VA THAO LUAN
Thu nh4n gen celB tir DNA 136 gen cila C.
thermocellum
A

DNA be gen dm C. thermocellum tach chiet
duqc co nOng de 480 ng/pl, vai di) tinh sach duqc
xac d jnh th8ng qua gia trj A260/A280 la 1,85.
Nhu v#3,, co the kit luan rang DNA be gen hoan
toan d am bao cho viec thu nhan gen trine tieu
celB. Gen celB, duqc khuech d oi tir be gen C.
thermocellum bang phan ung PCR bang cap moi
dac hieu ON917 va
vai kich thuerc
gen la 1639 bp. Sin pham PCR dirge phan tich
tren gel agarose 1% (Hinh IA) cho thay co 1 vach
DNA vai kich um& nam trong khoang 1.500 by
den 2.000 by dm thang chuin DNA. San pham
PCR sau khi tinh sach duqc xir ly vai enzyme cat
giai han la BamHI va Aatll va tinh sach !An hal
bang PCR purification kit (Qiagen, D ire). Trong
met chien thuat khac, vai plasmid gtic la pHT1219
va chien luqc ding h6a In-fusion (Clontech, Mg),
gen celB duqc thu nhan bang cap moi ON917 va
ON918. Ket qua dien di agarose (Hinh I B) cho
thay sin pham PCR cho vach duy nhat cao han vj
tri 1.500 by ciia thang chuan DNA, tuang ung vai
kich thuerc 1g thuyet la 1.641 bp. Nhu vay, chimg
toi da thu nhan thanh ding gen celB tit DNA be

gen dm C. thermocellum, san sing cho viec tao
plasmid pHT43-ce/B va plasmid pHT1219-ce1B.
Mon gen celB thu nhan duce chi chits phan ma
h6a cho lii hoot tinh va dockerin domain: Phan
gen ma hoa cho do an trinh ter tin hieu tiet, bao
g6m
27
a.a d
Au
tien
MKKFLVLL1ALIMIATLLVVPGVQTSA dtrqc
loci 136 vi chung toi mut% sir ding phlin trinh ti1r
tiet ciia plasmid khung soon pHT43 va pHT1219.
B

7000
4000
2500
1500
1000
700
400

by M I
7000
4000
2500
1500
1000
700

400

Hinh 1. Sin pham thu gen celB bang phan Ong PCR tit DNA tx) gen C. thennocellum. (A) thu gen bang cap m6i ON917 vi
celB_AatIl_R (sin pham 1.639 bp) cho &mg boa plasmid pHT43-celB. (B) thu gen bang cap moi ON917 vi ON918 (sin
pham 1.641 bp) cho ding hie plasmid pHT1219-celB. Sin pham PCR duvc chay tren gel agarose 1%, Gieng 1: sin pham
PCR thu gen celB tir DNA b8 gen C. thermocellum, M: thang DNA ZipRuler Express 2, Thermo Scientific, M9).

Kam tra plasmic! tai t4 tiny tao thanh
Cac plasmid khung soon pHT43 vi pHT1219 sau
khi duqc xir bang enzyme cat gieri han BamHI va Aatll

duce tinh sach vi ch thAn bj cho cac phim,img tao plasmid
tai to hqp. Ching toi tao plasmid tai to hqp th8ng qua
phucmg phip not bang DNA T4 Ligase vi In-fusion vai
chien luqc dirge trinh bay trong bang 1. Tien hanh sang
967


Pham Luang ThAng et al.
loc clang bien nap bang phucrng phap PCR khuAn lac,
san pham dirge phan tich tren gel agarosc 1% va so
sanh kich thuerc veri thang chuan DNA (Hinh 2).
Cac plasmid tiem nang dirge tach chiet va tie!)

tic sir dung phucmg phip giii trinh tit de xac nhan.
Ket qua dat. duce la sirtucrng (long 100% giira trinh
tu IS1 thuyet veri trinh to nhan du qc tir cone
Macrogen (Han Quoc) khi phan tich bang phan mem
Clone Manager 9.0.

