作 者 :孔旭东1; 2; 郁惠蕾1; 周佳海2; 许建和1
期 刊 :生物加工过程 2013年 11卷 01期 页码:77-86
关键词:手性合成;环氧水解酶;结构基础;分子生物学;
摘 要 :环氧水解酶能应用于外消旋环氧化物的动力学拆分或对映归一性水解制备光学纯的环氧或邻位二醇,具有广阔的应用前景。近年来,多个环氧水解酶晶体结构的报道使人们对它的结构基础有了更深入的理解。随着基因信息的增长,分子生物学和蛋白质工程技术的发展大大简化了大量克隆表达多样性环氧水解酶的过程,降低了环氧水解酶分子改造的难度,为新型具有工业应用潜力的环氧水解酶的开发提供了技术支持。本文综述了环氧水解酶的结构与机制以及近年来环氧水解酶重组表达及分子改造的研究进展。
全 文 :!11
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1
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2013
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ChineseJournalofBioprocessEngineering
Vol.11No.1
Jan.2013
doi:10.3969/j.issn.1672-3678.2013.01.014
!"#$
:2012-11-07
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,Email:
jianhexu@ecust.edu.cn
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JOPI
:1672-3678(2013)01-0077-10
StructurebasisofEHsanddevelopmentofnovelEHsasbiocatalyst
KONGXudong1,2,YUHuilei1,ZHOUJiahai2,XUJianhe1
(1.StateKeyLaboratoryofBioreactorEngineering,EastChinaUniversityofScience&Technology,Shanghai200237,China;
2.StateKeyLaboratoryofBioorganicandNaturalProductsChemistry,ShanghaiInstituteofOrganicChemistry,
ChineseAcademyofScience,Shanghai200036,China)
Abstract:Epoxidehydrolases(EHs)werewidelyusedinthesynthesisofchiralepoxidesanddiolsby
kineticsresolutionorenantioconvergenthydrolysis.Recently,withthecrystalstructuresofEHs,more
detailsabouttheircatalyticmechanismwereobtained.Withthedevelopmentofbioinformatic,molecular
biologyandproteinengineering,moreEHswerecloned,expressedandengineered.Thisreviewfocused
mainlyonthestructurebasisofEHsandthedevelopmentofnovelEHsasbiocatalyst.
Keywords:chiralsynthesis;epoxidehydrolase;structurebasis;molecularbiology
Cå
,
Ýt*i`n÷dà¯QR
。
æjÔǺ).yß1QR
,
îýns
Kßn
,2
{©ßKß0`n1R
、
õö*+
qçR±±ë!`¥
。
QR¶ôTqÐ
¶wUÑYYæjòpUÏ{j£JÀ¡Y
、
6
、
6
、
n
、
§ñ
、
Xâh
,
!uÖâd×
«z.«`QRmç
,
mn
、
_n
、
abÇÛº
T@l)tâO1=.«`hiÓª
。
¡ùi
QR0xSAr&U6`hòd×.«
`YnβÏtÐñ?À,Tѵ、_
dѵ
、
¾sѵâ ×"l{
[1-2]。
©î
,
QR¶
ôåßòijXOÒn
(L
ê&
)、
XÓÔI
n
(
Õ}Ö
)、
µé×n
(
}µúØ
)、
ÙÚ`
añ
(Frontalin)
ââ
[3]。
QR¶ôTUжwUÑYYòµ*
Ï{&VTO
,
T((ôÔo*ôT
ÔST`ywÚn¶ôT
、
¥§ÔST`¶ô®
¯
、
¶ôx|Ô
(epoxidehydrolases,
n
EHs,EC3.
