Tuyn tp Bo co Hi ngh Sinh vin Nghin cu Khoa hc ln th 8 i hc Nng nm 2012

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NGHIN CU THU NHN V TO DNG GEN M HA CELLULASE
T VI KHUN BACI LLUS SUBTI LI S
RESEARCH AND CLONING A GENE ENCODING CELLULASE FROM BACILLUS
SUBTILIS
SVTH: ng Th Chu Thu
Lp 07SH, Khoa Ha, Trng i hc Bch Khoa, i hc Nng
GVHD: Th.S Ng Thi Bch Vn
Khoa Ha, Trng i hc Bch Khoa, i hc Nng
TM TT
Enzyme cellulase c ng dng nhiu trong mi nng trng, nng nghip, cng
nghip, y hc,.Tuy nhin ti Vit Nam vic sn xut ch phm enzyme ny vn cha thc s
hon thin. Trong nghin cu ny chng ti tin hnh to dng on gen m ha endo-1,4-beta-
glucanase thuc h enzyme cellulase t vi khun Bacillus subtilis. on gen sau khi to dng
thnh cng c bc u biu hin trong chng E.coli BL21(DE3) cm ng bng IPTG 1mM.
Hot tnh ca enzyme c kim tra bng phng php thy phn trn mi trng thch cha
CMC (carbo-metyl-cellulose) 1% v phng php ng kh Bermeld. ng thi chng ti kim
tra kch thc ca enzyme mc tiu bng in di SDS-PAGE. Kt qu ny s lm nn tng cho
vic sn xut ch phm enzyme cellulase phc v trong nhiu ngnh cng nghip sau ny.
ABSTRACT
Cellulase enzyme plays a critical role in many fields such as agriculture, industry,
medicine, etc. However, production of this enzyme still haven't been completed. In this study, we
carried out cloning the gene endo-1,4-beta-glucanase which is coding for the biosynthesis of
cellulose on the Bacillus subtilis DNA. The cloning gene is represented on the expression system
of E.coli BL21(DE3) and induced with 1mM IPTG. The producing enzyme is test for activity on the
medium with CMC (carbo-metyl-cellulose) 1% and the reducing sugar Bermeld. As the same time,
we check the size of the target enzyme by SDS-PAGE. This result will base for the manufacturing
cellulase which is using for many industry application.
1. t vn
Enzyme cellulase c ng dng trong nhiu lnh vc: nng nghip, cng nghip,
y hc, mi trng, . Trong nng nghip v cng nghip thc phm: enzyme ny c
dng ch bin thc n cho vt nui, ci thin gi tr dinh dng ca thc n gia sc, gia
cm. Trong y hc: cellulase c dng trch ly cc cht t cy thuc, to nn nhng
bc pht trin vt bc cho ngnh cng nghip dc phm. Ngoi ra, cellulase cn c
ng dng trong quy trnh to cn sinh hc hng n vic to ra ngun nhin liu thay th
dn cho ngun du m ang ngy cng cn kit [4].
Cc chng vi khun nh Bacillus subtilis, vi nm nh Tricodeman,c kh nng
sinh enzyme cellulase mnh. Trong ti ny, chng ti tin hnh thu nhn v to dng
gen m ha cho endo-1,4-betaglucanase t vi khun Bacillus subtilis. ti nu c khai
thc trit s l bc tin quan trng, l c s hng n vic thu nhn v sn xut
ch phm enzyme cellulase thng mi vi gi thnh hp l.
2. Kt qu nghin cu v kho st
Tuyn tp Bo co Hi ngh Sinh vin Nghin cu Khoa hc ln th 8 i hc Nng nm 2012
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2.1. Kt qu thu nhn DNA b gen t Bacillus subtilis
thu nhn gen m ha cho enzyme endo-1,4-betaglucanase, chng ti s dng b
gen ca vi khun Bacillus subtilis lm mch khun. Theo cc nghin cu trc c tin
hnh ti phng th nghim cng ngh sinh hc i hc Bch Khoa Nng chng vi
khun ny c chng minh c kh nng sinh enzyme cellulase mnh. Do vy, u tin
chng ti tin hnh tch chit DNA b gen bng phng php SDS-kim. Kt qu thu
c in di trn gel agarose 1% nh sau:

Hnh 1 Kt qu tch DNA b gen Bacillus subtilis
Ging M: Thang DNA 100bp (Biorad)
Ging 1: DNA b gen Bacillus subtilis
2.2. Kt qu nhn bn on gen endo-1,4-betaglucanase
Trn c s DNA thu c, chng ti tin hnh phn ng OE PCR (overlap
extension PCR) thu nhn on gen mong mun . Phn ng PCR u c tin hnh hai
phn ng vi hai cp mi F1 v R1, F2 v R2 vi chu trnh nhit: 95
0
C trong 5 pht, 94
0
C
trong 1 pht, 52
0
C trong 45 giy, 72
0
C trong 1 pht 30 giy, 72
0
C trong 10 pht (lp li 35
chu k) thu c hai on gen c kch thc khong 900 bp, 700 bp. Phn ng PCR th
hai s dng sn phm ca hai phn ng PCR u lm nguyn liu (template) chy vi chu
trnh nhit nh trn 25 chu k sau b sung mi F1&R2 chy tip tc 30 chu k vi mc
ch thu c on gen c kch thc 1600 bp [2]. Mi F1, R2 c thit k c v tr ct
ca enzyme BamHI v XhoI tng ng. Cc kt qu PCR c in di kim tra trn gel
agarose 1% nh sau:







A B
Hnh 2 Kt qu PCR on gen endo-1,4-glucanase
Ging M: Thang DNA 100bp (Biorad)
Ging 1A: on gen th nht 900 bp
Ging 2A: on gen th hai 700 bp
Ging 1B: on gen endo-1,4-betaglucanase 1600bp
T kt qu in di cho thy chng ti
thu c on gen endo-1,4-betaglucanase hon
chnh vi kch thc khong 1600 bp. on gen
thu c s tip tc thc hin quy trnh dng ha
vo Escherichia coli DH5.

T kt qu in di cho ta thy chng ti thu nhn c DNA b
gen Bacillus subtilis nguyn vn. DNA c th tip tc s dng
thc hin nhn bn thu gen mc tiu.

1000 bp
Tuyn tp Bo co Hi ngh Sinh vin Nghin cu Khoa hc ln th 8 i hc Nng nm 2012
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2.3. Kt qu to dng v kim tra plasmid ti t hp pET28a(+)endo-1,4-betaglucanase
2.3.1 Kt qu bin np plasmid ti t hp pET28a(+)endo-1,4-betaglucanase vo
Escherichia coli DH5
Trong ti ny chng ti chn plasmid pET28a(+) lm vector mang gen mc tiu
vi hai v tr ct ca enzyme BamHI v XhoI ti v tr to dng a bi, on gen c
thit k c mang v tr ct ca hai enzyme ny. Do vy, u tin chng ti x l plasmid
pET28a(+) v on gen endo-1,4-betaglucanase bng hai enzyme ct hn ch BamHI v
XhoI. Phn ng ct c thc hin 37
0
C trong 4 gi. Sau thc hin phn ng ni to
vector ti t hp nh enzyme ni T4 ligase iu kin nhit 4
0
C qua m. Sn phm
ni c ha bin np vo t bo E.coli DH5 sau c sng lc trn mi trng LB
c b sung kanamycin (30 g/ml) 37
0
C, qua m.

A B C
Hnh 3 Kt qu bin np vector ti t hp vo Escherichia Coli DH5 (hnh A).
Kt qu bin np plasmid pET28a(+) vo Escherichia Coli DH5 chng dng (hnh B).
Kt qu cy tri Escherichia coli DH5 kh np chng m (hnh C).
Kt qu hnh 3 cho thy chng ti bin np thnh cng vector ti t hp vo
chng DH5. Sau bc ny, chng ti s tin hnh sng lc cc khun lc ti a thch
hnh A thu c chc chn dng c cha vector ti t hp.
2.3.1. Kt qu sng lc chng Escherichia coli DH5 c mang vector ti t hp
pET28a(+)endo-1,4betaglucanase
chn ra c khun lc c cha vector ti t hp pET28a(+)endo-1,4-
betaglucanase. Chng ti tin hnh sng lc bng phng php tch chit DNA tng s
[3].

