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Asymmetric synthesis of(R)-2-chloro-1-(3-chlorophenyl)ethanol by carbonyl reductase-producing strain Candida ontarioensis

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇



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ChineseJournalofBioprocessEngineering
Vol.10No.3
May2012
doi:10.3969/j.issn.1672-3678.2012.03.004
OPQI
:2011-09-20
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:1672-3678(2012)03-0017-06
Asymmetricsynthesisof(R)2chloro1(3chlorophenyl)ethanolby
carbonylreductaseproducingstrainCandidaontarioensis
ZHANGBeihua,NIYe,SUNZhihao
(KeyLaboratoryofIndustrialBiotechnologyoftheMinistryofEducation,SchoolofBiotechnology,
JiangnanUniversity,Wuxi214122,China)
Abstract:AcarbonylreductaseproducingstrainCandidasp.PT2Awasselectedfromstraincolectionof
ourlaboratoryandwasidentifiedasCandidaontarioensisby18SrRNAsequencing.Thefermentation
conditionsandbioreductionconditionsfortheasymmetricsynthesisof(R)2chloro1(3chlorophenyl)
ethanolbyCandidaontarioensiswereoptimizedastemperatureof30℃,initialpHof65,rotationspeed
of180r/min,andcelconcentrationof200g/L.Afterfor72hreaction,thee.e.andtheproductyield
were995% and99%,respectivelyonsubstrateconcentrationof10g/L,whereastheyieldwasdropped
to843% onenhancedsubstrateconcentrationof30g/LUsingC.ontarioensiswholecelspretreated
with4g/LCTABfor20minat4℃,theproducte.e.andtheyieldreached999% and975% onsub
strateconcentrationof30g/Latshortenedreactiontimeof24h
Keywords:asymmetricreduction;Candidaontarioensis;cetyltrimetylammoniumbromide(CTAB);per
meability;(R)2chloro1(3chlorophenyl)ethanol
  
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20,
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89wx)
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tuK
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20,
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250mL
KT®>89wx)s

*
30℃、180r/min
"89
20h。
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i
18SrRNA

  
Û`9y),E‘’§}:扑’
Candida
sp.PT2A
),E
DNA,
ÿ
CF:5′TCCTCTAAATGAC
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êqÈXE
[15]。
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2mL
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100mL,
~F
—
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=n(MZ:¶)n(
½¾°¶
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ee
ð

AR-AS
AR+AS
×100%
(s
:AR—S¹ºiž R uÁ:¶>̈?;
AS—S¹ºižSuÁ:¶>̈?。
2 
†‡%ˆ)
21 Candidasp.PT2A
i
18SrRNA
`‚=’
  
Ë0,P5Ø>
CandidaspPT2A
>
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rRNA
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(GenBank
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Can
didaontarioensis
g*;Ai`

QRNêÎ(

,^
‡
CandidaspPT2A
A£ó
Candidaontarioensis。
q
1 Candidasp.PT2A
Æóm
Candida
‚iæç2ÚA
Fig.1 Phylogenetictreeindicatingtheestimated
relationshipsbetweenstrainCandidasp
PT2AandrelatedspeciesofgenusCandida
22 Contarioensis
de˜ÇÈiÙÚ
221 
wx)½¾
pH
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pH
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ð

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Â
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pH
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pH
ó
65。
q
2 
ƒ„KŽ
pH
C.ontarioensis
de˜ijk
Fig.2 EfectsofpHoncarbonylreductaseproductionof
C.ontarioensis
222 
ŒU089:V>?@
  
89ŒUí?@4–Ÿ M—~89:V>
×·,¿ÉA

,^

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—–_)`aVV®>?@

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1。
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òÅ
:OD660~V®Ç~30℃<Îû,89ŒU0³
X:¶
ee
ðÔQ?@

Ë̌U/
30℃
`û•
40℃,OD660~V®ÇÞê"ú。,^ü£ÎÈ8
9ŒUó
30℃。
 
1 
ɝC.ontarioensis
de˜ijk
Table1 Efectsoftemperatureoncarbonylreductase
productionofC.ontarioensis
ŒU
/℃ þV®

(U·L-1)
ee
ð
/% OD660
20 003±0001
25 003±0001
30 2259±113 >999 025±0013
35 2023±101 >999 016±0008
40 1625±081 >999 014±0007
 
223 
­S³™089:V>?@
  
VW9;­S³™0
Contarioensis
89>Ÿ
 …U–_)`aVV®>?@

°¶
10g/L)。
4–…UË̳™>`û9GPÊ

È­S³™
‹*
180r/min
<

_)`aVV®ËÌ­S³™
91 
!

#
   
Uÿ!ø

_)`aV:M4
Candidaontarioensis
bc
(R)2
d
1(3
de)

[P
>PÊÍPÊ

È­S³™û*
180r/min
J¾"ú

ü£­S³™ÿ
180r/min
óá

ì™è
êè
)。
zÞ­S³™089ËÌs>¡Dÿ–·
—ÏQÌ×·?@

23 Contarioensis
“\>78

;
1 (3
;

øÄ+ÚÇÈiÙÚ
231 
Ó+
pH
090+`a

d
-1 (3
d
e)

