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Optimization of Morgarella morganii esterase production and its application in synthesis of chiral intermediate of Pregabalin

摩氏摩根菌产酯酶条件优化及在普瑞巴林手性中间体合成中的应用



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ChineseJournalofBioprocessEngineering
Vol.10No.6
Nov.2012
doi:10.3969/j.issn.1672-3678.2012.06.003
STUM
:2011-11-29
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CCTCCM2011175
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(g/L)
ªãä½
150,
wxy
70,Na2HPO410,Fe2(SO4)301,
zB
80100。
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|

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:1672-3678(2012)06-0012-06
OptimizationofMorgarelamorganiesteraseproductionandits
applicationinsynthesisofchiralintermediateofPregabalin
FUDejin,ZHENGRenchao,ZHENGYuguo
(InstituteofBioengineering,ColegeofBiologicalandEnvironmentalEngineering,
ZhejiangUniversityofTechnology,Hangzhou310014,China)
Abstract:Inordertodevelopaneficientandinexpensivebiocatalyticprocessfor(S)2carboxyethyl3cy
ano5methylhexanoicacid,keyintermediateofPregabalin,productionofesterasebyMorgarelamorgani
CCTCCM2011175wasoptimizedbysinglefactormethodandorthogonaltest.Theoptimalmediumcompo
sitionswereasfolows(ing/L):glucose150,beefextract70,Na2HPO410,Fe2(SO4)301,and
Tween80100Theesteraseactivityreached10710U/L,whichwas15timeshigherthanthatbeforeop
timizationKineticresolutionsofrac2carboxyethyl3cyano5methylhexanoicacidethylesterusingwhole
celsofMmorganiCCTCCM2011175afordedtheproductin45% conversionand94% e.e.Itprovided
apotentialypromisingbioprocessfortheproductionofchiralintermediateofPregabalin.
Keywords:Morgarelamorgani;esterase;Pregabalin;kineticsresolution
  
L@
(esterase,EC311X)
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ulLN3–{|AXÍZYm«/8YA–î
N=

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L@OL•–/Ó
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ÌUÖgZ[YL@§½x¸&
ÓA–
[1]。
ÓA–L@A–îN–X4TGô–
3XY],"“
[2]。
  
PQRS
(Pregabalin)
NFp4
(3S) 3
½e


eb«
[34],
# γ ½q«(GABA)¶W
rL=GÏÐs

tuvwóô
[56]。
ë7

PQR
SÖgNA6¬­–yóÃ!uÛ

üÖ×xèà
y@XWaóGA–îN

…^Rz

m7

{

eb«7L
(CNDE)
Ðày@XWaó
G{|AX
(S)2
m7

{

eb«

üTGUVWВm

{|

|N†òPQRS
[7]。
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üNF @“}Ef
[ày@Yg³~/XWaóG

tuõv}³
7A

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°gõv^Rz
(CNDE)
Yày@

L@ó@¸

X4PQRS@“3XÖ×YF²

¤ÿŸ˜”
ƒ„UöaNò™

h°XWaóGõv
CNDE
YL@6AB
MorgarelamorganiCCTCCM2011175
(^ 1)[8]。
j#

üBhL@²@ ~•

…/[&
ÖgN25

  
¡£¤YëY#tun
MmorganiCCTCCM
2011175
6@CDMN

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6Ö×ýŒþ

n
1 MmorganiCCTCCM2011175
óo
CNDE(1)
‚ƒíîïðñ£mòÙ
(2)
Fig.1 MmorganiCCTCCM2011175catalyzedresolutionofCNDE(1)tochiralintermediateofPregabalin(2)
1 
vw%xy
11 
ÄÅvw
111 

  
L@6AB
MmorganiCCTCCM2011175
$
aìÖghFA–Ö⣤ñ”ƒ„UöaNò

–YÉ&UãˆÝ„…–YÉUó

‰Š
)。
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„…
  
±T„…
(g/L):
ˆ‰4
100,
-.Ý
20,
>?Þ
10,(NH4)2SO420,K2HPO420,NaCl
10,FeSO401。
  
Á¦„…

â±T„…Ít

  
éꄅ

O±T„…Uáè
15% ~
20%
ßà

12 
ÄÅxy
121 
BW„…
  
éꄅ
:30℃
„…
24h。
  
Á¦„…

Kéê9YBÁçÁÃƯJ4
50
mL
Y
250mL
íî~U
,30℃、150r/min
„…
24h。
  
5-„…

KÁ¦„…/
4%(
Wƒv½

ç
ÁJçÁÃíî~U
,30℃、150r/min
„…
36h。
122 
A–J¯Œ
  
¢
10mL
5-¯™ó‹9=

×ÅBW

D[A
ʺ{
(085%)
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,95℃
Œ*ÃëHJ

…45
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/
g/L
ã

D˜_£]X

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123 
@ E¯Œ"“
  
@ Œa
:30℃、pH80
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1μmolnŽ!ñ¾Y@JŒa4.p@ Q