Hnh 4 Kt qu sng lc chng DH5 mang vector ti t hp
Ging M: Marker 100 bp (Biorad)
Ging 1,3,4,5: Cc dng DH5 mang plasmid pET28a(+)
Ging 2: Dng DH5 mang plasmid ti t hp pET28a(+)endo-1,4-betaglucanase
2.3.2. Kt qu quy trnh tch thu plasmid ti t hp v kt qu ct kim tra plasmid ti t
Qua kt qu in di trn, ti ging 2 l dng c
mang plasmid ti t hp v kch thc plasmid ti t hp
khong 7 kb do vy s nm trn so vi vch plasmid
bnh thng c kch thc 5,4 kb. T chng ti s thu
nhn c dng cn tm ti ging s 2. Dng ny sau s
c nui tng sinh trn mi trng LB/Kanamycin
tin hnh tch thu nhn plasmid ti t hp.

Tuyn tp Bo co Hi ngh Sinh vin Nghin cu Khoa hc ln th 8 i hc Nng nm 2012
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hp pET28a(+)endo-1,4-betaglucanase:
Sau khi xc nh chnh xc dng DH5 c mang vector ti t hp. Chng ti
tin hnh nui tng sinh khun lc v thc hin quy trnh tch thu nhn plasmid ti t hp.
Tip theo, chng ti thc hin phn ng ct plasmid ti t hp thu c bng enzyme
BamHI v XhoI. Phn ng ct c thc hin 37
0
C trong 4 gi. Sn phm tch thu nhn
plasmid v sn phm phn ng ct kim tra c in di kim tra trn gel agarose 1%.

Hnh 5 Kt qu in di kim tra plasmid ti t hp v ct plasmid ti t hp
Ging M: Thang DNA 100bp (Biorad)
Ging 1: Kt qu sn phm ct plasmid ti t hp
Ging 2: Kt qu sn phm ct m vng plamisd pET28a(+)
2.4. Kt qu th hot tnh enzyme endo-1,4-betaglucanase thu c
2.4.1. Kt qu nh tnh kh nng sinh enzyme endo-1,4-betaglucanase
gen mc tiu c biu hin, chng ti tin hnh ha bin np vector ti t hp
trn vo chng biu hin E.coli BL21(DE3). Chng ny c cm ng bng IPTG 1,5mM
37
0
C trong 4h. Kh nng thy phn enzyme c kim tra bng phng php cy chm
im trn mi trng thch CMC 1%.

Hnh 6 Kt qu nh tnh kh nng sinh enzyme celulase ca EcoliBL21
2.4.2. Kt qu xc nh hot enzyme sinh ra bng phng php ng kh
Ngoi nh tnh kh nng sinh enzyme ca chng ti t hp. Chng ti s dng
phng php ng kh ca Bermeld nh lng hot tnh enzyme. Trc tin chng
ti xy dng th tng quan gia nng glucose chun v gi tr OD540nm. Da vo
phn ng ca thuc th DNS vi cc dung dch chun glucose, cng mu bin i
c xc nh bng phng php o mt quang bc sng OD
540nm
. T gi tr
OD
540nm
cc nng khc nhau, chng ti xy dng c th th hin s tng quan
gia nng glucose v gi tr OD
540nm
Nh vy plasmid sau khi x l bng enzyme cho hai
vch tng ng vi vch pha trn l plasmid pET28a(+) c
kch thc 5,4 kb, vch di l on chn c kch thc
khong 1600 bp. So vi plasmid pET28a(+) c ct m
vng ging 3 th vch plasmid sau khi ct ging 2 c kch
thc tng ng. iu chng t gen mc tiu c gn
chn vo plasmid pET28a(+).

T kt qu thu c chng ti kt lun vector
ti t hp pET28a(+)endo-1,4-betaglucanase khi bin
np vo chng E.coliBL21(DE3) c hot tnh cellulase.
Chng ti s tip tc s dng chng ny thc hin
cc th nghim kim tra hot tnh enzyme cellulase.

