Ảnh hưởng của đuôi poly histidine lên khả năng biểu hiện tiết endoglucanase a của clostridium thermocellum trong bacillus subtilis WB800N

Tap chi Cong ngh.e Sinh hoc 13(1): 161-167, 2015

CiJA DUN POLY-HISTIDINE LEN KHA NANG BIEU HIEN TIET
ANH
ENDOGLUCANASE A ODA CLOSTRIDIUM THERMOCELLUM TRONG BACILLUS
SUB77LIS WB800N
Phom Luang Thing, Nguyin Huang Ngqc Pinning, Than Thj Phtning Trang, Tian Linh Thireic,
NguyAn Dtrc Hoang
Truerng Dgi hoc Khoa hoc it nhien, Dai hoc Quoc gia Thanh pha Ho Chi Minh
Ngity nhan bai: 26.10.2014
Ngay nhan clang: 04.12.2014
TOM TAT
Endoglucanase A (CeIA) t6. Clostridium thermocellum li enzyme chit, nhiet nem trong phirc he
cellulosome c6 kha Wing phin cit cellulose met each hieu qui. De c6 the a ding thu nhOn enzyme Ce1A,
enzyme nay phii dtroc dung hqp vai duoi tinh the vi bieu hien trong met ching chit an toin, nuoi city vi c6
khi ruing tiet tot protein ra mai truing nuoi city. Trong nghien dm nay duoi tinh the poly-Histidine vi. chimg
chit Bacillus subtilis dirge sir dung nhim khio sat sir anti huding cis duel tinh the nay len khi ning bieu hien
vi hoot tinh dm enzyme CeIA. Cic duoi tinh the poly-Histidine v6i so luqng amino acid Histidine khic nhau
(4xHis hoc 5xHis hoc 6xHis) duqc thiet ke dung hop tai ctiu C cua enzyme CeIA. Mut de bleu hien vi hoot
tinh cua cite protein di to hop ductc dinh gia thong qua kha ning gin cut Ni-NTA cue duoi poly-Histidine vi
dual
hoot tinh ph'in giai CMC to thanh dtr6ng. khir cua CeIA. Ket qua phin tich thong ke ye anh htremg
poly-Histidine len CeIA cho thiy, cite dieu kien nhiet de, nOng de chit cam (mg khic nhau deu cho thiy duoi
poly-Histidine dung hqp tAti diu C cis CeIA c6 inh hu6ng len sir bleu hien cfing nhu hoot tinh cua enzyme
nay. 0µ the li so lugng a.a His cing nhieu thi mire de bieu hien vi hoot tinh cua enzyme cing giim nhung khi
fling gin dm protein dung hqp len cut Ni-NTA cing chit va hieu qui.

Tir klia: Bacillus subtilis, bleu hien tiet, CeIA, Endoglucanase A, poly-Histidine
M6 DAU
Cellulosome dm Clostridium thermocellum la
mut trong nhiing phirc he enzyme cO kha fling phan

cat Vet cellulose, trong dO Endoglucanase A (CeIA)
la mOt trong nhang thanh phin chit yeu cua phirc hg
cellulosome (Demain et al., 2005), (Fontes va
Gilbert, 2010), (Gold va Martin, 2007). De bleu hien
va thu nhan CeIA tir chimg vi sinh vat Gram ducrng,
ky khi va ua nhiet C. thermocellum, chung chit bieu
hien Bacillus subtilis la del tuonF tiem fling nhit vi:
(i) Wang thing ve mat ciu true to bao vi ca hat cling
la vi Ichuin Gram ducmg; (ii) da co he thOng plasmid
ton tai dOc lap pHT43 (MoBiTec), cho phep bieu
hien protein tai to hop ding tiet ra mai hieing (Phan
et al., 2006), (Simonen va Palva, 1993); (iii) dieu
kien nuoi city dun gian, chi phi thip nhung hieu qua
tao sin phim va sinh khoi cao gip nhieu lin so vol
viec nuoi cay va thu nhan CeIA tren C. thermocellum
(Schumann, 2007); (iv) la chung an toan GRAS
(General Regarded As Safe) duqc cong nhan bai
FDA (Foods and Drugs Administration), My

(Schumann, 2007). Trong nghien ciru khfic, cluing
toi da bieu hien thanh c8ng CeIA tai to hop ding tiet
ten B. subtilis 1012 va B. subtilis WB800N. Trong
nghien ciru nay, duoi tinh the poly-Histidine duqc
dung hop h dau C dm protein CeIA nharn img,clung
trong viec tinh the CeIA. Tuy nhien, do ban chat cua
mang nguyen sinh chat, cfic dpi phan hi sinh hoc
mang dien tich duong nhu du8i tinh che polyHistidine co the se bi .han the di chuy'en qua ming
trong qua trinh tiet. Anh htreyng bai so lucmg His
trong duoi poly-Histidine la 4, 5 NIA 6 a.a duqc khao
sat trong nghien ciru nay.