[O>?@
  
Y

êè1
30℃
<9;Ó+½¾
pH
0`
aЇ>?@

/Y

òÅ

9;Ó+
pH
0:
¶>
ee
ðÔQ?@

:¶>ÉoBUÇó
999%
ÿ‹

~
pH50~65
lÛ²

:¶>:šô‹`
õö

!~
pH65~75
lÛ²zùåÍÖ>³X
íÃ

/*:š~
pH65
<{Îû

΄}~
pH65
ó³XЇÎÈ
pH。
ÆÇzÞ

Ó+
pH
0_)`aV>WX®¯QA£>?@

q
3 
BCD
pH
C.ontarioensis
“\>78

;
1(3
;
øÄe,Æe
e.e.
Eijk
Fig.3 EfectsofbuferpHonproductyieldande.e.
forasymmetricreductionof2chloro1(3chloro
phenyl)ethanonebyC.ontarioensiswholecels
232 
³XŒU090+`a

d
1 (3
de
)

[O>?@
  
~
pH65
Ї–ê"

VW³XŒU090+
`aЇ>?@

ÂÃÄY
4。
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òÅ

:¶>
ee
ðˆ›~
999%
ÿ‹

9{³XŒU>?@

³XŒU0:¶:š>?@9Þê

~
30~45℃
l
۲Lj›~
98%~99%。
/*
35℃
<>:šä
þ
30℃
<‘û1
02%,
,^}~
30℃
óЇ>
ÎȳXŒU

233 
­S³™090+`a

d
1 (3
de
)

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¿ÉA

,^

VW­S³™0WX

d
1 (3
q
4 
ɝC.ontarioensis
“\>78

;

(3
;
øÄe,Æe
e.e.
Eijk
Fig.4 Efectsoftemperatureonproductyieldand
e.e.forasymmetricreductionof2chloro1
(3chlorophenyl)ethanonebyContarioensis
wholecels
de)

[O`aЇ>?@

ÂÃÄY
5。
/Y

òÅ

ËÌ­S³™>‘û

³XЇ–ê>·—
횑û

:¶>:š9GPÊ

ȳ™1*
180
r/min
<:š{Îûð

,^

ÚYVÿ·—í
š

¡D~:¶³Xš>?@

ü£
180r/min
óÎ
È­S³™

q
5 
FG+HContarioensis
“\>78

;
1(3
;
øÄe,Æe
e.e.
Eijk
Fig.5 Efectsofrotationspeedonproductyieldand
e.e.fortheasymmetricreductionof2chloro1
(3chlorophenyl)ethanonebyC.ontarioensis
wholecels
234 
4–—˜…U090+`a

d
1 (3
de)

[O>?@
  
Ї–ês>4–—˜…UZz¤ý³XÐ
‡>V˜

,^

VW4–—˜…U090+`a

d
1 (3
de)

[O>?@

Y
6),
4–—˜
…U‹*
200g/L
<

:¶>:šËÌ4–…U>
PÊÍPÊ

ÍÈ4–—˜…U1*
200g/L
<

:
¶:š9«ZX


ee
ð9{4–—˜…UZ
02
M
 

 
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10
"
 
X>?@Lj›~
999%
ÿ‹

,^

ü£`aÐ
‡–êsÎÈ4–—˜…Uó
200g/L。
q
6 
`•ÒœContarioensis
“\>78

;
1(3
;
øÄe,Æe
e.e.
Eijk
Fig.6 Efectsofcelconcentrationonproductyieldand
e.e.forasymmetricreductionof2chloro1
(3chlorophenyl)ethanonebyC.ontarioensis
wholecels
235 CTAB
G˜090+`a

d
1 (3
d
e)

[O>?@
q
7 CTAB
½Contarioensis
“\>78

;
1(3
;
øÄÆe
e.e.
Eijk
Fig.7 EfectsofCTABpretreatmentconcentrationon
producteeandyieldforasymmetric
reductionof2chloro1(3chlorophenyl)
ethanonebyContarioensiswholecels
  CTAB
íAîQí>Ÿ :¯©ª}

·G
CTAB
09yŸ Èæ⩪

òÿQí×åZŸ
 K>:¯

ú‹°¶

:¶>·—Tn

SÍÊ
ÑU–Ÿ WXЇ>™š
[16-17]。
VW
30g/L
°
¶<9;
CTAB


⩪—˜…U

~
4℃
‘
’"0
C.ontarioensis
Ÿ Èæ⩪
20min
Þ

0
90+`aЇ>?@

Y
7)。
/Y

òÅ

ËÌ
CTAB
G˜>‘û

:¶>:š9G‘û

È
CTAB
—˜…Uû*
4g/L
<

:¶>:šJ¾"ú

È
CTAB
—˜…Uó
12g/L
<

:š/
989%
ú‹ó
971%,^
‰

È
CTAB
—˜…Uû*
8g/L
<

:
¶>
ee
ðîJ¾"ú

S
100%
"úó
955%。
:¶>
ee
ð="ú>a,òFíËÌ
CTAB
—˜
…U>‘û

4–Ÿ >:¯9G‘û

Aæˆ

 ²>Mª$%8MZX

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~
4g/LCTAB
óÎÈ©ª—˜…U

24 CTAB
IJKÏÐCandidaontarioensis
°
±öÚi“\>78

;
1 (3
;
øÄiLM23
  
0FG
CTAB
⩪~詪>
Contarioensis
Ÿ WX>ЇÈÌÈæþÍ

ÂÃÄY
8。
/Y

òÅ

èì
CTAB
©ª>U–Ÿ WX>ЇÈæ
72h
Þ

:¶>:š~
ee
ði†ó
843%
~
999%;
Íì
CTAB
⩪>Ÿ WXЇ
24h
Þ

:¶>:š~
ee
ði†{
975%
~
999%。
0¢’>³XÈ棯
HPLC


Y
9),
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ee
ð{
999%
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/^òÅ

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