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Development of biocatalytic process for t-butyl 6-cyano-(3R,5R)-dihydroxylhexanoate

生物催化法合成6-氰基-(3R,5R)-二羟基己酸叔丁酯



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ChineseJournalofBioprocessEngineering
Vol.11No.1
Jan.2013
doi:10.3969/j.issn.1672-3678.2013.01.006
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E.coliBL21/pCDFDuetgdh cr X
¹[ZPQKrX


(5R)


úÇ:();ë
S*ûå2Ú


(3R,5R)
3&Ç:();

hi[

TLa"b
485g/L、
ýþÿd«?[b¬“F
1∶1、
’“
28℃、pH70
‚ƒ¸
,800g/L6

(5R)


úÇ:();>?ûå
2h
ˆ

«?WXY
RZ
990%,
G?
d.e.
c¶·
995%。
T]^_`a
,NADP+
"bSrXùYb4cø"

DEF
:6

(3R,5R)
3&Ç:();

úÇûåQ

ýþÿ!"Q

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JOPI
:1672-3678(2013)01-0029-06
Developmentofbiocatalyticprocessfortbutyl6cyano
(3R,5R)dihydroxylhexanoate
XIAOLi,WANGYajun,CAOZheng,LIUZhiqiang,ZHENGYuguo
(EngineeringResearchCenterofBioconversionandBiopurificationoftheMinistryofEducation,InstituteofBioengineering,
ZhejiangUniversityofTechnology,Hangzhou310014,China)
Abstract:Acoexpressionstrain,E.coliBL21/pCDFDuetgdhcrX,caryingcrXandgdhgenes,
displayinggooddiastereoselectiveRketoreductaseactivitytowardstbutyl6cyano(5R)hydroxy3
oxohexanoate.Efectsoftemperature,pHcelloadingandconcentrationsofglucose,substrate,NADP+on
biotransformationeficiencywereinvestigated.Resultsindicatedthatundertheoptimalconditionsof
28℃,pH70,massratioofglucosetosubstrateat1∶1(w/w),80.0g/Ltbutyl6cyano(5R)hydroxy
3oxohexanoatewascompletelyconvertedtotbutyl6cyano(3R,5R)dihydroxylhexanoatein2hwith
d.e.over99.5%.Inaddition,nosignificantimpactofNADP+ concentrationwasobservedinthetest
rangeontheasymmetricreductionoftbutyl6cyano(5R)hydroxyl3oxohexanoate.
Keywords:tbutyl6cyano(3R,5R)dihydroxylhexanoate;carbonylreductase;glucosedehydragenase;
coexpression
  
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E.coliBL21/pCDFDuet gdh cr X,
÷ø
z}T
E.coliBL21/pCDFDuetgdh cr X
[º)
ڍn5Ã
(5R) 1
°±
(3R,5R) 2,
•h*+

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G
1 
û§¨c(
E.coliBL21/pCDFDuet gdh cr X
®tàÞß
(5R)1
‹Œ
(3R,5R)2
Fig.1 Schemefor(3R,5R)2synthesisthrough
asymmetricreductionof(5R)1with
E.coliBL21/pCDFDuetgdhcrX
1 
QRSTU
1.1 
c(
E.coliBL21/pET28a cr X
U
E.coliBL21/
pCDFDuet gdh cr X
撓)t‚@&)+
23*¿À

””

1.2 
“”%†"
»‡`a0ùŒ
(g/L):
efg
100,
hiš
50,NaCl100,
¼Ò
200。
LB
`a0
(g/L):
¾efg
100、
hiš
50、
NaCl100。E.coliBL21/pET28a cr X
`a0½
%n(s
30μg/mL•–ƒñ,E.coliBL21/
pCDFDuet gdh cr X
`a0½%n(s›x^
œ³
50μg/mL4ƒñ。
1.3 
)C*+
4Ė

•–ƒñ
、IPTG
âJÀ

T=
TaKaRa
VW
;NADP+、NADPH,
æ|}>VWáf
;(3R,
5R) 2,
T=
Toronto
T@°l23VW
;(5R) 1

}^
735%),
撓ŽH¿ntÀÁ˜ëVWÂ
Ã

¾efgÇhiš

T=
Oxoid
VW

•ÚT@
JÀ]Ö,w¯¨²

1.4 
“”TU
{Ä~°±

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Šò{jó˜
100mL
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500mL
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4h`a

¬ßž¯r
50%
`ò{^ò{jó
˜
100mL
4h`a0`
500mL
R€–

Øj‚
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a
2h
nl
OD600Ö08õö,(
s
IPTG
FCÀ
28℃
FC