VAT LIEU VA PHIJONG PHAP
Ttio cac plasmid tai t6 hqp
Cac plasmid pHT1454 (CeIA-4xHis), pHT1455
(Ce1A-5xHis) va pHT 1456 (Ce1A-6xHis) duqc tao
thanh tir plasmid khung stRm IA pHT1202 mang trinh
to tin hieu tiet cua gene amyQ tir Bacillus
amyloliquefaciens va promoter manh Pgrac212
(Phan et al., 2013). Phucmg phi!) dong hOa duqc sir
161


Pham Lucmg Thing et al.
ding la In-fusion yeti cac cep mei thu gene d#c hieu
cho celA dung hqp duel 4xHis (ON915/0N916E),
5xHis
(ON915/0N916D)
va
6xHis
(ON915/0N916C) (phg lgc Si). Ket qua deng hea
duqc kiem tra bang phuong phip giai trinh tlr vai
Op mei ON314 va ON1207.
To cluing B. subtilis tai to hip
Cac plasmid tai to hop pHT duqc Wen nap vio
B. subtilis WB800N (Nguyen et aL, 2011) bang
phucmg phap bien nap to nhien theo quy trinh Wang
din cua MoBiTec (MoBiTec GmbH Company,
2012). Khang sinh ch9n 19c hi chloramphenicol
neng dO 10 µg/ml. Sau 24 gib, 6 6 37°C, cac khan
lac dv tuyen duoc sang 19c lai bang PCR khuan lac

vii c(ip moi duqc sir ding la ON707 va ON916, sin
phim to thanh CO kich thus* la 1479 bp.
Khoo sit kha fling bieu hi§n tat CeIA (ra tren
vbng phan giii
Cac chung tiem nang duqc nuoi cay tren dia
thach LB-Cm10 (10 pg/m1 chloramphenicol) chira
0,5% CMC va chit cam ung IPTG v&i cac ming de
khic nhau 0; 0,01; 0,05; 0,1; 0,5 va 1 mM. U 24 gib
6 37°C, nhuem thuec thir de Congo 1% trong 5 phut,
Ilia bang NaCI 1 M. Kha nang phin giii CMC cang
cao thi yang phan giai cang len (Yoon et al., 2007).
Xic nh#n kha nang bieu hi§n flit CeIA trong djch
nuoi ay B. subtilis
Te boo B. subtilis WB800N mang plasmid tai to
hip duqc nuoi cay trong moi trutmg LB long 6
37°C. Khi m#t de to boo did gia trj ODA khoang 0,8
— 1,2, cam ling vol IPTG 6 ming de la 0,1 mM. Sau
khi cam ung ducrc6 gia, tien.hanh thu 1 ml mAu dick
net, tira TCA non de cuoi la 5% (Rajalingam et
al., 2009) lei tien hanh rhan tich SDS-PAGE va
Western Blot vei khang the the da deng khang CeIA.
Killing the thu dip dung hop enzyme, Horseradish
peroxidase (HRP) duqc phat hien bang co chat
3,3',5,5'-tetramethylbenzidine (TMB).
Xic nh.n sv din t31 cua dubi tinh the 4xHis,
5:His vi 6xHis
Sic ky ai hrc là ky thu#t tinh the dva tren trong
tic ac hieu cua ion Nit' yen a.a His (Uppsala,
2002). Trong de, phan tir imidazole to do se earth
tranh veri His trong viec bam len cot Ni-NTA. Ming

de imidazole can thiet cho viec dung ly cling cao,
tuong tic dm duoi tinh the veri pha tinh Ni-NTA
cang Ian. Cac duOi dung hip 4xHis, SxHis va 6xHis
se duqc kiem tra sir hien dien thong qua danh gia kha