Bang 1. Chian Ittqc dong hda tao plasmid pHT43-celB via pHT1219-ce/B.
Ten gen

celB (1)
celB (2)

Kfch
thteerc

Plasmid
khung
su*n

Chien
thuat to
&mg

Plasmid san M61 PCR
phAm
khuen loc

celB_Aall I_R

1639 by

pHT43

T4 DNA
Ligase

pHT43-ce/B

ON917
ON918

1641 by

pHT1219

In-fusion

M61 PCR thu gen
ON917

pHT1219-

celB

Kich
thutoc

ON1209
ON918

1738 by

ON707
ON918

1743 by

7110

ww

1743

— ID

IMP

ins

7911

A
B
Hinh 2. Kat qua PCR khan lac cac plasmid pHT43-ce/A (Hinh A) va pHT1219-ce/B (Hinh B). Cac gieng 1, 2, 3, 4 va 5 la
cac khan lac dy, tuygn, M la thang DNA ZipRuler Express 2, Thermo Scientific, M9).

Khao sat kha nang bieu hien ctia celB trong mai
trtrirng ran
Khuan lac dun B. subtilis mang plasmid pHT43celB va pHT1219-ce/B sau khi sang 16c duce cham
sang dia LB-Agar-Cm, b6 sung 0,5% CMC va IPTG
veti n6ng dO 0,5 mM u 24 gi6 a 37°C. Kha nang bleu
hien cira CeIB dirge danh gia thong qua ban kinh
wing phan giai co, chat trong mai tnremg. Ket qua

khao sat dirge trinh bay trong hinh 3.
Quan sat ket qui kha nang bleu hien cellulase B

(Hinh 3) to thay ca hai chimg B. subtilis mans
plasmid pHT43-ce/B va pHT1219-celB deu xuat
hien vong phan giai va Ian hun so veri mau khung
dirge cam irng bang IPTG. Dieu nay chirng to hai
chimg B. subtilis mang plasmid pHT43-ce/B va
pHT1219-celB deu c6 kha nang Neu hien tiet EndoI ,4-B-glucanase B ra mei tnremg nuoi ch.
IPTG

pHT43-ce/B

pHT1219-celB

Hinh 3. Kha nang phan giai CMC cua hai chting B. subtilis mang plasmid pHT43-celB va pHT1219-celB ten dia thach.
968


Tap chi Cong nghe Sinh hoc 13(3): 965-971, 2015
Mem tra kha 'tang tiet Endo-1,4-ll-glucanase B
bang phwang phip SDS-PAGE
De khao sat kha nang tiet protein tai to hqp ra m8i
titan long, hai chring B. subtilis chfra plasmid
pHT43-ce/B va pHT1219-ce1B duqc nudi cAy trong
moi trureng LB vai khang sinh Chloramphenicol
37°C va tee to lic 250 vong/phiri. Thu matt 0 gi6 khi
OD600 dot 0,8 va bit diu cam ung vai IPTG a nOng (le
0,5 mM, tiep ttic thu mau sau 2, 4, 6, 8, 10, 12 va 14
gia nuoi cAy. Thi nghiem &Km tien hanh tuang tir voi
mau chimg am 111 B. subtilis 1012 mang plasmid
pHT43 duqc cam ung IPTG va thu mau sau 14 gia
nu6i city. Ket qua thi nghiem Quqc trinh bay a hinh 4.

Ket qua (lien di SDS-PAGE . cho ditty so vai
mau d8i chirng am, cac voch tai to hqp xuat hien 6
thei diem 10 gib va 12 gia sau cam Ong 6 cac 2
chung B. subtilis 1012/pHT43-ce/B va B. subtilis
1012/pHT1219-celB. Cac voch nay tuang ung veri

kich thtrac 61 kDa va 53 kDa so yeti thang chttin
protein. Trong de, vach 61 kDa tucmg ung vai kich
thtrac ly thuyet dm protein Ce1B sau khi loci b6 trinh
tv tin hieu tiet SamyQ. Cau trite dm enzyme CelB
bao gem 2 domain chinh la loi hoot tinh (kich thtrac
53 kDa) ca kha nang plan cat ca chAt cellulose vo
Qinh hinh va dockerin loci 1 c6 vai tre lien ket CeIB
yen protein gia d6 CipA trong qua trinh hinh thinh
phut hg cellulosome. CeIB sau khi &Km tiet ra ngoai
mai truang nu6i cAy se bj protease ngoai bao dm B.
subtilis 1012 phan cat va dockerin loci 1, IA domain
rieng biet yea loi hoot tinh so bj looi be ra khei cau
trite cria Ce1B. Tiep theo, de cO cai nhin re) ham ve 2
voch cAu trim CeIB khi bieu hien tren B. subtilis
1012, chung toi tien hanh phan tich Western blot yeti
khang the dic hieu khang CeIB duqc nhin tir nhenn
nghien ciru cria giro su Pierre Beguin, met trong
nhiing nh6m elAu tien bieu hien thanh cong CeIB tai
to hqp (Petit et al., 1985).