3.2.3)
STîý¶ôT`x|®¯o©Û
dRx|
。EHs
fgjÜmª
、
|
、
ÙÚÇ
ó&â A*&ß0
。
_y
20´
µ
70
$
õ4/¶ôx|ÔZæjÜmªß0ç
R¶ô×x`õöº
[4]。
&ß0`´
«&ºT
:
Ümªß0@|çR¶ôT
`|çº
、
|ÇÙÚÊU}¶ô×`
¯Ä`õöUkpqÊUó&¶ô×3ï`
õöâ
[5]。20´
µ
90
$õnó&Îï
EHs`
fg23|§
EHs
Îï`¼½
,
<QR¶ô
T&`hiÖ23ä/
。
ÕÖ&
STÀ
,EHs
h¿Ú«AÅÄÇÛÔ
、
Ô
`Îïfg
、
©àáR:â}/
,
æefghi
jQR¶ôTqUÑYY`
。
÷ø´«!
EHs`
á¿Uº
、
E
EHs`
ïàq*÷âO
&u§
EHs
&STi~`235
。
1 EHs
XÊS°±
1.1 α/βùú4²³EHs
!d"
EHs`
R+á¿
(ArEH,PDBID:1EHY)
j
1999
æ
Nardini
â
[6]
4/
,
ÎïjÝÝ8B
%
(AgrobacteriumradiobacterAD1),
UÜmªÎï
`
sEH
¾ïR
。ArEH
Åα/βx|Ô-v,S
Tá¿æα/βx|ÔÞßá¿~Ç1"Äá¿~
ù
(
ð
1a),
Î=j α/βá¿~` Asp107、His275U
Asp246
*ù`STRTßÑj~
2
"á¿~cÕ
,
Cå*4/`à¯
EHs
]Åj~d-v
。
¹bß
á¿`èéÕÉ×Ù§ÑjÄá¿~`
2
"
ø,
Tyr152/Tyr215
ST*+UÜáÇÛ9¶
ôжÕi
。
AUc¾¿
,Argiriadi
â
[7]
èé§Î
ïj~
(Musmusculus)` MssEH(PDBID:1CQZ)
b
ßá¿
,
`
C
|U
ArEH
×2`α/βá¿~U
Äá¿~
,
8>Äá¿~α helix` ¿ã
dÙ¥q
,
µ`
2
"
Tyr`
ÑØ«zdô
,
d
õþ§dÊU§STh
。
¹á¿1RÑ/
Ñj
1
"
L
µé
,
U*ST`È4ÒÓ,¶ô
T«zà
。
`
N
|5T}
1
"ÚST
º`á¿~
,
MssEH
Y_ß`-.«Õi
。
!
3
" α/βx|Ô×` EHá¿ZÎïjÞ½
(Aspergilusniger)` AnEH[8],
8>UÜmªó
Sß
EHs(mEH)
¾ïRÈ:
,
µcáâÙá¿~
,
¨©Zd{ò{R`
EHs。AnEH
×2j
mEH
`
hE
N
À|Ñã:~
,
!α/βx|Ôá¿~`Üà
б "Ô`α helix¶ÌÄá¿~`
äà
,
!u,Äá¿~Uα/βx|Ôá¿~Èå
`æ
。
!
2004
$l~ç÷
3
"α/βx|Ô
×
EH
bßá¿eèé
,¯
qZÎïjK
(Homo
sapiens)` HssEH(PDBID:1S8O)、
Îïj8ç
(Solanumtuberosum)` StEH(PDBID:2CJP)
ÇÎï
já£%
(Mycobacteriumtuberculosis)` MtEHA
(PDBID:2BNG)。
HssEH
U
mssEH
«z¦2
,
æ
N
|`èTË,Ôá¿~Ç
C
|`¶ôx|Ôá
¿~ù
。
=
20´
µ
70
$õ
EHs
e4/n
,EHs`
ST
âpy̧fgEs`23
[9]。
Ì
1993
$
Lacourciere
â
[10]
Ù§
H182O¶·DóSß EHs
(ÉVTnwÝÝRÏ
,
d¾¿á§í
¿_4.`¥§Ôbßá¿ÇSTâ°pq
U
mEH
½t`¦2R
,
á0`/*éòy
`STâ
(
ð
1(d))。
Asp
STh`!