1.5 
,4ž‹Œ74uši
4h~
9000r/min、4℃
–—
5min
³;%ß

&œÌx

x^¯r
085%)
ÅÆ%’

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ùi
100mmol/L、pH70K2HPO4 KH2PO4ÍÎ~¯s


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º)ÉÊ~
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:ƒ–—
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1.6 E.coliBL21/pET28a cr X、E.coliBL21/
pCDFDuet gdh cr X
-.n‘p‹Œ
(3R,5R) 2
  
4h~
9000r/min、4℃
–—
5min,
³;%ß

%’:i&œÌx
(085%)
ÅÆ

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KH2PO4 K2HPO4ÍÎ~,bcdUÜpx^¾
1∶1
œ(

%ßx^œ³
970g/L,380r/min
VT

ST*+–”,
pH
ŒÙ”
70
õö

Ù¿C9

J9iƒxNY„…

–—

t™~:i:ž~¦
§Ý¯¨å™

¯qùi
E.coliBL21/pET28a cr X
U
E.coli
BL21/pCDFDuetgdh cr X
[º)ST‹Œ
(3R,
5R) 2,
“&STÀ:i
E.coliBL21/
pCDFDuet gdh cr X
[º)¿

bcdœ³
、pH、
³

ܜ³
、NADP+、
%ߜ³
(5R) 1
VT
°qw
(3R,5R) 2d.e.
ª`€

1.7 
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(5R) 1、(3R,5R) 2
q•¥P¿ß:i”
•
HPLCLC 20AD
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Ъ¦Ö¬ßž¾
1∶3
ì°`NXx{~

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­9^
20μL,å™ÇÈ210nm,ύ
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w
(3R,5R) 2d.e.
ª¬’
(1)


d.e.=
A(3R,5R)-A(3S,5R)
A(3R,5R)+A(3S,5R)
×100% (1)
’–
:A(3R,5R).-(3R,5R) 2w`·ž;
A(3S,5R).-(3S,5R) 2¿Ew`·ž。
ÜVT°η¬’(2)6®。
η= 1-cVc0V
( )

×100% (2)
’–
:c0.--±Ü(5R) 1œ³,mol/L;c.-
hn
(5R) 1
œ³
,mol/L;V0.-y±h~ß
ž
,L;V
.-_›hªßž
,L。
2 
XYSZ[
2.1 E.coliBL21/pET28a cr X
S
E.coli
BL21/pCDFDuet gdh cr X
-.n‘p
ç"
(3R,5R) 2
  
”
30℃、pH70
56D
,E.coliBL21/pET28a
cr X
U
E.coliBL21/pCDFDuet gdh cr X

)ST
(5R) 1
ڍn5Ãáâ(.
1。
æ.

ò
ó

¥.ì%$
E.coliBL21/pCDFDuetgdh cr X
STž°ë!:j(d`³05ÃÔ%$
E.coli
BL21/pET28a cr X,
~Ubcd¥HÔÊU`Û
ԛ&–x¦Ð

§
1 
®¯c(t
(5R)1
®tàÞß5wx
Table1 Comparisonof(5R)1asymmetricreductioneficiencybetween
E.coliBL21/pET28acrXandE.coli/pCDFDuetgdhcrX
%$
ρ(Ü)/
(g·L-1)
y±hƒ°

(g·L-1·min-1)
h¿Õ


VT°


d.e.
ª


E.coliBL21/pET28a cr X 10.0 0.065±0.054 10 99.0 >99.5
E.coliBL21/pCDFDuet gdh cr X 50.0 0.653±0.048 1 99.0 >99.5
 
2.2 E.coliBL21/pCDFDuet gdh cr X
§¨
©ª5«p
2.2.1 
&ȽË`™Ù

37℃
D`a
10h
n

¬ßž¯r
5%

{^òs
LB
4h`a0–

™Ù%$`&ȽË

áâ¡ð

*-

æð

òó

%$Å*
2h
ї
#n­sr&È#
,`
a
7h

OD600ìÌ58
õö

G
2 
c(˜­¹º
Fig.2 GrowthcurveforE.coliBL21/pCDFDuetgdhcrX
2.2.2 
FCÀ
IPTG
œ(¿Õ
”Ú¾&Ș¼¯q(s›œ³Ö
010
mmol/L` IPTG,28℃
FC
10h。
FCÀ
IPTG
œ(
¿ÕÔ1`€(ð
3。
æð