B. subtilis 1012 pHT43-ce/B
4 6
8
10 12

kb M (-) 2

B. subtilis 1012 plIT1219-ce1B
2
4
6
8
10 12 (gib)

80
CelB
60
50
40
30
25

1

■Wle

CelB

Hinh 4. Kha nang tiet CeIB cua chUng B. subtilis mang plasmid pHT43-ce/B v9 pHT1219-ceIB tren m6i trutmg long bang
SDS-PAGE. (-): dich nu6i cay B. subtilis mang plasmid pHT43 sau 14 gid cam Ling IPTG 0.5 mM: 2. 4, 6. 8. 10. 12 gitr thu
mau sau khi duvc cam Ong bang IPTG 0,5 mM.

Ket qua lai Western blot
Ket qua lai yen khang the clic hieu khang CeIB

duqc trinh bay trong hinh 5, trong d6, c6 the thAy
duqc protease ngoai bao di phan cat enzyme CeIB tir
kich thy& thuyet IA 61 kDa xueng kich thuac 58
va 53 kDa, twang Ong v6i domain lei hoot tinh. Cac
vach tin hieu lai là d ec hieu via mau d of chimg
khong c6 bit kj, tin hieu lai nao hinh thanh. Ben
conh Q6, cAu true CeIB cung cap boi ngfin hang dir
lieu Uniprot va Pfam cna EMBL-EBI (European
Molecular Biology Laboratory - European
Bioinformatics Institute) cho thay c6 2 domain rieng

biet là loi hoot tinh va Dockerin loci 1. Trong Q6,
Dockerin loci I to thanh bai 2 yang lip loi ciut
motif gan Canci va lien ket vai 101 hoot tinh CelB
bing met clop as c6 ciu ft-6c clan gian. Dva vac) tin
hien lai thu Qu qc teen ming nitrocellulose, c6 the
thay Qtr qc CeIB v6i kich thu6c 61 kDa duce bieu
hien 6 ca 2 chung B. subtilis trong khao sat, bj
protease phan cat thanh 2 vach kich thy& la 58 kDa
va 53 kDa. Trong de, voch 58 kDa lA CeIB bj phan
cat 1 phAn domain dockerin tool 1, ngay Gal vi tri
giila 2 motif gan Canxi va voch 53 kDa là cAu truc
lei hoot tinh CeIB Qi lo0i bo holm toil) dokerin loci
1, vai cau trite dtr &An trong hinh 5B. Deng that ket
969


Phom Lutrng Thing et al.
qua nay con cho thay plasmid pHT43-celB cho hieu diem bit diu bleu hien tiet tot CeIB la 4 gier sau khi
qua tiet tot ham plasmid pHT1219-ce/B. Va theri cam irng bang 0,5 mM IPTG.

B. subtillt 1012
B. subtills 1012
pHT43-ce/B
pHT1219-ce/B
M 2 4 6 8 10 12 2 4 6 8 10 12 (506)

A

It

CelB - Dockerin Poll
CelB - 1/2 Dockain
CelB

Glycoside hydrolase superfamily (53 kDa)

Dockerin

Iola I (8 kDa)

CclB Dockerin full

as.

CelB 1/2 Docker's'
CelB

Hinh 5. Ket qua lai Western blot v6i khang the thd chuyen Wet cho CeIB. Hinh A: ket qua lai v6i meu krang Ong 100 pl djch
nuoi cey B. subtilis mang plasmid pHT43-ce/B va pliT1219-ce/B ding v&i chung dei chi:mg mang plasmid pHT43 khOng
mang gen (-). Hinh B: du true protein CeIB tren ngan hang di), lieu Uniprot va cac kieu ceu trot du, down la san phem cat

cua cac protease ngoai bac) tit b6i B. subtilis. Trong de, c6 2 domain chInh la domain host tfnh (kfch thirOc 53 kDa) va
Dockerin loci 1 (8 kDa).

