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Asymmetric reduction of t-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate by carbonyl reductase

羰基还原酶不对称还原(R)-6-氰基-5-羟基-3-羰基己酸叔丁酯



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ChineseJournalofBioprocessEngineering
Vol.11No.1
Jan.2013
doi:10.3969/j.issn.1672-3678.2013.01.004
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:1672-3678(2013)01-0017-06
Asymmetricreductionoftbutyl6cyano(5R)hydroxy3carbonylhexanoate
bycarbonylreductase
CAOZheng,WANGYajun,XIAOLi,LIUZhiqiang,ZHENGYuguo
(EngineeringResearchCenterofBioconversionandBiopurificationoftheMinistryofEducation,InstituteofBioengineering,
ZhejiangUniversityofTechnology,Hangzhou310014,China)
Abstract:tbutyl6cyano(3R,5R)dihydroxyhexanoate,akeychiralbuildingblockforatorvastatin,was
synthesizedthroughasymmetricreductionof(R)6cyano5hydroxy3carbonylhexanoatecatalyzedby
carbonylreductasecoupledwithglucosedehydrogenase.Theoptimizedbiocatalyticconditionswere
obtained:(R)6cyano5hydroxy3carbonylhexanoate1470g/L,glucose1282g/L,celloading150
g/L,thepH65,and30℃.Undertheseconditions,(R)6cyano5hydroxy3carbonylhexanoatewas
completlyconvertedfor6h.WithoutexogenousadditionofNADP(H)andwithtbutyl6cyano(3R,
5R)dihydroxyhexanoatethed.e.valuewasover99.5%.
Keywords:asymmetricbioreduction;atorvastatin;tbutyl6cyano(3R,5R)dihydroxyhexanoate;
coenzymeregeneration
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Fig.1 Carbonylreductasecoupledwithglucose
dehydrogenaseasymmetricreductionof
(R)6cyano5hydroxy3carbonylhexanoate
((5R)1)totbutyl6cyano(3R,5R)
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`a0
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LB
`a0
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a0ùŒ
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a0½%n(•–ƒñl›x^œ³Ö
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:i
LB
`a0
,`
a0ùŒ
(g/L):
¾efg
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hiš
50、NaCl100、
(NH4)2SO450、SA20、KH2PO4136、K2HPO4·
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ÜVT°η¬’(2)6®。
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E.coliBL21/pET28a cr3
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¿

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(5R) 1
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100g/L
¿

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20min
¿

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S
E.coliBL21/
pET28a gdh4
c.陗t
(5R)1
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100g/L
56
D

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(5R) 1
ڍn5Þ°`€-j.
1。
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òó

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×Ù
E.coliBL21/pET28a cr3
U
E.coliBL21/
pET28a gdh4
x^¾Ö
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Ƴ05ÃÔ%ß
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bcd¥HÔ%ßx^œ³
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Table1 EfectsofbiomassratioofE.coliBL21/pET28acr3andE.coliBL21/pET28agdh4on
(5R)1asymmetricreduction
m(CR3)∶
m(GDH4)
ρ(GDH4)/
(g·L-1)
h¿Õ


ρ[(3R,5R)2]/
(g·L-1) ρ

Ü
)/ρ(%ß) VT°/% d.e.ª/%
4∶1 2.5 6~7 87.86±0.93 16 100 ≥99.5
4∶2 50 5 89.69±0.81 13.3 100 ≥99.5
4∶3 7.5 5 90.82±0.79 11.4 100 ≥99.5
4∶4 100 5 90.63±1.65 10 100 ≥99.5
4∶6 150 4 89.32±0.96 8 100 ≥99.5
2.3 
c.A™t
(5R) 1
®tàÞߝÄ5wx
%ß՜³ñò€Ún5Ýhƒ°

í
E.coliBL21/pET28a cr3
U
E.coliBL21/pET28a
gdh4
x^¾Ö
4∶2
¿%ß՜³¬ð

*-­s
23

#Ìáâ¯qæ¡ð

*-

æð

òó


hûÖ¿

VT°
100%,d.e.
ª‚j
99.5%。
&
‹;ú%¾q%ߜ³ST`€

:i%ß
x^œ³Ö
150g/L`
56ÕÖST56

G
3 
c.ê¿st®tàÞß¼@5wx
Fig.3 Efectsofcelloadingsizeon(5R)1
asymmetricreduction
2.4 
Åprst
(5R) 1
®tàÞߝÄ5wx
  
h³€Ôh`ƒ°

ST³
(5R) 1
ڍn5Þ°`€-jð
4。
æð

òó

VT³
26℃
¿
,(5R) 1
ÿ[h‰«
8h。
á:³l
35℃
n

hƒ³("