Tuyn tp Bo co Hi ngh Sinh vin Nghin cu Khoa hc ln th 8 i hc Nng nm 2012
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Hnh 7 th th hin tng quan gia nng glucose chun v gi tr mt quang 540nm


T kt qu OD ca mu th c enzyme cellulase (OD
540nm
= 0,306), mu th khng
c enzyme (OD
540nm
= 0,015) v ng chun, chng ti nhn thy hot ca enzyme
cellulase ca mu thu c l: x = 242,5 g/ml. iu chng t dch enzyme thu c
c hm lng enzyme cellulase cao nn kh nng thy phn ng tt.
2.4.3. Kt qu in di SDS-PAGE mu enzyme thu c
H enzyme cellulase c trng lng khong 50-55 kDa, nh gi kt qu to
dng chng ti tin hnh in di dch enzyme sau khi ta bng phng php in di SDS-
PAGE vi gel polyacrylamide 10%. Kt qu cho hnh nh sau:

Hnh 8 Kt qu in di SDS-PAGE dch enzyme cellulose sau khi ta bng acetone
Ging M: Thang chun protein(Fermentas)
Ging 1,3 : Dch enzyme thu c t E.coliBL21(DE3)
Ging 2,4: Dch enzyme cellulose thu c t dng E.coliBL21(DE3) biu hin
T kt qu thu c cho thy sau khi biu hin chng ti thu c phn ln l h
enzyme c kch thc nm trong khong 50 55 kDa ph hp vi kch thc ca h
enzyme cellulase. Vy chng ti to dng v bc u biu hin thnh cng on gen
m ha enzyme cellulase trong chng E.coliBL21(DE3).
3. Kt lun
Qua qu trnh nghin cu chng ti to dng thnh cng on gen m ha
enyme cellulase t Bacillus subtillis trong E.coliBL21(DE3). Bng cc k thut sinh hc
phn t chng ti xc nh on gen to dng l on gen mong mun. ng thi,
chng ti tin hnh th hot tnh ca on gen c to dng bng cc phng php cy
chm im, ng kh bc u xc nh hot tnh ca chng to thnh v kim tra
kch thc enzyme thu c bng in di SDS-PAGE.
Nhng th nghim trn l c s tip tc tin hnh kho st cc yu t nhit ,
mi trng, nng cht cm ng, sao cho thu c lng enzyme cellulase l ln
nht. Nhm mc ch phc v cho vic tin hnh sn xut ch phm enzyme cellulase
thng mi phc v cho th trng.
Tuyn tp Bo co Hi ngh Sinh vin Nghin cu Khoa hc ln th 8 i hc Nng nm 2012
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TI LIU THAM KHO
[1] Trn Th Ha, Phm L Thanh Mai, Hng Phc Ton, Nguyn Quc Bnh, Tng
hp cc cDNA m ha hai enzyme endoglucanase 1,2 ca Trichoderma Reesei bng
phng php overlap extension PCR (OE-PCR), Trung tm Cng ngh sinh hc
thnh ph H Ch Minh.
[2] Heckman, K.L and L.R Pease (2007), Gene splicing and mutagenesis by PCR-driven
overlap extension, Nat Protoc, 2(4):924-32
[3] GUO Xu-Dong, MAO Shu-Yan, HOU Dong-Xia, BOU Shorgan, A rapid method for
Preparation of plasmid DNA for secreening recombinant clones, Chinese jounarl of
Biotechnology, (2007) 23(1), 176-178.
[4] Wang Li, Wei-Wei Zhang, Ming-Ming Zang, Yu-Lin Chen, Cloning of
Thermostable Cellulase Gene from newly isolated Bacillus subtilis and its expression
in Escherichia coli, Mol Biotechnol, (2008) 40:195-201
[5] Deliang Yang, Haibo Weng, Minge Wang, Weihua Xu, Yaozhao Li, Huiling Yang,
Cloning and expression of a novel thermostable cellulose from newly isolated
Bacillus subtilis strain I15, Mol Biol Rep, (2010)37:1923-1929 DOI 10.1007/s11033-
009-9635-y.