òó

”r&
Èå#(s
IPTG
­sFC

%ßÔ1›j:x
û
,¨
©àᔍr&Èå#

ò{n`a
2h(
¦
h
OD600Â08),(sIPTG­sFC。
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3 IPTG
/ð¸´t4u5wx
Fig.3 Efectsofinduceraddingtimeon
CRXandGDHactivities
2.2.3 IPTG
i^
.ù%4h`a
2h,¯
q(sÚ¾œ³
IPTG,
28℃
FC
10h。IPTG
i^Ô1`€(ð
4。
æð

òó
:IPTG
`›œ³”
030~070mmol/L
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&ST„‹Œ

¬0
(3R,5R)
Y­0 ,®O-
./0¿

Ô11”,:xû

¨©

×Ù_4
`FCÀ^œ³Ö
050mmol/L。
G
4 IPTG
A™t4u5wx
Fig.4 EfectsofIPTGconcentrationon
CRXandGDHactivities
2.2.4 
FC¿Õ
FC¿ÕÔ1`€(ð
5。
æð

òó

FC
7~13h
1—˜÷:`Ô1

µÈ

Ô1u@Á

~òºZæj¿Õ*È

efx
௯|*ô

¨©

_*FC.ì¿ÕÖ
13h。
G
5 IPTG
01¸´t4u5wx
Fig.5 Efectsofinductiontimeon
CRXandGDHactivities
2.3 E.coliBL21/pCDFDuet gdh cr X
‘p
(5R) 1
®tàÞß©ª5«p
2.3.1 
bcdi^`€
”
500g/L(5R) 1
56D

bcdi^Ü
VT`€(ð
6。
æð

òó

íbcdUÜ
x^¾ÚÁj
1∶1
¿

Üòpÿ[VT

):Ü
œ³`bcdÔf˜þë,°Õi

!ÅÆ
³;

àibcdUÜpx^¾Ö
1∶1
œ(

2.3.2 pH
`€
”
500g/L(5R) 1
56D

h
pH
ÜV
T`€(ð
7。
æð

òó

”
pH60~75
./
0

VT°‚j
990%,
)Ž`É@àáR“Úy
pH
`€
,d.e.
ª‚j
995%。
í
pH
Ö
70
¿
,(5R)

VTƒ³_"
,¨
©

VT
pH
_›àá
70。
G
6 
ëìí¿st
(5R)1
®tàÞß5wx
Fig.6 Efectsofglucoseconcentrationon(5R)1
asymmetricreduction
G
7 pH
t
(5R)1
®tàÞß5wx
Fig.7 EfectsofpHvalueson(5R)1
asymmetricreduction
2.3.3 
h³`€
”
800g/L(5R) 1
56D

h³Ü
VT`€(ð
8。
æð

òó

³Ú€³0
5ÃÔ`ÞßàáR

<=³`>:
,d.e.
ª±›
‚j
995%,
”³Ö
25
Ç
28℃
¿
,800g/L
`
Üòpÿ[VT

µ
25℃
VT`ƒ³*j͕

¨©

h³àá
28℃。
G
8 
¼@rst
(5R)1
®tàÞß5wx
Fig.8 Efectsofconversiontemperatureon(5R)1
asymmetricreduction
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ܜ³`€
”%ߜ³
970g/L
56D

ܜ³Ü
VT`€(ð
9。
æð

òó

Üx^œ³Ö
800
Ç
1000g/L,
h
2h
0

Üòpÿ[VT

íܜ³ìÌ
1200g/L
¿

<=¿Õ`ÑÈ

Ü
¡˜B^$Ñ

¾¿
1000mg/L
`
NADP+
h`€

4/”Äaº)VT*+–
,NADP+
ZŸœ(hƒ°`€Ú‚

~òºUîïRÛ
ԃ„µ*º)™­sº)x˜Ð
[12-13]。
2.3.5 
%ßi^`€
%ßi^÷‚+³€ST`ž°qVT
°

”ptìÓ01t

ài
485、970、1455g/L
`%ߜ³

ijھܜ³`VT23

áâ
(.
2。
æ.

òó

á:%ß`i^

ºÿ[VT
`ܜ³W¦h}e(
。485g/L
%ߺ”
2h
    
G
9 
}~¿st
(5R)1
®tàÞß5wx
Fig.9 Efectsofsubstrateconcentrationon(5R)1
asymmetricreduction
0ÿ[ST
800g/L(5R)1
ڍn5Ã
,970g/L
%ßòp”
2h
0ÿ[VT
1000g/L
Ü

w
(3R,5R) 2d.e.
ª‚j
995%。
§
2 
c.¿s

}~¿st
(5R)1
®tàÞß5wx
Table2 EfectsofE.coli/pCDFDuet gdh cr Xloadingsizeandsubstrate
concentrationon(5R)1asymmetricreduction
ρ(%ß)/
(g·L-1)
ρ(Ü)/
(g·L-1)
h¿Õ