162

nang gin Ni-NTA bang sic ky ai
Neng dO
imidazole c3nh tranh tang clan theo se Wong a.a His
trong du8i dung hop 4xHis, SxHis va 6xHis.
Dinh gii anh huteng se Wong a.a His trong duoi
dung hop len khg nang flit CeIA cilia B. subtilis
Chung B. subtilis WB800N duqc xic nh#n
bieu hien tiet CeIA vol eau trac day de dm 4, 5 va
6 His trong du8i dung hqp. Tien hinh nuoi cay va
cam ling bleu hien CelA dun hqp 6 cac ming de
chat cam ung IPTG tir 0,1 den 1 mM. Djch nuoi
cay t6 2 gib den 24 gia sau cam trig duqc thu
nh#n de danh gia hoot tinh theo quy trinh cal bien
cua IUPAC (International Union of Pure and
Applied Chemistry) (Ghose, 1987). Phucrng phap
th8ng ke sir dung cho danh giit sv khgc bit ye kha
nang bieu hien la ANOVA met chieu ket hop plan
tich Tukey's significant difference, sal so chip
nhOn la 5%.
KET QUA
Tao chfing B. subtilis c6 mang plasmid tai to hop
Cac khuan lac do tuyin B. subtilis duqc nuoi cay
tren moi trutmg chen 19c LB-Cm10 sau 24 gib se

duqc kiem tra sv hien dien cua plasmid tai to hop
bang cep moi ON707 va ON916 veri san phim PCR
co chira gene celA dv kien là 1479 bp. San phi.=
PCR duqc kiem tra tren gel agarose 1% (Hinh 1) cho
thay cac khuin lac duqc hra chip deu cho sin pham
PCR dung %raj kich thuOc dg doin. Day se la cac
khuin lac duqc sir ding trong viec sang 19c kha nang
phan gal CMC 6 buOc tiep theo.
Kha nang phan giai CMC cua die chiing B.
subtilis tai to hop
Vangphan giii CMC duqc hinh thinh khi CelA
t6 cac khuan lac duqc flit ra moi trueng xung quanh
phan cat cAu tire CMC, yang moi trubng thiu co
chit se khong bit mini veri dung djch Congo red 1%.
Ket qui trong Hinh 2 cho they dueng kinh Wong
phan giii tirog can theo ding de chit cam ung IPTG.
Tuy nhien, ket qua Wong phan giai cho they kha nang
ti6t CelA auk chimg B. subtilis WB800N kh6ng hoin
toin tuyen tinh vai ruing de chit cam ung IPTG, hieu
qua t3o enzyme Qat t6i da 6 ming •10 chat cam ung la
0,1 mM.


Tap chi Gong ngh" Sinh hac 13(1): 161-167, 2015

Bacillus subtilis WB800N
by

pHT1456
pHT1455

pHT1454
M KL I KL2 KL I KL2 KL1 KL2

5000
2000
1479 by

1200
800
500

Hinh 1. San phArn PCR khuech dal vOng gene celA tip khuen lac B. subtilis WB800N mang plasmid tai to hop pHT1454,
pHT1455 va pHT1456.

Bacillus subtilis WB800N
0,00 0,01 0,05 0,10 0,50 1,00 (mM IPTG)

•

•

•

• •

•
• •

•

••

•

0
•

0

• •

0 0
O

0

• 0 0
10

Hinh 2. yang phan giai CMC cua cac chi:mg tai to hop B. subtilis WB800N mang cac plasmid pHT1454, pHT1455 va
pHT1456 b cac ming dO chat cam 0,ng IPTG va dieu Van nhiOt dO nu6i cay. Chung vi khuen mang plasmid pHT43
(MoBiTec GmbH Company, 2012) khong mang gene ma h6a cho CeIA duvc sir dyng lam doi chCrng am.

phan CeIA tai to hgp trong clich, nuoi cay vi sinh vet.
Dieu nay dirge the hien qua ket qua SDS-PAGE
Hinh 3 vai vach protein tai to hop (vi tri ki hieu bang
B. subtilis WB800N la claing chu cid duac dot cham mau trang) a kich thuac khoang 50 kDa, tucmg
bien 8 loai protease ngo4i bao, tuy nhien, cac
ang vai kich thuac 1Y thuyet ciia protein CeIA (49,14
protease con lai ciia no van co kha nang phan cat met kDa) cho tai vach 40 kDa, twang drig vai domain lei