,1U。
  
ÇZW ]¾

nŽ!qLY78<¯
80μL,TrisHClF  ¯ (pH 80,50mmol/L)
1720μL,B>¯200μL,ÌU7837Wƒv½
4
4%,
nŽ!qL7ä³4
01mmol/L。

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5min,405nm
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[9],
D˜_£]
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124 
±V¢£
  
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125 MmorganiCCTCCM2011175
XWaóG
õv
CNDE
  
ÇZW ˜X4
10mL
“«ºF¯
(10
mmol/L,pH80),
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05g,
0–ä³
100mmol/L。
31 
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# cd°A

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30℃
{ü¾”•ÇZ

Œ§¢H
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A¢9=¯
200μL,t§áè7«7L800μL,O—Å
”•È9Çv2!

A¢9˜:;Í

áèµJù{
Na2SO4*™ï°|šÍA«vw。
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6–n›WuJ%Y¯Œ
  
œ
GC 14C
šÍA«J
,G TA
TGž®;
šÍA«K
(30m×025mm×012μm)。vwC
D


135℃,
Áš
He,
ÅJ
10mL/min,
°HJ
1μL,vŲ 40∶1,N¯ÈE°HŸì³*4
220℃。
6–n›WuJ%
(e.e.)
Î
e.e.=
AS-AR
AS+AR
ãà

³U
:AR4R ¨Ý6–Y êƒ;AS4 S ¨Ý6
–Y êƒ

6 – n › W a ó Ž
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ln[1-C(1+e.e.p)]
ln[1-C(1-e.e.p)]
ãà

³U
:e.e.p46–Yn›WuJ%;C4PNŽ。
PNŽ
(C)
Î
C=AX6–Yä³
±T0–Yä³
×100%
xãà

127 


6–ä³Y¯Œ
  Agilent6890
šÍA«J
,HP 5
š´GA«K
(30m×032mm×025μm)。vwCD:Kì
130℃,
/
5℃/min
â¡¢ìÃ
165℃,
Áš4
N2,
ÅJ
1mL/min,
°HJ
1μL,vŲ50∶1,N¯È
E°HŸì³*4
250℃。
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‹Œ%)
21 C
­‰ôõ
  
€tÓA–¡ÃY@ iŒ™n

¸Yf[
­©¼€Ít

°qrs6@

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10
Á

¸
nBWA‚E6@Yrs

‘œ]^

ñO

$^

S_

ˆ‰4—áJ4
100g/L
§

@ EA–J
rÍn~g

v¥l˜
4580U/L
/
35g/L。
qt
JÛÜ4n@ rs~¿

jS/žŸ¡gA–
J

ñ/a¢ˆ‰44

¸

22 N
­‰ôõ
  
ÓA–O:;

¸Y„…US/¢çf
[½«/Ìf:;£N–UY¼Á€t‘¨Y
‹¤

3XA¥ñ¦¾Y>?H/Ìf®¯–H

ù¾R”4v|x3X

ؘy:f&ÓA–A
‚

¢£U

¸—áJ4
50g/L,
Ìn²@ E
n
2 C
­ž
MmorganiCCTCCM2011175
«¬|f¿‰Ÿ 
Fig.2 Efectsofcarbonsourcesongrowthandesterase
productionofMmorganiCCTCCM2011175
A–JYrs‘œ^
3。
$^

S_

µf

(NH4)2SO4、NH4ClAù; N¸n@ EA–Jr
s€žŸ

E±Ý

>?ÞA:;

¸žŸ¡g
A–JE²@ 

ÌU/E±Ý4

¸§

²@ 
3g
(6250U/L)。
n
3 N
­ž
MmorganiCCTCCM2011175
«¬|¿á‰Ÿ 
Fig.3 Efectsofnitrogensourcesongrowthandesterase
productionofMmorganiCCTCCM2011175
23 
G߯à‰ôõ
  