m(
Ü
)/m(


(g·g-1)
y±hƒ°

(g·L-1·min-1)
VT°

% d.e.ª/%
4.85 80 2 16.49 0.830±0.014 99 >99.5
4.85 100 6 20.62 0.696±0.088 85.2 >99.5
9.7 100 2 10.31 1.174±0.044 99 >99.5
9.7 120 6 12.37 0.869±0.004 89.2 >99.5
14.55 120 1 8.25 2.184±0.004 99 >99.5
14.55 140 1.5 9.62 1.276±0.148 99 >99.5
3 
X
 
[
³05ÃÔ

bcd¥HÔ¥.ì%$
E.coli
BL21/pCDFDuet gdh cr X,
º¥PàáR5Ã
(5R) 1
‹Œ
(3R,5R) 2。
”
28℃、pH70、970
g/L


bcdU
(5R) 1
x^¾
1∶1`
56D

1000g/L(5R) 1
”
2h
0òÿ[eVTŒ
(3R,
5R) 2,
w
d.e.
ª‚j
995%。
Uš&E
Pichia
guiliermondiCCTCCM2011386
¦¾
,E.coliBL21/
pCDFDuet gdh cr X
./0}Í`1RÇÞß
àáR

—˜.«`)thiåJ

ƒ„JK

[1] PfeferkornJA,BowlesDM,KisselW,etal.Developmentofa
practicalsynthesisofnovel,pyrolebasedHMGCoAreductase
inhibitors[J].Tetrahedron,2007,63(34):81248134.
[2] CasarZ.Historicoverviewandrecentadvancesinthesynthesisof
superstatins[J].CurOrgChem,2010,14(8):816845.
[3] WolbergM,VilelaM,BodeS,etal.Chemoenzymaticsynthesisof
thechiralsidechain ofstatins:application ofan alcohol
dehydrogenasecatalysedketonereductiononalargescale[J].
BioprocessBiosystEng,2008,31(3):183191.
[4] ScheflerJL,BeteV,MortreuxA,etal.Preparationand
stereoselective hydrogenation of chiral (4hydroxy
tetrafuranylidene)carboxylates:anewformalentrytofunctional
antiandsyn3,5dihydroxyesters[J].TetrahedronLet,2002,43
(15):26792682.
[5] XuZN,JingK J,LiuY,etal.Highlevelexpressionof
recombinantglucosedehydrogenaseanditsapplicationinNADPH
regeneration[J].JIndMicrobiolBiotechnol,2007,34(1):
8390.
[6] YangW,XuJH,PanJ,etal.Eficientreductionofaromatic
ketoneswithNADPHregenerationbyusingcrudeenzymefrom
33 
!

#
  
‡
 
ˆâ

&ST„‹Œ

¬0
(3R,5R)
Y­0 ,®O-
Rhodotorulacelsandmannitolascosubstrate[J].BiochemEng
J,2008,42(1):15.
[7] NiY,LiCX,ZhangJ,etal.Eficientreductionofethyl2oxo4
phenylbutyrateat620g/Lbyabacterialreductasewithbroad
substrate spectrum[J].Adv Synth Catal,2011,353(8):
12131217.
[8] NiY,LiCX,WangLJ,etal.Highlystereoselectivereductionof
prochiralketonesbyabacterialreductasecoupledwithcofactor
regeneration[J].OrgBiomolChem,2011,9(15):54635468.
[9] BrutigamaS,DennewaldaD,SchürmannbM,etal.Wholecel
biocatalysis:evaluationofnew hydrophobicionicliquidsfor
eficientasymmetricreductionofprochiralketones[J].Enzyme
MicrobTechnol,2009,45(4):310316.
[10] KratzerR,PuklM,EggerS,etal.Wholecelbioreductionof
aromaticαketoestersusingCandidatenuisxylosereductaseand
Candida boidini formate dehydrogenase coexpressed in
Escherichiacoli[J].MicrobialCelFact,2008,7:112.
[11] 
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=[W

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[12] Zhang J,Witholt B,Li Z.Coupling of permeabilized
microorganismsforeficientenantioselectivereductionofketone
withcofactorrecycling[J].ChemCommun,2006,4:398400.
[13] ZhangJ,WitholtB,LiZ.EficientNADPH recyclingin
enantioselectivebioreductionofaketonewithpermeabilizedcels
ofamicroorganismcontainingaketoreductaseandaglucose6
phosphatedehydrogenase[J].AdvSynthCatal,2006,348(4/
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