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Heterologous expression and characterization of enoate reductase from Aspergillus oryzae RIB40

米曲霉(Aspergillus oryzae)RIB40中烯酮/烯酯还原酶的异源表达及性质分析



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2013
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ChineseJournalofBioprocessEngineering
Vol.11No.1
Jan.2013
doi:10.3969/j.issn.1672-3678.2013.01.008
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:2012-11-07
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,-./014567
(973
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(2011CB710801)
)*+,

â­}
(1987—),
L
,¸
«ôõK

PQ23&

23OP

&STU-§T)

ö÷þ

TÁK
),
23ü
,Email:zhu_dm@tib.cas.cn
U¹ˆ
(Aspergilusoryzae)RIB40
?VW

V|
Þß45€¬§¨71é>¢
…„†
1,2,
‡tˆ
2,
S
 
‰
2,
Z
 
[
2,
\]^
2,
…Š‹
1,
_`
2,
abc

(1.
fgdh/0 56230e

fg
300457;
2.
NDd0e fg2E56hiFGH 2EjDk23lmn

fg
300308)
B
 
C

ž„w
(Aspergilusoryzae)RIB40
ÇÎ{
DNA
FŽ

@A
PCR
èõ‘’‘;ûåQ
(AspER)
ÇÎ
(asper)
ˆ“”.[Z•a
pET32a(+)
–

T¶FL
BL21(DE3)
¡žR‰—˜[Z

@A
NiNTA
™üšRŠ‹
›Xˆ

Œ›“œ_
19
³

œ²YF
6062%。
=®æ+‡ˆŠ‹hižŸ
,AspER
ž3ra—˜ T

3
[®‹[¡QFLM·
NADPH
<¢XûåQ

šô
pH
F
70~80,
šô’“F
40℃。
S

¾‘’
Kmü
kcatc®£F(245±036)mmol/Lü(44±04)×10
3s-1。
«?R®‹€AspER
S¤Ay¥WUõ¦>?l
^_
õ¡S

§Ç¤Ay¥We.e.
c¨_·
99%。
DEF

ëS*ûå

‘’

‘;ûåQ

©ª«šQ
?G>HI
:Q78;Q81    
JKLMN
:A    
JOPI
:1672-3678(2013)01-0041-06
Heterologousexpressionandcharacterizationofenoate
reductasefromAspergilusoryzaeRIB40
ZHANGHailing1,2,GAOXiuzhen2,CHENXi2,RenJie2,FENGJinhui2,
ZHANGTongcun1,WUQiaqing2,ZHUDunming2
(1.ColegeofBiotechnology,TianjinUniversityofScience&Technology,Tianjin300457,China;2.NationalEngineeringLaboratory
forIndustrialEnzymes,TianjinInstituteofIndustrialBiotechnology,ChineseAcademyofSciences,Tianjin300308,China)
Abstract:ThegeneencodingenoatereductaseofAspergilusoryzaeRIB40,asper,wasamplifiedfromthe
genomeDNAandclonedonpET32a(+).EncodedproteinwasexpressedasasolubleforminE.coliBL21
(DE3).RecombinantnativeproteinwaspurifiedbyNiNTAafinitychromatography.Thenativeenzyme
wasNADPHdependentandexistedasadimerdeterminedbygelfiltration.Theenzymeexibitedmaximam
activityatpH7.0topH8.0at40℃.Thepurifiedenzymewasactivetomaleimideanditsderivatives.The
conversionandtheenantiomericexcesstoward2methylmaleimidewerebothhigherthan99%.
Keywords:asymmetricreduction;enoatereductase;oldyelowenzyme
  
yw`ڍn(Hhòp¾¿¶s”"Q
R–—

”ڍn‹Œ;<–Z«z.«`dם
h

Z‹Œ÷ÏÛºT)l`ª«õö

CåpA
ÅSTÀÖø*`AÅSTڍn(Hhhi
Öfg

µZAÅST*+*‰«`AÅSTÀ
Ó[¸¹

Hz…ù…ú

w–$Ñ.AÅ

wl
¿Eyë°âë°§•hi
[1-2],


y-5ÃÔ
gZd×òpÇì/ C Cڍn5Ã`&S


òpUAÅSTlÍ
[3]。
Cåùiyû

y
-5ÃÔST`˜Óª`h‚1Zi[º)Î
STÿŒ`
[4-7]。
ùió&º)8>òp:Þß
àáR}5ÃÏ{ α,β ÚÐǝoû,µZ5
à C CôÇ C Oô`T@àáRµz9,~
Z¨Öº)–5“”Y¥HÔ