ç3™¦S/%4@ G€Y˜XXvŽ@
Yr =

”q¢çrs@ G

OÓA–YA¥ 
LU§¨],îA

ç3™¦Y—áJ4
01g/L,
_£‘œ^
4。
$^

S_

£—á
Mg2+
§

BW
A‚3)

q—á
Fe3+
§²@ 3g


5585
U/L,
ؘ

aóö„…U—á
Fe3+。
24 
ö÷¼|¡Û᣼‰ôõ
  
§½L@4_’@

Ì_’=+,#©ª*

íq«N*LA*àN3–
[10]。
˜^

Bê G=
S/tuˆ`à41
A
 
–
 
á
 
Ö
 
u
 
â
  
!
10
"
 
1—
‘?
;2—Al3+;3—Zn2+;4—Mg2+;5—K+;6—Mn2+;
7—Fe3+;8—Na+;9—Ba2+;10—Fe2+;11—Ca2+;12—Cu2+
n
4 
G߯àž
MmorganiCCTCCM2011175
«¬|f¿‰Ÿ 
Fig.4 Efectsofmetalionsongrowthandesterase
productionofMmorganiCCTCCM2011175
@

ày@O®¯IUYtjGrs@ 

t§.
Bê G=ŽS/%4

¸
[11]。
ؘ

ª9—á
100g/L
©ª*

íq«N*L

«¬«

­à
«
[12]、
®ì
80、
®ì
20、Triton 100
/E0–
CNDE
n@ EA–JYrs

‘œ]^

ñO

$^

S_

®ì
80
/­à«žŸ¡g²@ 

v¥l˜
8599
/
8392U/L。
n
5 
ö÷¼|¡Û᣼ž
Mmorgani
CCTCCM2011175
«¬|¿á‰Ÿ 
Fig.5 Efectsofinducersandsurfactantsongrowth
andesteraseproductionofMmorgani
CCTCCM2011175
25 
ø­‰ôõ
  
“Rf#®¯¯«

“àAA–hv¦Y˜
XRf



g“«N3–Eå§@ G€
Ž¾,“RfY1â

ª9¼Á“¸E̘3Y
rs

‘œ]^

ñO

—áJ4
10g/L。
$^

S_
:Na2HPO4žŸ¡g²@ EA–J,v¥l
˜
9054U/L
/
44g/L。
1—
‘?
;2—K2HPO4;3—Na2HPO4;4—NH4H2PO4;
5—K2HPO4+KH2PO4;6—Na2HPO4+NaH2PO4;
7—K2HPO4+NaH2PO4;8—K2HPO4+NH4H2PO4
n
6 
ø­|ø­ùdž
Mmorgani
CCTCCM2011175
«¬|f¿‰Ÿ 
Fig.6 Efectsofphosphorussourcesongrowth
andesteraseproductionofMmorgani
CCTCCM2011175
26 
}~Oœ
pH
‰ôõ
  pH
#rsBWA‚E6@Y],Øf

ugŽ
u•Y
pH
S’“BWYA‚¶˜k†

6@Ž
sÍZ”É

4™:Œµ¬Y
pH,
[
10mol/L
HCl
/
NaOH
K„…Y
pH
v¥l4
40、50、
60、70、80
/
90,
çÁ5-
36h
ï
,¯
Œ@ /
A–J

‘œ]^

ñO

$^

S_

£„…±
T
pH
4
70
§

L@Y²@ 3g
(9130U/L)。
n
7 
^›œ
pH
ž
Mmorgani
CCTCCM2011175
«¬|f¿‰Ÿ 
Fig.7 EfectsofdiferentinitialpHofmediaongrowth
andesteraseproductionofMmorgani
CCTCCM2011175
27 
úûÑÅ
  
a¢ˆ‰4
(A)、
E±Ý
(B)、
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80(C)3
p
n6@rs~hYØf/E.p‘?ê
(D)
°|’
Øfí{Y±V¢£

‘œB
1。
±V¢£v
wB
2。
51 
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IJIKB6L@CDMNEOPQRSTGUVW3XUYZ[
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úûÑÅÄŋŒ
Table1 Resultsoforthogonalexperiments
¡Z
ρ/(g·L-1)
ˆ‰4

E±Ý

®ì
80

‘?