Phan tich SDS-PAGE ve Western Blotting eau

&tic CeIA dung hop

163


Plum Luang Thing et al.
hoot tinh cua protein Ce1A (39,4 kDa). Ket qui nay
duqc xac djnh loi bang phial tich Western blot v6i
khang the dac hieu cho protein CeIA. V6i cac voch
dim, ro vi chi xuAt hien 6 phan (loon tiet 6 cac
chung B. subtilis mang plasmid pHT1454, pHT1455
vi pHT1456, kit qui lai cho thiy twang tac protein —
khang the là (lac hieu, kheng c6 bit kk tin hien miu
nio too thinh 6 djch nuoi ciy chung doi chimg B.
subtilis mang plasmid pHT43.
Ben conh Q6, 6 cac gieng tuung ring v6i cac chimg

mang plasmid tai to hqp, 2 voch cau trac cua CeIA
too thinh la tuong tIng yeti kit qua (lien di SDSPAGE li domain loi hoot tinh vi CeIA Cray dti kh6ng
bj phan cit. Trong Q6, tin hieu lai cua Ce1A Ichong bi
phan cat (GH8 — Doc 1) cao ham Ilan so v6i loi hoot
tinh (GH8). Kit qua phan tich cho thiy CeIA tai to
hop khi bieu hien tren B. subtilis WB800N tuy bi
phan cit nhung phin lan protein dung hop van gill
duqc cau truc nguyen vgn v6i duel poly-Histidine 0
diu C vi c6 the tinh the duvc bang sac kji ai luc voi
Ni-NTA.

Bacillus subtilis WB800N

A
M

.s'2) 414:c•b`55.
iz4

0 4.41 "SI°

.i<4

kDa
85

kDa

O

60

70
55

50

40

40

H8 - Doc I
GH8

35
30

B

Glycoside hydrolase family 8 (GH8) - 39,4 kDa

Dockerin
loci I
CD 0
0

0

Doc I - 8 kDa
Hinh 3. Phan tich kha nang bibu hien CeIA trong mei Wong long. A, kat qua phan tich SDS-PAGE (tral) vii Western blot vol
khang the the da dong khang CeIA (phai) mau djch tiet CeIA dung hqp tir B. subtills WB800N tai thoi diem 6 gist/ sau khi
cam (mg IPTG 6 a:mg de 0,1 mM 6 37°C; B, cau trim protein CeIA va cac domain tren ngan hang (16 lieu Uniprot.

Anh hir0ng clia se !Iron His trong duoi tinh chi
len kha nang barn len cOt Ni-NTA
Sic kj, ai lgc dua tren tuang tac kh6ng ceng hOa
tri gift Nei" va vong imidazole trong cau trim dm
His. SO lucmg His trong duel tinh the cing nhieu,
Omn; tac cia protein v6i pha tTnh Ni-NTA cing cao
vi nong de chat conh tranh imidazole tu do sir dung
trong dung dich dung ly cing nhieu. Tien hinh do

hoot tinh cac phan doon dung ly theo nang dO tuyen
tinh cua imidazole MY 5 cho den 250 mM, kit qui cho
thiy djch nu6i ciy B. subtilis WB800N mang
plasmid pHT1456 (ma hOa cho CeIA dung hqp duel
164

6xHis) bit diu cho hieu qua dung ly tot tir nong de
imidazole la 80 mM trong khi Q6 CeIA dung hqp yen
duoi 5xHis (plasmid pHT1455) bit dau cho hien qua
dung ly 6 40 mM imidazole; vi CeIA dung hqp v6i
duoi 4xHis (plasmid pHT1454) hiu nhu khong cho
!lieu qua gin cot tot, v6i hoot tinh enzyme do Cluqc 6
hiu het cac phAn Croon dung ly deu rat thip. Ket qui
nay cho thiy, luc tucmg tic gifra protein dung hqp
vai cot Ni-NTA tang din theo s6 Wong His trong
duoi tinh the dung hop (Hinh 4).
Doi v6i tramg hqp CeIA dung hqp 4xHis, khi
tien hinh do matt tru6c tinh the (Queng khir too