!uw&Lw

CôÕw°@Á
[8-9]。
Ö§zp~dÇü

@ $
ÎKdýPjyû

y-5ÃÔ­s¯–²Tn
!þÜÝq•hi23

!u_‚ë³}ˆp
{Lh`4&

~Ú7eéh`T@ÇÞßà
áR

u)5‚‚e(§w°
[10-13]。
÷ø
AspergilusoryzaeRIB40
–`yû

y-
5ÃÔ­s§ï.ì

ÿϲT`ef­sÔ@
RxqÜÝ`¯¨

1 
QRSTU
1.1 
QR’•–
1.1.1 
%$ÇxS
佃
A.oryzaeRIB40,
æ¼¹œ)‚@!2MN
áf

h´%‚R‘%
E.coliDH5α、E.coliBL21(DE3)
Ç·¸ˆß
pUC19,
æ–,?@")tÔ,-)+ì
Ó\”“

.ìˆß
pET32a(+),
T=
Novagen
VW

1.1.2 
`a0
YPD
`a0
(g/L):
hiáC
5,
efg
10,
bcd
20,KCl2,KH2PO41,MgSO4·7H2O05,
FeSO4·7H2O002;pH55。
LB
`a0
(g/L):
hiáC
5,
efg
10,
NaCl10;pH70。
X&ñ100μg/mL,‚ƒñ
(Amp)。
1.1.3 
´«JÀÇä¬
ë°R0xÔ
KpnⅠ、EcoRⅠ、SmaⅠ,T=
Fermentas
VW
;PrimeSTARHSDNA
_‹ÔÇ
DNA
ÔòJÀW

T=
TaKaRa
VW
;RNA
Ô

T=
Sigma
VW
;DNA
]šY³JÀW

T=vÔ&T?;

«
T

˜ëVW
;NAD(P)H
Ç
NAD(P),
T=U,
Codexis
VW

*˜`Ü

T=
AlfaAesar
o
Sigma
Aldrich
VW

£,¹§À
GelRed,
T=
BiotiumInc
VW

xSáCJÀWÇefÙ^JÀW

T=«
T#ÖJÀ˜ëVW

–—â¯qT=U,
Eppendorf
Ç
Thermo
VW

:/‡åâ

T=I,
APV
VW

ef²TäÇ
Superdex20010/300GL,
T=U,
GE
VW

ԗä
SPECTRAMAXM2e,
T=U,
MD
VW

z¦§Ýä
Agilent7890,
T=MQ^VW

QRÏ
CPChiraSilDEX
(25m×025mm×025μm),T=I,VarianVW。
1.2 
TU
1.2.1 
0¨ù
DNA
`áC
A.oryzaeRIB40
%$Å
YPD
~ß`a0`a
2d(30℃、200r/min)
n
,4000r/min4℃
–—
10
min
³;%ß

%ß$Çn~2ȚÀ

{j
600μL³|~(50mmol/LNaOH,1mmol/LEDTA,
1% TritonX 100)
–.Ë

“(sߞ¯r
1%β
%0NY
;65℃

1h
n
,12000r/min
–—
10
min;
t™iM V;C

Én

+Y3Ý
DNA,
*#
DNA
3Ýi}˜
100μg/mLRNAÔ`.°x

,37℃

1h

DNA
ºÿ[{|

1.2.2 

asper`
·¸
Ôê
A.oryzaeRIB40


y-5ÃÔ0¨
(GenbankID:XM_001727598.1)
½ŒÍ6¶Ö
:5′
GGGGTACCGACGACGACGACAAGGGATCCATC
TCTTCCACATC 3′(
D7Ëà¯Ö
KpnⅠÔxÑ/,
Þßà¯ÖR(ÔÔxÑ/

Ç
5′CGGAATTCCT
AAAGTGCACGCCAGAACT 3′(
D7Ëà ¯ Ö
EcoRⅠÔxÑ/)。p A.oryzaeRIB400¨ù DNA
ÖDü
,PCR
‹e56Ö
:94℃
Ï+R
5min;98℃,
30s;55℃,45s;72℃,100s;30
"¶

