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Expression of carbonyl reductase in Bacillus subtilis with different promoters

不同启动子调控羰基还原酶基因在枯草芽胞杆菌中的表达水平



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ChineseJournalofBioprocessEngineering
Vol.11No.3
May2013
doi:10.3969/j.issn.1672-3678.2013.03.009
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:1672-3678(2013)03-0046-06
ExpressionofcarbonylreductaseinBacilussubtiliswithdiferentpromoters
WANGYanna,ZHANGLiang,DINGZhongyang,SHIGuiyang
(NationalEngineeringLaboratoryforCerealFermentationTechnology,
KeyLaboratoryofIndustrialBiotechnolgyoftheMinistryofEducation,JiangnanUniversity,Wuxi214122,China)
Abstract:InordertoachieveitshighexpressioninBacilussubtilis,carbonylreductasegenemldhwas
amplifiedbyPCRwiththetemplateofchromosomeDNAfromMorganelamorganiCMCC(B)49208
andconnectedwithpromoterPQandpromoterp43toobtaindiferentexpressionplasmidsPHYp43
mldh,PHYPQmldh,PHYp43p43mldh,andPHYp43PQmldhSubsequently,theserecombined
vectorsweretransformedintoBsubtilisWb600SDSPAGEanalysisofrecombinants.Andwholecelular
biotransformationshowedthattherewasdistinctivediferenceamongthefourrecombinants,inwhichthe
BsubtilisWb600(PHYp43p43mldh)couldobtainthehighestconcentration(1421mg/L)ofd
pseudoephedrineinconversionsolutionandthemoleratioofsubstratewas7825%
Keywords:carbonylreductase;Bacilussubtilis;promoter;dpseudoephedrine
  
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p43
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PQ,
ʼ^2h5=ýe

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ƸX¶3Õô†àΒÿeó©{[¢h3}
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O
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òóô
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(g/L):
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10,
JÈp¤
5,
NaCl10;
Ëî¹eòóôPc(ã
15g/L
Ä`k

1×105Pa
Å[
20min。
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{[hòó

f²P

(ãUÛ!
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æ–‡
25μg/mL‚Y±ª[h“ü。
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DNA
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(E)
O

ÓÎN/
(PE)
ˆŽ§3ŠèY¢–‡Š&
ˊ¨+
;1
ýô

à:ôìN
(MAK)
Œ
Ë

Î
NADH、
õö÷³xÎ
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èY
Sigma
ï
ð

d:¤‡ŠfŖ×ÙlÛhñí×O&ˆ
¤

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RS
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DNA


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pET28amldh
½h6hv
mldh
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mldhup(GCCG
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O
SmaI
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TACCTATAATGGTACCGCTATCACT),
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­bÊËÎ
BamHI
O
SalI
ÎÚ9é

[ÿZ‘½
1&)&)+jÊïð•“

123 
±ª[hά¨
N¤¸å±ªÿeó©{[̈XC[Š

M
ãÂj
Tet(25μg/mL)h LB¿eòóô,37℃、
200r/min
òó
12h。


‰
1%
hMd–ÌM¯
Âj
Tet(25μg/mL)h LB¿eòóô,37℃、200
r/min
òó
12h。
WšÓÔ[e

‚ŽW%&¿
(02mol/LKH2PO4Na2HPO4%&¿ pH75)ÊÙ
74 
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10h。
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10min
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O
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hC[ŠMdž
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òó
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[¿
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’
37℃、200r/min
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Q
1%
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MŸ±jòó¯
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ò
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ŠŠ

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_`Qab
21 PHYp43mldh
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Q
Morganelamorgani
Þne
DNA
Å µ

Q
mldhup
O
mldhdown
Å[
,PCR
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h
mldh
3Êôžv

׈¡‚
BamHI
O
SmaI
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BamHI
O
SmaI
óÎÚhýe
PHY
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¶M

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EcoliJM109
ÏÇب©

’Âj
25μg/mLh Tetyåuµ½“ü¸å̈
X

”•±ªæÛ
PHYp43mldh。
p¤æÛ
PHYp43mldh,BamHI
O
SmaI
óÎ
Ú

$Ü[Ï3@46
1。
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á7

óÎÚ¡æ
3Yôx
,5165bp
hvÅýe
PHYp43
h0ß

1101bp
hvÅ6hôž
mldh
h0ß

M—
ÐÑ
DNA;
1—
æÛ
PHYp43mldh
~
BamHI
O
SmaI
ÎÚ3@
D
1 
‘lf?^#
PHYp43mldh
;ŠH*+
Fig.1 Identificationofrecombinantexpressionvector
PHYp43mldhbyenzymedigestion
22 PHYp43PQmldh
Å
PHYPQmldh
;OP
221 PHYp43PQmldh
hʼ
›œ


PMDPQGm
‚
NcoI
ÎÚ

Àu

¢¡¯
ŽWˆ

5Åýe

2PQ
Morganelamorgani
Þn
e
DNA
Å µ

Q
mldhup
O
mldhdown
Å[

‚
pfu
Î
PCR
ãuæ
1101bp
h
mldh
3Êôžv

׈¡Yzlv¶M

¶M×̈
EcoliJM109
Ï
Çب©


20μg/mLhGmyåuµ,“ü¸å
̈X

”•±ªæÛ
PMDPQmldh。
p¤æÛ
PMDPQmldh,BamHI
ÎڐXí
.