Tv chi COng nghe Sinh hQc 13(1): 161-167, 2015
thanh la 0,498 mg/ml) va djch rim qua OA (du6ng
khir tao thanh la 0,190 mg/ml), ket quA cho thiy hoot
firth enzyme 6 cac phan don nay cao han rat nhieu
so vai djch dung ly tt 01, (du6ng khir tao thanh là
0,096 mg/ml). Kat qua nay cho thiy Ai Ivc lien ket
girra duoi dung hqp 4xHis vai Ni-NTA rat kern,
lucmg protein bj ilia tr8i cang nhieu, hoot tinh

enzyme trong cac phan down dung ly cang thap. Nhu

vay, cac ket qui phan tich tren deu chi ra rang CeIA
dung hqp yeti du8i poly-Histidine du ce bj phan cat
mOt phin do enzyme ngoai bao cua B.subtilis
WB800 nhung chi mOt phin nho 6 diu N, phan con
loi cua ciu trite protein van con chira phin 101 hoot
tinh va duoi poly-Histidine.

0.3
0.25
E

E 0.2
• pHT1454

0.15

• pHT1455
pHT1456
—0.05

I C
5

10

k
20

40

80

160

250

isItng dO Imidazole (mM)
B. subtilis WB800N duvc sir
Hinh 4., DO thj hoat tinh enzyme tir cac phan doan dung ly theo nong do imidazole. Djch tiet

dung gen len cac cdt china hat Ni-NTA, sau d6 viva va dung ly beri cac nong d6 imidazole khac nhau. Cac phan (loan protein
dung ly duvc sir dung de xac djnh host tinh phan cat cellulose the hien qua lung duVng khii tao thanh.

Anh hiking cda s6 hrQng His trong duel tinh the
len kit* fling tat cita B. subtilis
Qua trinh chuyen vi tren ming (translocation) co
vai tre vo ding quan trong trong con duirng tiet 6 vi
sinh vat. Vii ban chat tich dien duang cita a.a
Histidine, cac duoi tinh the it nhieu anh huang len
qua trinh chuyen vi ten ming, din. den tic nghen
kenh tiet va han the khi rang bieu hien protein
ngooi bao. Ket qua phan tich khA nartg bieu hien
bang ANOVA mOt chieu ket hqp Tukey's significant
difference duqc tom tat trong bang 1.
Co the thiy hoot tinh enzyme too thanh giam din
theo chieu tang dm s6 Itrqng His trong duoi dung
hqp 6 hitt het cac dieu kien cam Ong IPTG. Cv the,
plasmid pHT1454 vai duoi 4xHis cho hieu qua bieu
hien cao nit& (xep loci A) va duoi 6xHis cho hieu
qua bieu hien thip nhat (xep tool B) 6 diet' kien nong

d8 chat cam (mg tir 0,05 den 1 mM IPTG. Cl nong de

IPTG the!) (0,01 mM), viec bleu hien CeIA tai to hqp
dien ra chain han, qua trinh tiet dien ra a toe dO vtl'a
phii, do 86, kh8ng con anh huang cua duOi tinh the
len qua trinh fiat. Mat Mac, ket qua bieu hien dm
pHT1455 ma Ma. duoi 5xHis dung hqp CeIA cho
thiy hieu qua bieu hien nim 6 mire trung binh,
khong that sir khac biet Ian so vai duoi 4xHis
(plasmid pHT1454) 6 hAu het cite nong dO IPTG cam
(mg tir 0,01 den 0,5 mM. Sr klac biet giira duoi
4xHis va 5xHis chi the hien ro khi sv bleu hien flat
toi da 6 1 mM IPTG. Tuy nhien, khi tien hanh thi
nghiem nu8i ciy 6 30°C, CA 3 chung B. subtilis
WB800N mang cac plasmid deu tang truong rat
chum (s6 lieu khong ding 1)6) nhung hieu quA tiet
cao han han 6 37°C. Ben conh d6, khi phan tich
ANOVA mOt chieu ket hqp Tukey's significant
difference cho dieu kien nuOi ciy 30°C, khOng co bit
kS, inh htrang nao cua duoi His len viec tiet cua
CeIA dung hqp (phi lic S3).
165


Pham Luang Thong et al.
Bang 1. Phan Lich anh hvong cua du6i poly-Histidine len bleu hien

tier c0a CeIA cue chi:mg B. subtilis WB800N bang phan
tich ANOVA va x4p nhOm Tukey's significant difference. BO dCr lieu phan tich bao gem gia tri hoot tinh CeIA do duvc ft': 2
den 24 gi& sau cam Crng v&i n8ng dO cam Ung IPTG ff.? 0,01 mM den 1 mM & 37°C (phy lyc S2). A: nhOm cac dieu kien cho

min bieu hien va hoot tinh coo; B: nhOm cac dieu kien cho min bleu hien va hoot tinh thap; AB: nhOm cac dieu kien cho
min bieu hien va hoot tinh trung binh. Trong do, cac chi) cal khac nhau the hien ket qua phan tich ANOVA va x6p nhOm
'Deng Tukey's significant difference v&i de tin cey le 95%.