‹ewi
}˜£,¹§À
GelRed` 08%
¼Òd]šþ×å
™“Y³

²Tn`
DNA
»¼ÔòÌ
pUC19(SmaⅠ
(Ôx

ËRˆßt

ÔòwVs
E.coliDH5α£
yoº)–

”}˜
100mg/L
øi‚ƒñ
、40mg/L
Xgal
Ç
100μmol/LIPTG`LBûüt37℃`a16
h,
<â‹àf§·¸­s
PCR
ӝ

)R·¸xS
­s™½Ç½Œ¯¨

*ÚÖ
pUCasper。
1.2.3 asper
0¨.ìˆß`¿ÀUef
AspER
`
.ì²T
.ùxS
pUCasper
Åë°R0xÔ
KpnⅠÇ
EcoRⅠˆTnÔò̾90xԈT`pET32a(+)
t

#Ì`.ùxS
pET32a(+)asper
Vs
E.coli
DH5α£yoº)–,”}˜ AmpXR` LBûü
t
37℃
`a

áxSn­súÔxӝ

z)Rx
SVTÌ
E.coliBL21(DE3)
–#Ì.ù)+%

z(%Šò{j}˜
Amp` LB
`a~`J’
–
,37℃
`a*Ã

p
1%
ò{^ò{j}˜¾9X
24
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&ñ`
800mLLB
`a0
,37℃
D
200r/min
+«`
al
OD600Ö06~08¿(s01mmol/LIPTG,37

FC.ì
6h。
`an`%ߔ
7000r/min
–—
15min
`56³;nÅƓ.ËjÍÎ~
A(20
mmol/LTrisHClpH74,250mmol/LNaCl)
–

:/
‡åÉʺ)

*#º)³|~”
4℃

14000
r/min
–—
30min
nCt™
,045μmo™*on­
sjDž¨²TC`ef

,-œ³»³Å¥G#C
`ef

*#C`ef­s
SDSPAGE
å™

“Ôê
efÙ^JÀW`~ÕÐ+™Ùefœ³

50%
SA”“”
-20℃
–±i

1.2.4 
Ô1™Ùqª2@Êr™Ù
AspER
Ô1R`—Õå™:ióºüh

”Ô—
ät­s

hßÁÕߞÖ
200μL,T}5mmol/L

¶ yû
(DMSO
{|
),05mmol/LNADPH
Ç
100
μg/mLAspER。hßÁ`ì°:i20mmol/LTris
HCl
ÍÎ~
(pH74)。
h`4&p(s
NADPH
ÕÖ


hµ*Ԍ/™
25℃
D
340nm
Æɪ`+T
ΙÙԍÜ`1R‚‘
。NADPH
)µˆÉÁrÖ
622/(mmol·cm)。
d"Ô12(Ñ
(U)
ÙÒÖÓ¯Ô0
ˆ»
1μmolNADPH*‰`Ô^。
AspER
Ü

¶ yû`ª2@Êr™Ù
O„¾9¬ËtuÔ1¯¨O„­s
。2
¶ y
û`œ³./ÍÙÖ
01~10mmol/L。
hƒ³
ÙÒÖӯԈ»
NADPH
`0)µr

:iú1
r„6®
KmÇVmax。
1.2.5 
Ô_;…o`×Ù
AspER
”v>…oD`_;…oµ*ዯÄz
–έs×Ù

*i…¨ÏÖ
Superdex20010/300
GL,
Ъ¦Ö
50mmol/L
Ë,ÌÍÎ~


150
mmol/LNaCl)。
—ÕefÖ
:Ovalbumin(43×104),
Conalbumin(75×104),Conalbumin(158×105),
Feritin(44×105),Thyroglobulin(669×105)。
Ôê
—ÕefUC`ef`”Ñߞc®
AspER
`¦¯
Äx^

1.2.6 pH
Ǎ³Ô1`€
µ*™ÙÚ¾
pH
ÍÎ~–
AspER
`Ô1

×
Ù¹ef`_4
pH。
:i`ÍÎ~¯qÖÏÐ,
Ë,ÍÎ~
(pH22~80)
Ç
TrisHCl
ÍÎ~
(pH
72~94)。
_4h³gZµ*™ÙÚ¾³
D`Ô1×Ù