6

90
80
7

spHT43-ce/B
• pHT I 219-celB

§ 6°

-a

Kt

T. 4

0
:2 30
b
'0
S 10
0

4
6
8

10
12
Thbi gian nuoi coy sau cam img IPTG

14 (gi6)

Hinh 6. Kat qua do hoot tlnh cellulase B tiet ra mai taterng nuoi cey cua chung B. subtilis mang plasmid pHT43-celB va B.
subtilis mang plasmid pHT1219-celB.

K4m tra host tinh ctia dich nuoi cav
Tu ket qua kiem tra hoot tinh (Hinh 6), chung to
djch nuoi coy cua ca hai cluing B. subtilis 1012 deu
c6 hoot tinh phan buy CMC, tuy nhien, khong co sq
khac Net Ian ve kha nang phan giai giiia 2 chiing
bleu hien B. subtilis 1012 mang plasmid pHT43-ce/B
va chung B. subtilis 1012 mang plasmid pHT1219celB. Float tinh cellulase cao nhat la 12 gi6 sau cam
(mg IPTG, va ket qua nay phi' hqp vai ket qua phan
tich SDS-PAGE ding nhu la .Western Blot. D Ong
thai, ket qua nay ding cho thay rang, 101 hoot tinh
CeIB van cho kha nang phan cat CMC, do do, hoot
tinh CeIB khong bi anh twang nhieu bai protease
ngooi bio ctia B. subtilis 1012.
970

KET LUAN

Tir cac IC& qua dot duqc a tren, chiing Ceti ket
luan rang di tach dong va bleu hien thanh tong gcn
CelB tir C. thermocellum DSM 1313 dual clang tiet 6
B. .subtilis va cho hoot tinh. phan giai CMC dot 80

U/mL. Ket qua nay g6p phan cho viec tim hieu sq
bieu hien enzyme tai to hqp dam le huemg den viec
MIA() sat sts bieu hien him hqp cellulosome CO hoot
tinh trong B. subtilis trong cac nghien cau tiep theo.
Liri cam cm: De tai Ave ho trot beri du an JICA-JST
can Tricang Doi hoc Bach khoa. Dgi hoc Quiic gin
TP. Ho Chi Minh. ma so JICA-JST-2012-06.


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Riederer A, Takasuka TE, Makino S, Stevenson DM,
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CLONING AND EXPRESSING ENDO-1,4--GLUCANASE B FROM CLOSTRIDIUM
THERMOCELL UM IN BACILLUS SUBTILIS
Pham Luong Thang', Pham TM My Tien', Tran Quoc Tuan', Nguyen Le Tuan Anh', Nguyen Hoang
Ngoc Phuong', Phan TM Phuong Trangl, Nguyen Thi Nguyen2, Nguyen Dinh Quan2, Phan Dinh Tuan z,
Tran Linh Thuoc', Nguyen Duc Hoang1421
University of Science, Vietnam National University, Ho Chi Minh City
of Technology, Vietnam National University, Ho Chi Minh City

2University

SUMMARY
Vietnam is one of countries having the most abundant agricultural waste with more than 40 milion tons of
straw produced annually. For complete reuse of this cellulose substrate, bioethanol industry is founded and
able to solve the world energy crisis problem. In a typical bioethanol producing process, the cellulose
hydrolyzing ability of cellulase enzymes is a vital issue. From that reason, we expressed recombinant Endo1,4-13-glucanase B (CelB) from anaerobic Clostridum thermocellum in potential host trains Bacillus subtilis.
Firstly, gene celB was isolated by PCR reaction from C. thermocellum DSM1313 DNA chromosome. The
recombinant plasmid pHT43-celB was generated by T4 DNA ligase reaction while pHT1219-celB was created
by In-fusion cloning technique. The transformants were checked by colonies PCR and confirmed by
sequencing. Bacillus subtilis 1012 strains carrying plasmid pHT43-celB or pHT1219-celB were used to study
the expression of CelB in both halo technique on agar plate and activity measurement by inoculating in liquid
medium. The results showed that in the 0.5 mM of IPTG inducer concentration, recombinant B. subtilis cells
could hydrolyze CMC substrate in the plate. In addition, SDS-PAGE combined Western blotting and
enzymatic activity assay results demonstrated that B. subtilis can secrete active CelB, equal to 80 Unit/ml of
cultured medium.
Keywords: Bacillus subtilis, Endoglucanase B, Clostridium thermocellum, cellulose, plasmid pIIT

Author for correspondence: E-mail:
971