Plasmid

pHT1454

IPTG (mM)

T6ng gnat

0,312 A

pHT1455

0,301 A
pHT1456 0,271 B

0,01

0,05

0,1

0,5

1,0

0,256 A

0,248 A

0,308 A

0,317 A

0,337 A

0,281 AB
0,265 B

0,309 AB

0,330 AB
0,291 B

0,349 A
0,331 A

0,234 A

KET LUAN
Duoi tinh ch6 poly-Histidine da &roc sir dung tir
rat lau va cho thay hiau qua cao trong viac thu nhan
protein nai bao. Tuy‘ nhien, viac sir dung duoi tinh
che His cho san xuat protein ngoai bao van clura
throe danh gia cu the va nghiem tits. Th8ng qua danh
gia kha nang tinh che Ce1A tiet tai to hop tir B.
subtilis WB800N, nhom nghien ciru da cho thay tiam
nang img dung dm duoi tinh the nay trong viac thu

nh'an protein ngoai bao. Ben canh d6, bang cac phan
tich th8ng ke ANOVA don gian, chimg,toi da chi ra
rang so Itrong His trong du8i tinh che anh twang
khong nh6 len kha nang tiet cua vi sinh vat, cu the la
B. subtilis WB800N trong nghien ciru nay. So luong
His tang, cho hi'au qua tinh che cao nhtmg han che
kha nang Melt hian, dac bit la 6 cac dieu kin cam
img bieu hien mph nhu nhiat da 37°C, 'tong dO chat
cam img IPTG cao I mM. Ket qua nay c6 gia tri
tham khdo quan trong cho viac chop lua phuang
phap, chitin lugc tinh che protein ngoai bao dirge
bieu hi6n a B. subtilis.
Liri cam oil: Erd tai duvc thifc hien nha su ho tra
kinh phi ciia See Khoa hoc va Cong nghe Thanh pha
Ha Chi Minh, nem 2012-2013. Ma so:
178/2013/HD-SKHCN.

0,277

B

0,295

B

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Tap chi Cong nghe Sinh hoc 13(1): 161-167, 2015

THE EFFECT OF POLY-HISTIDINE TAG ON SECRETION ABILITY OF
ENDOGLUCANASE A FROM CLOSTRIDIUM THERMOCELLUM IN BACILLUS
SUBTILIS WB800N
Pham Luonj Thang, Nguyen Hoang Ngoc Phuong, Phan Thi Phuong Trang, Tran Linh Thuoc, Nguyen
Duc Hoang
University of Science, Vietnam National University, Ho Chi Minh City
SUMMARY
Endoglucanase A (CeIA) from Clostridium thermocellum is a heat-stable enzyme in the cellulosome
complex, able to hydrolyze cellulose effectively. This study aims to investigate the influence of poly-Histidine
tag fused with CelA on the secretional ability in Bacillus subtilis. Histidine is a positive charge amino acid,
therefore protein fused with poly-Histidine tag may cause traffic jam when the fusion protein translocate
through the membrane. To confirm this hypothesis, a series of 4, 5 and 6 Histidine amino acid tags were
generated at the C-terminus of Ce1A. The expression of extracellular CelA fusions was identified based on the
presence of histidine fusion tag by immobilized nickel ion affinity chromatography. Then, the expression levels
of the fusions in B. subtilis WB800N were evaluated by determination of enzyme activities. The ANOVA
combined Tukey's honest significant difference analysis was employed for comparisons of histidine-tag effects
in many CelA secreted conditions such as inoculated temperature and IPTG inducer concentrations. The results

showed that constructs containing more histidine have lower expression levels — this confirmed the proposed
hypothesis.
Keywords: Bacillus subtilis, CeIA, Endoglucanase A, poly-Histidine, secretion

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