³./Ö
25~45℃,5℃
Öd"
ÕÖ

Ö§r3•³ØÙR

dٜ³`ef{
~”Ú¾³D&Ã
100min,
Ó
20min
C9

É

!dÉC9¿&Ã
10min),
å™ef”
25℃
D
`$ÚÔ12

p&Ãå`Ô12Ö
100%)。

™ÙO„]¬Ë
1.2.4
—ÕÔ1™Ù56™Ùtu
Ú¾56`Ô1

Ó"h

"ûs

1.2.7 
Üݯ¨
¬Ë
1.2.4
Ô1™ÙO„ìÓ\/˜`
α,βÚÐdz0âT‹­sÔ1Rå™pr3
AspER
`ÜÝ

;ÌÜx{R¥

*˜`Ü
]{|ÌYZ0[(DMSO)
ߞ¯r
1%
–

•
ŸŒ¯{|”
pH70
Ë,ÌÍÎ~

1.2.8 
ÜVT°Çw
e.e.(
o
d.e.)
ª`™Ù
AspER
˜1R`QRÜ`ÞßàáR`¯¨
:ibcd¥HÔºT`Ûԛ&ßÁ­s
。1mL
hßÁ
(100mmol/L
Ë,ÌÍÎ~
,pH70)
–T}

bcd
9mg,D
bcd¥HÔ
1mg,β NADP1mg,
AspER150μg,Ü2mg。ÍØ`h”37℃、200
r/min`
56Dh
24h
ni¦¾ßž`Z0®O0
q;C

*#˜â¦iƒxú,ÜÇonz¦§Ýä
å™ÜVT°Çw
e.e.(
o
d.e.)
ª

2 
XY’Z[
2.1 AspER
5§¨’Gp
;Ìefò{.ìq²T`‰«

”ˆß¿
À*+–:i§
pET32a(+)
t`
Histag
Ç
Trxtag。
AspER,
”
E.coliBL21(DE3)
–pò{‘’.ì

Å
NiNTA
²T

ef²³á:
19
†

Y³°Ö
6062%;
Å¥n`efÅR(Ôxp

|2‹.ì
`—3n

Õ12á:Ì
53265U(
.
1),
¾²Tå
DzTnŠxp

|—3¿1:
,¨
©æÖef

|2‹.ì`—3€§ef`1R
[14]。
Ôêef
`ø0,$0rϙef(߂‘Ö
471×103,
U
SDSPAGE
å™áâ

ð
1)
@2

Ôê¯Äï]š…
¨áâc®
,AspER
`¦¯Äx^Ö
91×104,
æ
©òc?¹ÔpY_ߑ’“”

íp
NADPH
Õ
ÖÛÔ¿

Ô`12ÂÖ
NADH
ÕÖÛÔ¿Ô12`

†


AspER
ÖÒØj
NADPH
`ôT5ÃÔ

§
1 AspER
5GpXY
Table1 PurificationofAspER
¾12

(U·mg-1)
Õ12


²T
†r
Y³°


šÔ
0.885 40.858 1 100
NiNTA
²T
1.680 24.766 1.90 60.62
R(Ôx4
3.368 53.265 3.80 121.05
34 
!

#
  
â­}â

佃
(Aspergilusoryzae)RIB40
–yû

y-5ÃÔ`ï.ìqRx¯¨
M—
—Õef
;1—
šÔ~
;2—Ni NTA
²Tn`ef

3—
R(Ôxp²Tnef

|2‹.ì`—3n`ef
G
1 AspER
5
SDSPAGE
þšXY
Fig.1 SDSPAGEofAspER
2.2 
45XY¼@
pH、
rs’rsZi1
ð

Ö
AspER
Ô`_4h
pH、
³q³
ØÙRáâ

æð

òó

p

¶ yûÖÜ

AspER
”
pH70~80
¿./0§_:1R

Áj
pH50`
56DÔÿ[#1

ð
2(a))。
Ô`_4
h³q³ØÙRO‡
,AspER
”
40℃
./0
§_:1R

ð
2(b)),
”
25~40℃
./&Ã
100
min
nÔ1ØÙ


45℃

10min
nÔ1#
˜z@
40%,40min
nÔ0Âÿ[#1

ð
2(c))。
2.3 
4»ÒvӃKši
Ôêú1rð

ð
3)