$Ü[Ï3@46
2。
‘6

á7

ÎÚ¡æ3
3813bp
O
1126bp2
ôv

›;
mldh
íG~ã
ýe
PMDPQGm。
M—
ÐÑ
DNA;1—PMDPQmldh
~
BamHI
ÎÚ3@
D
2 
‘lf?^#
PMDPQmldh
;ŠH*+
Fig.2 Identificationofrecombinantexpressionvector
PMDPQmldhbyenzymedigestion
84
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11
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da

p¤±ªæÛ
PMDPQGm
‚
SmaI
Î
Ú

pUÓ
1224bp
hv
PQmldh,
pUÓ$Ü[
Ï3@46
3;
K2ó‚
SmaI
ÎÚhýe
PHYp43
֦M

̈
EcoliJM109
ÏÇب©


25
μg/mLhTetyåuµ,“ü¸å̈X,”•±ª
æÛ
PHYp43PQmldh。
p¤æÛ
PHYp43PQmldh,
ñ­‚
SmaI
O
BamHI
ÎڐX

$Ü[Ï3@46
4。
‘6

á
7
:SmaI
ÎÚ"¡
,1224bp
hvÅ
PQmldh,
5165bp
hvÅ
PHYp43,
áX;
PQmldh
“|
¶Mžýe
PHYp43
½
;BamHI
ÎÚ"¡
,6266bp
Å
PHYp43mldh
h0ß
,123bp
Å
PQ
h0ß

Á
bôxÁßpq”•a^ž


SmaI
O
BamHI
ÎÚ3@

›;
PQmldh
íG~ãýe
PHYp43。
M—
ÐÑ
DNA;1—PMDPQmldh
~
SmaI
ÎÚ3@

2—PQmldh
æÛ
DNA
D
3 
_`
PQmldh
;a™!
Fig.3 GelextractionofPQmldh
222 PHYPQmldh
hʼ
p¤æÛ
PHYp43PQmldh,
‚
PstI
ÎÚ

pU
Ó
5032bp
hv

¢¡’öۈ

̈
Ecoli
JM109
ÏÇب©

>ÂY
25μg/mLhTetyåu
µ

“ü¸å̈X

”•±ªæÛ
PHYPQmldh。
p¤æÛ
PHYPQmldh,
‚
PstI
ÎÚ

$Ü[Ï
3@46
5。
‘6

á7
:1356bp
ÅÚjhÂ
p43
hv0ß
,5032bp
Å
PHYPQmldh
h0ß

23 PHYp43p43mldh
;OP
p¤æÛ
PHYp43mldh,
‚
BamHI
ÎÚ

À
u

¢¡¯ŽWˆ5Åýe

QæÛ
PHYp43
Å 
µ
,p43up
O
p43down
Å[

‚
pfu
Î
PCR
ãu
æ
290bp
hƸX
p43
5Åv

Yz¶M¡Ìˆ
EcoliJM109
ÏÇب©

’Âj
25μg/mLTeth
M—
ÐÑ
DNA;1—PHYp43PQmldh
~
SmaI
ÎÚ3@

2—PHYp43PQmldh
~
BamHI
ÎÚ3@
D
4 
‘lf?^#
PHYp43PQmldh
;ŠH*+
Fig.4 Identificationofrecombinantexpressionvector
PHYp43PQmldhbyenzymedigestion
M—
ÐÑ
DNA;1—PHYp43PQmldh
~
PstI
ÎÚ3@

2—PHYPQmldh
~
PstI
ÎÚ3@
D
5 
‘lf?^#
PHYPQmldh
;ŠH*+
Fig.5 Identificationofrecombinantexpressionvector
PHYPQmldhbyenzymedigestion
yåuµ½“ü¸å̈X

”•±ªæÛ
PHYp43p43mldh。
p¤æÛ
PHYp43p43mldh,BamHI
O
SmaI
óÎÚlvX

$Ü[Ï3@46
6。
‘6

á
7
:5165bp
hvÅ
PHYp43
h0ß

9
1391bp
hvÅ
p43mldh
h0ß

›;ƸX
p43
íG~
ãýe
PHYp43mldh。
24 
‘lT;*+
  


d±ªæÛ
PHYp43mldh,PHYPQ
mldh,PHYp43p43mldh
O
PHYp43PQmldh,
ˆŽ
Ìÿeó©{[¡§±ª[êë¿lv
SDS
PAGE
‹>¨©&̈.