²@ 

(U·L-1)
1 1(2) 1(6) 1(5) 1 605.9±3.4
2 1 2(7) 2(10) 2 764.0±10.3
3 1 3(8) 3(20) 3 653.8±25.9
4 2(5) 1 3 2 849.9±18.9
5 2 2 1 3 737.0±19.2
6 2 3 2 1 871.4±36.5
7 3(10) 1 2 3 989.0±6.2
8 3 2 3 1 1052.2±35.6
9 3 3 1 2 831.9±5.8
¡
2 
úûÑŋŒoŠ
Table2 Analysisoforthogonalexperimentsresults

ˆ‰4

E±Ý

®ì
80

‘?

K1 674.6 8150 8432 7250
K2 8195 8511 8153 8750
K3 9578 7857 8933 8520
R 2832 654 499 1498
  
±V¢£vw

B
2)
BC

ØfY+Ñ°¡±
Ñ4
A(
ˆ‰4
)、C(
®ì
80)、B(
E±Ý
),
¼Øf
Y3O˜34
A3B2C3。
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A(
ˆ‰4

Y3O—áJ4
100g/L,
–
Ðl˜™a¢Y

p{UY3g%

ؘ°.}
£¤™áሉ4—áJn@ Yrs

‘œBC
£ˆ‰4—áJl˜
150g/L
§

@ l˜3h%
10710U/L。
ؘ

ò63M„…˜X
(g/L):
ˆ
‰4
150,
E±Ý
70,
®ì
8010,Fe2(SO4)3
01,Na2HPO410。
  
n/9±V¢£‘œ°|"Pvw

B
3),
‘
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ˆ‰4E®ì
80
nüBh6L@[:ž
ŸYrs

28 
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20h
°è`Œ#

A–Jl˜
45g/L。
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(ˆÝY
A‚²–Ý
,30h
§l˜
10183U/L。
¡
3 
xýoŠ‹Œ
Table3 Analysisofvarianceresults
Øf ¤P"/ y$³

²
F005 žŸG
A 120288079 2 32024 398 
B 6437365 2 1714 398
C 3756189 2 1000 398
D 39116400 2 10414 398 
³P
3756189 11
n
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MmorganiCCTCCM2011175
3 M.morganiCCTCCM2011175
þ
†
CNDE
ØÙôõ£™š
31 MmorganiCCTCCM2011175
ØÙôõ£
™š
CNDE
  MmorganiCCTCCM2011175
Є…MN

L@²@ ¡gï

°.}ª9™ÌXWaóGõ
61
A
 
–
 
á
 
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u
 
â
  
!
10
"
 
vCNDE
Yœ
(^ 9)。
$^

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üL@n
CNDE
[:~)YXWaóG

O0–ä³4
100
mmol/L
§

A–îNÇZ
24h
ï

PNŽh&
45%

Êg%4
50%),

(S) 2
m7

{

eb«ªFZ³
(ee)

94%
/9

n›W
aóŽ
(E)
gl
75。
n
9 MmorganiCCTCCM2011175
ÿ!‡ˆ
þ†
CNDE
ØÙôõ£™š
Fig.9 RestingcelsofMmorganiCCTCCM2011175
catalyzedstereoselectivehydrolysisofCNDE
32 MmorganiCCTCCM2011175
"¸#£
#$
  
4°.}£¤
MmorganiCCTCCM2011175
Y
ÖgNZ[Í%

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Mmorgani
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YîNœ
(^ 10)。
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10
S
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 ¡0–ä³Yág

6–ä³ -9¢

£0–
ä³4
400mmol/L
§

6–7ä³gl
120mmol/L,
6–ªFZ³*YTO
94%
/9

n
10 
§¨"$̱
Mmorgani
CCTCCM2011175
‰þ†%
Fig.10 TimecoursesofCNDEhydrolysiscatalyzed
byMmorganiCCTCCM2011175under
diferentsubstrateconcentrations
4 
‹
 
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tu„…QØfMNE±V¢£

:ŒIJ
IKB
MmorganiCCTCCM2011175
6L@3M
„…
(g/L):
ˆ‰4
150,
E±Ý
70,Fe2(SO4)3
01,
®ì
80100,Na2HPO410。O˜CD¾@ 

10710U/L,
A–Jl˜
45g/L,
v¥²\T
„…CD¾¡g™
25´
/
15´ 。
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