AspER


¶ yû
`
KmÇ kcat¯qÖ(245±036)mmol/LÇ(44±
04)×103s-1。
3­èéæóyû

y-5Ãԍ

¶ yû`
KmÖ 001~55mmol/L
[15-19]。
AspER


¶ yû`
KmÇ kcatUÎïj
Lactobaciliscaseistr.Zhang
`yû

y-5ÃÔ
(LacER)
¦@
,LacER
Z
NADH
ÒØE

u
AspER
È
ýPjp
NADPH
ÖÛÔ
[22]。
2.4 
45}~€1
i²Tn`
AspER
Ú¾{×`α,β ÚÐÇ
³0T‹

×

¶û×

¥Î„[6q•r&


â,Ç-â

­s§ÜÝïà

p
AspER


¶ yû`¾1ÙÒÖ
100,
6®¹Ô*˜Ü
`¦Ô1

.
2)。
æ.

òó
:AspER
òpS
T¶…yû` C Cúô`5Ã,¶tCõ0`Ñ
؍ST1R˜÷‚`€

¡

Z0¶yûò
pe5Ã



Z0¶yûgf˜1R

¹Ô
ÒÓv`Çû˜ST1R

µÚºSTL0C
õ`α,β ÚÐǝ、û、â,q•-` C Cúô
`5Ã
。AspER
¥Î„[6q•r&˜:
G
2 pH
’rst
AspER
4u5wx74uZi1

Ú

[ãVWÜ}~

Fig.2 EfectsofpHandtemperatureonAspER
enzymeactivitiesanditsstabilities
G
3 
\\K)GUN]
AspER
4»ÒvӃK
Fig.3 LineweaverBurkplotofAspER
`1R

Cõ0`ÑØq‚‘Ô1˜Ú¾+³`

。AspER
5õ¥Î„[6Ç5õ¥Î,6

pqL0¥Î„[6ÇL0¥Î²6˜¦2`
1R

µ¥Î„[6Ç

Z0¥Î„[61˜
44
&
 

 
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:`1R

u¥Î,6Ç

Z0¥Î,69f˜
1R

lj
AspER
¥Î„[6Ç¥Î,6./
0¡©˜7/ã`è

Cå5ڙ8

§
2 AspER
5}~¡hiXY
Table2 SubstratespectrumofAspER
Ü w ¦12
/%
100

18.2

23.0
7.1
103.5





Œ.
Ü w ¦12
/%

541.0
125
112.1
213.3
129.2


214
129
2.5 
ÅpĒ
e.e.(^ d.e.)
Æ>¢
i
AspER
Br "˜1R`α,β ÚÐdz
0T‹.§STh

p—ÕÜLjýßw
֍Ë

iz¦§Ýä噧Žd`VT°Çw
`
e.e.(
o
d.e.)
ª
[20](
.
3)。
æj

Z0¶y
ûw`ˆýߔѿÕò@

ÚºzŽd¯Ð

*pƒ„×ÙÜVT°Çw
e.e.
ª

æjf˜
5õ¥Î,6`ˆýß

*pf˜å™5õ¥Î,
54 
!

#
  
â­}â

佃
(Aspergilusoryzae)RIB40
–yû

y-5ÃÔ`ï.ìqRx¯¨
6`háâ

æ.

î0
:AspER


Z0¥Î
„[6—˜:`VT°ÇÞßàáR
(e.e.
ª]
:j
99%);


a9û`VT°:j

a9û

µ—˜@2`ÞßàáR



L0¥Î„[6`
VT°8>:

µÞßàáRÁ

§
3 AspER
‘pÞß¼@5-./01
Table3 Stereoselectivityofbioreduction
catalyzedbyAspER
Ü VT°
e.e.(
o
d.e.)
ª
2methylmaleimide >99% >99%
R carvone >99% 75.5%
S carvone 89.6% 77.4%
2phenylmaleimide >99% 10.4%
 
3 
X
 
[
p佃
(Aspergilusoryzae)RIB40
0¨ù
DNA
ÖD:

Œ·¸

.ìǯ–²T0d{y
û

y-5ÃÔ
AspER。
¹Ôòpùi
NADH
Ç
NADPH
ÕÖÛÔ

µ
NADPH
˜:`ÞßR

AspER
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[1] TutleJB,OueletSG,DavidW.Organocatalytictransfer
hydrogenationofcyclicenones[J].JAmChemSoc,2006,128
(39):1266212663.
[2] OueletSG,TutleJB,DavidW.Enantioselectiveorganocatalytic
hydridereduction[J].JAmChemSoc,2005,127(1):3233.
[3] WiliamsR,BruceN.Newusesforanoldenzymetheoldyelow
enzymefamilyofflavoenzymes[J].Microbiology,2002,148(6):
16071614.
[4] SwiderskaMA,StewartJD.Asymmetricbioreductionsofβnitro
acrylatesasaroutetochiralβ2aminoacids[J].OrgLet,2006,
8(26):61316133.
[5] WadaM,YoshizumiA,NodaY,etal.Productionofadoublychiral
compound,(4R,6R)4hydroxy2,2,6trimethylcyclohexanone,by
twostep enzymatic asymmetric reduction[J].Appl Environ
Microbiol,2003,69(2):933.
[6] KataokaM,KotakaA,HasegawaA,etal.Oldyelowenzyme
fromCandidamacedoniensiscatalyzesthestereospecificreduction
oftheC Cbondofketoisophorone[J].BiosciBiotechnol
Biochem,2002,66(12):26512657.
[7] OhtaH,KobayashiN,OzakiK.Asymmetricreductionofnitro
olefinsbyfermentingbakers′yeast[J].JOrgChem,1989,54
(8):18021804.
[8] MülerA,HauerB,RoscheB.Enzymaticreductionoftheα,β
unsaturatedcarbonbondincitral[J].JMolCatalB:Enzymatic,
2006,38(3/4/5/6):126130.
[9] HalM,HauerB,StuermerR,etal.Asymmetricwholecel
bioreductionofan [alpha],[beta]unsaturated aldehyde
(citral): competingprimalcoholdehydrogenaseandC Clyase
activities[J].Tetrahedron:Asymmetry,2006,17(21):
30583062.
[10] ManganD,MiskelyI,MoodyTS.A threeenzymesystem
involvinganenereductaseforgeneratingvaluablechiralbuilding
blocks[J].AdvSynCatal,2012,354(11/12):21852190.
[11] BrennaE,GatiFG,ManfrediA,etal.Enoatereductase
mediatedpreparationof(S)methyl2bromobutanoate,auseful
keyintermediateforthesynthesisofchiralactivepharmaceutical
ingredients[J].OrgProcResDev,2012,16(2):262268.
[12] YantoY,WinklerCK,LohrS,etal.Asymmetricbioreductionof
alkenesusingenereductasesYersERandKYE1andefectsof
organicsolvents[J].OrgLet,2011,13(10):25402543.
[13] MuelerNJ,StuecklerC,HauerB,etal.Thesubstratespectraof
pentaerythritoltetranitratereductase,morphinonereductase,N
ethylmaleimidereductaseandestrogenbindingproteininthe
asymmetricbioreductionofactivatedalkenes[J].AdvSynCatal,
2010,352(2/3):387394.
[14] 
;É<

Ôò=
.N
À|—3Kúô5efhã/`
€
[J].
&œ@è
,2009,25(
e>
1):371372.
[15] ChaparoRiggersJ,T Rogers,E VazquezFigueroa,etal.
Comparisonofthreeenoatereductasesandtheirpotentialusefor
biotransformation[J].AdvSynCatal,2007,349:15211531.
[16] Fitzpatric,K T,NAmrhein,MacherouxP.Characterizationof
YqjM,anoldyelow enzymehomologfrom Bacilussubtilis
involvedintheoxidativestressresponse[J].JBiolChem,2003,
278:1989119897.
[17] FrenchCE,BruceNC.Purificationandcharacterization484of
morphinonereductasefrom PseudomonasputidaM10[J].
BiochemJ,1994,301:97103.
[18] FrenchC,NicklinS,BruceNC.Sequenceandpropertiesof
pentaerythritoltetranitratereductasefrom Enterobactercloacae
PB2[J].JBacteriol,1996,178:66236627.
[19] BrownBJ,ZDeng,PAKarplus,etal.Ontheactivesiteofold
yelowenzyme[J].JBiolChem,1998,273:32753.
[20] GaoX,RenJ,WuQ,etal.Biochemicalcharacterizationand
substrateprofilingofanewNADHdependentenoatereductase
fromLactobaciluscasei[J].EnzymeMicrobTechnol,2012,51:
2634.
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