241 SDSPAGE
ñí
¤±ª[O§3[hêë¿lv
SDSPAGE
ñ
94 
!

# mØ»{
:^
2ƸX¨zÕô†àÎôž’ÿeó©{[¢h5=tu
M—
ÐÑ
DNA;1—Pp43mldh
~
BamHIandSmaI
ÎÚ3@
D
6 
‘lf?^#
PHYp43p43mldh
;ŠH*+
Fig.6 Identificationofrecombinantexpressionvector
PHYp43p43mldhbyenzymedigestion
í

3@46
7。
‘6

á7

±ª[’
425×104
Kæ3;
K(
[13]
)*hÕô†àÎ
h0ß÷Ñ

9
BsubtilisWb600
O
BsubtilisWb600
(PHYp43)
’
425×104
Kϙx

›;
p43
O
PQ
ƸX“|QƸí
mldh
ôžh5=

1~6—Wb600、Wb600(PHYp43)、Wb600(PHYp43mldh)、
Wb600(PHYp43p43mldh)、Wb600(PHYp43PQmldh)、
Wb600(PHYPQmldh)
±ª[êë¿
;M—
ÐÑÀf
D
7 
‘lTbcB;
SDSPAGE

Fig.7 SDSPAGEofrecombinantstrainsbrokenliquid
242 
±ª[Öhά¨
¤

d^25=ýeh±ª[
BsubtilisWb600
(PHYp43mldh),BsubtilisWb600(PHYp43p43
mldh),Bsubtilis Wb600(PHYp43PQmldh),
BsubtilisWb600(PHYPQmldh)
lv>¨©Ìˆ.
¡h½«¿lv
HPLC
[Ï3@45
1。
‘5

á
7
:^
2ƸX+¨zÕô†àÎh5=ƒn^2

kPƸXh¨z5=ƒn3YC!ƸX

d¢±
ª[
BsubtilisWb600(PHYp43p43mldh)
lv>¨
©Ìˆ.

̈¿¢

ÓÎN/h‡\‚3


1421mg/L,
+̈*Å
7825%。
f
1 
‘lT;Šª*+
Table1 Enzymeliveappraisalofrecombinantstrains
±ª[Ö
ρ(dÓÎN/)/
(mg·L-1)
+̈*


PHYp43mldh 772 4250
PHYp43p43mldh 1421 7825
PHYp43PQmldh 1364 7510
PHYPQmldh 547 3010
-*ƸXhèˆ

j}Qp3íÕô†àÎ
ôž
mldh
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9b½ìY¦(
Î

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gdh
h5
=–¶‚lÎhgÖuv

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!hST

M+‡、
òóôõ{

§dIJôõ
lvèˆ

Q#•žiçh3@

25 
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BsubtilisWb600
O

d±ª[h&[¸nŽ
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+N

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á7

§3[O±ª[&
[Þ#hƒˆór0q÷2
,BsubtilisWb600
’
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2h
l㧊&[#
,5h
§Š&[#3—

9

d±ª[
BsubtilisWb600(PHYp43mldh),
BsubtilisWb600(PHYp43p43mldh),Bsubtilis
Wb600(PHYp43PQmldh),Bsubtilis Wb600
(PHYPQmldh)
÷g§3[ñ­s¡íÄ
1、0、1
O
1h,
§Š&[#3—Y
5~6h
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ôžhpãÖÔj’Ã0+\½STž[eh&
LLM

D
8 BsubtilisWb600
őlTU;5ր
Fig.8 GrowthcurvesofBsubtilisWb600
andrecombinantstrains
05
&
 

 
(
 
)
 
*
 
+
  
!
11
"
 
26 
‘lTU;Q+N
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5

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±ª[Ö
BsubtilisWb600(PHYp43
mldh),BsubtilisWb600(PHYp43p43mldh),
BsubtilisWb600(PHYp43PQmldh),Bsubtilis
Wb600(PHYPQmldh)
’¶ãòó
60h
¡hm
´Ýåñ­Å
96%、97%、97%、94%(
5
3),
›;±ªæے
BsubtilisWb600
¢áQ݁m
´

ñ­üýòó
36
O
60h
P[ÖhæÛlv


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f
2 
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Table2 Stabilityofrecombinantstrains
òóPw


yå[ŠŠ
PHYp43mldh PHYp43p43mldh PHYp43PQmldh PHYPQmldh
12 100 100 100 100
24 100 100 100 99
36 99 98 100 99
48 98 98 98 97
60 96 97 97 94
3 
_
 
b
ʼh±ª5=ýe
BsubtilisWb600(PHY
p43p43mldh),
‘YƸXhkP5‚
,d
ÓÎN
/ז•žíilc–hp3

‚=ž
1421
mg/L,
+̈*Å
7825%,
Ʀ·ôžhpãÖ
Ôj’Ã0+\½STž[eh&LLM

±ªæ
ے
BsubtilisWb600
¢áQ݁m´

‚ƒHI

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