dõÕÖj£JÀ¯¶ô3ÃÄ
,
UÜ
-Õß
,
Ú
2
"$0
His
U
Asp
µ*êCx¯
Ä`xÄ
,
w&
OH-
j£¯-Õß4&x
|
,
ÝUÑYYÿSTh`!Yõ
。
x|*
+-Õß`³0ô*`ôÀÄUsÕ
tU@`
2
"´4ø0ù`ôë
,
!uØo
á¿
。
Ì
1999
$
ArEH
bßá¿`èédõ×
æ§~dâ
,
¾¿4/STh5^q
2
"Î=
ìÄá¿~`
Tyr,
µ*U¶ôôÃÄHôe
(þÀR
,
-=ÁÙÜáÑØÇÛ9¶ôÐ
¶`Õi
。
1.2
tV
A4ùú4
!d"«α/βx|Ô×EHs` bßá¿(Îïj
K`
LTA4
x|Ô
)
j
2001
$æ
Thunnissen
â
[11]
|¨
#Ì
,
ZCåèé`?dd"z¶ôx|nw&«U
ÑYY`
EH。
á¿Åjα α!üýÞß,æN|
á¿~
、
STá¿~Ç
C
|á¿~Rà¯ù
(
ð
1b)。
STá¿~aäefÔá¿ã
,
T}d"
Zn2+
áÑ/
,
¾¿fRy
A4x|ÔÇø0íÔ
1R
。
áDUÙ/¥+ìÓáâòp×ÙST
â
(
ð
1e),
h4&¿ìÑáj
His295、His299
U
Glu318
`
Zn2+
ÕÖ
Lwies
,*C¶ôж
,
w&`
C63
87
&
(
)
*
+
!
11
"
ÄæfRy`¥îRy^l
C12,375
1T`x¯ÄêC0ÿx|h
。
G
1 EHs
pÔ
Fig.1 Catalyticmechanismofepoxidehydrolases
1.3
´µV
1,2 EHs
2003
$
,Arand
â
[12]
|¨§Îïj¦û¦\%
(Rhodococcuserythropolis)
`ÏÐy
1,2 EHs
(limonene1,2epoxidehydrolase,LEH)` 3D
á¿
97
!
1
# ºÛHâ
:
¶ôx|Ô`á¿01qÔÐ4
(PDBID:1HS6),
Åjd×`
EHs,
7
149
"ø0,
。
Åj~d×
EHs`
bßá¿5Tá
£%
(Mycobacteriumtuberculosis)` MtEHA(PDB
ID:2BNG)
Ç4%
(Streptomyceslasaliensis)
Ê
U_q`
lsd19(PDBID:3RGA)。
d`á¿
ãZ
1
"æ
4
" α üýÇ6":³ï½`
βÞß*ùα/βð
á¿,ð`01"E
sef0à`Üá¢_
,
ST$0TÑj
Üà`"
Asp
UÑjøcÕ`
Arg,
¢_Ú
௴«æûxR$0*ù
(
ð
1c)。
U!d×
EHs
¦¾
,LEH
STâ°7T}dõh
(
ð
1f)。
Tyr53
U
Asn55
`Û9D
,
æ
Asp132
êCx`
1
"
xÄ
,
w&`
OH
j£¯¶ô
,
U©¾¿
Asp101
z
xÄ^ñ¶ôôÃÄ
。
há:n
Asp132
zx
ĵ*
Arg99
^Y
Asp101,
<ÔYÌy±
o
。
æj
LEH
µ*(õhÿx|
,
Ú^qÜUÔ`
Õß
o
,¨
©
LEH
×
EHs
òºòyxpî`j
£JÀ¡
N3
-、NO2
-、CN-、Cl-、Br-、I-、OCN-、
SCN-、HCOO-
âì/¶ôж
,
G#pYYpî`
w
[13]。
1.4 EHs
5-./01
jα/βx|ÔÞßEHsÇLEH×EHs,x
|îý¶ôT*+¾¿©àáRÇ
:~àáR¼½
。
ãÙ`
EH
òº}jST
R
Eo
S
EÜ`x|
,
©àáR
,
òijýÜ
s®¯
(
ð
2)。
¾¿j(``
R
EÜ
,
í
EH
¯Cõ` α3ÃÄ¿,w¿E4&òV
w&
S
EYY
,
uí¯Cõ`β3ÃÄ¿,w
¿EÑ&
R
EYY
。
¨©
,
íST{¿E`
Üp¦`ÑØàáRsx|¿
,
òì/
ý¶ôT`©ÛdRx|G#(d¿E`Y
Yw
。
~d*+òµ*EHs
ojn:iÔÇT@ST¶ôx|`O
ì/
。
¡ùi
Aspergilusniger
Ç
Bacilussufurescens
%$òôTLNys©ÛdRx|G#
89% e.e.