µÔ#1
/ãë!
,(5R) 1
VT°Ö
935%。
퍳‚j
40℃
¿

Ô#1¦.

hׯ͕

ð
4)。
8>
³Ô1˜‚`€

µZÔ¥P¿àá
Rƒþë€

w
d.e.
ª1”,”
995%
pt

¨©h³àáÖ
30℃。
G
4 
rst®tàÞß¼@5wx
Fig.4 Efectsoftemperatureon(5R)1
asymmetricreduction
2.5 
˜~‘p.g
pH
t
(5R) 1
®tàÞߝ
Ä5wx
  pH
WZ€&STh`¨ñcd

&S
TßÁ
pH

(5R) 1
ڍn5Þ°`€-j
ð
5。
æð

òó

í
pH
Ö
65
¿

hƒ³_"

VT°
100%,d.e.
ª‚j
995%;
í
pH
Ö
75
¿

hÌ
6h
¿

hìÌûÖ

VT°
634%,d.e.
ª‚j
995%。
¨©

h_4
pH
Ö
65。
2.6 NADP+
陿st
(5R) 1
®tàÞߝÄ
5wx
  
å#`ÏìÓ×ÙSTßÁÛÔÒØ×EÖ
NADPH/NADP+
E

­dõ§îïR
NADP+
x^œ³
(5R) 1
ڍn5Þ°`€

ð
6)。
æð

òó

ËùU
0025、005g/L
NADP+
ù
(5R) 1
VT°
100%,d.e.
ª‚j
995%,
~.þº)0ïRÛԆðÛԍ¶

ƒ
‰ÍÙîïRÛÔ

¨©

×ٝhßÁ–Úœ
(ÛÔ
NADP+。
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t®tàÞß¼@5wx
Fig.5 EfectsofpHvalueon(5R)1asymmetricreduction
G
6 NADP+
陿st®tàÞß¼@5wx
Fig.6 EfectsofNADP+concentrationon(5R)1
asymmetricreduction
G
7 
ëìíS
(5R)1
5¿s—t®tà
Þß¼@5wx
Fig.7 Efectsofglucoseconcentrationon(5R)1
asymmetricreduction
2.7 
ëìí¿st
(5R) 1
®tàÞߝÄ5
wx
  
bcdÕÖ
GDH
`ÕiÜ

•œ³
(5R) 1
ڍn5Þ°`€-jð
7。
æð

ó

íbc
dU
(5R)1
)µ¾ªÖ
1∶1、115∶1
¿

hƒ°¦
@
,6h
hÿ[
,d.e.
ª‚j
995%。
í
(5R) 1
U
bcdœ³¾ªÖ
14∶1、18∶1
¿

hƒ°•

‰
«
8h
Ü
(5R) 1
vºÿ[h

hyƒ³ë!


~òºUbcd¥HÔ4&§Ü,°˜Ð

¨©

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)µ¾Ö
1∶1。
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ùiïà“¿À`ÞßàáRß

12:`³
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E.coliBL21/pET28a cr3
U¿
À`bcd¥HÔ.ì%$
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1∶1,E.coliBL21/pET28a cr3
x^œ³
100g/L,E.coliBL21/pET28a gdh4
x
^œ³
50g/L,pH65,
³
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h¿Õ
6h,
(R) 6
¬0

­0

³0 ,®O-VT°
100%,
w
d.e.
ª‚j
995%。
¹O„ºðdy
:œ³`Ü

hƒ³"

É@àáR:

¾¿&
ST*+–Ú‰î(Ó[¸¹`ÛÔpòì/
ڍnh`:ƒ­s

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ƒ„JK

[1] 
ÚÛ

KñÜ

Ÿ¯×nX:Y/Õi`23­5
[J].
Ý
‚Þ;
,2008,23(8):604606.
[2] 
ßàá

âÞ

Ÿ¯×@Òn‡ˆã¨
[J].
t­mn

2005,26(7):313314.
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11601164.
[4] 
ä¼



Ÿ¯×nÕiÇÕiâ°`Žæç
[J].
݂
0?ÖC
,2006,23(1):57.
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[6] LiljebladA,KalinenA,KanervaL T.Biocatalysisinthe
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12 
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³05ÃÔڍn5Ã
(R)6
¬0

­0

³0 ,®O-
carboxylates:anewformalentrytofunctionalantiandsyn3,5
dihydroxyesters[J].TetrahedronLet,2002,43:26792682.
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