Ç
92%
`³°
[14]。
u|Îï`8ç
StEH[15]
Ç-ç
mbEH[16]
¯qò(`STôTLN
yÇK0ôTLNy`©ÛdRx|
。
Furstoss
â
[17]
ùi
Aspergilusniger
ÔU,x|`
Oiý`K0ôTLNy°±§¦h`
R
EYY
,
_p
99% e.e.
Ç
58%
³°§
R
Nifénalol。
G
2 EHs
¶·/01
Fig.2 Regioselectivityofepoxidehydrolases
2
®
EHs
~p+5¯E
2.1
¸M6/TU
=
20´
µ
90
$õ-Kdó&Îï`
EHs
s§Es`23
,
4/§ÓºðàáRx|¶
ôT`º%
、
V%%$
,
|§
EHs`
Ôï¼½
。
_y`ïà´«!æ`%$04å
EHs
1R
,
}jàáºð@|yw`%$
(
ºx|
æ©w&`¶ôÕß
)
oºdy¶ôTçR`
%$
。
up¶ôÜÕÖ?d
C
ï8BJ9
s¢;`aïàoûõá:ܳsóTï
à
,
ò¬ãÙÜ!=>õfgïà
EHs
w&
%$
。
=
1993
$-
,Mischitz
â
[18-19]
ïàÌdÓ
¶ôTx|Ô1R`º%%$
,
.§dÁè
é
,
T^%$`ÜݲÙ
、
ùi
EHs
s°
±D`QR
、EHs
ÁÙT
、EHs
ST`©Û
dRx|â
,
Ù¯þ§º%
EHs
â`
hid2
。
,`
Zocher
â
[20]
W¬w
EHs`
V
%%$s§¦h`)Õ
,
Aspergilusniger
.
ì`
EHs
©neEs23æì/§2lT
。
Zocher
â
[20]
Ù§
120
$4%ôTLNy`
1RqàáR
,
!ïàÌ
Streptomycesantibioticus
Tü4
©àáR
E
ªì
31,
)Uà¯cåèé
`%$lÍ`©àáR
。
,0_Ææ¼H
)@ùÀǽù!8Bïà#Ì
1
$º%
Bacilus megaterium ECU1001
Ç
1
$ V %
TrichosporonloubieriECU1040,
dL00xSA
qlÍ`©àáR
,E
ª¯qìÌ
478
Ç
658[21-22]。
GH@
Li
â
[23]
ùi
Pseudomonas
sp.
º)®¯
3
L0¶ôN¡Z,N-ì̧
95% e.e.sÇ25%`³°。)t@RS,
½ù¯#Ì`
Rhodococcussp.ML 0004
%$ò
08
&
(
)
*
+
!
11
"
ijSTx|² ¶ô ,&w
L
z{,
[24]。
l~ÖÅ
,
æ^ó&%$Ç|eïàÌ
¶ôx|Ô1R
,
ó&´«Î=j
Agrobacterium、
Aspergilus、 Bacilus、 Beauveria、 Corynebacterium、
Pseudomonas、Rhodococcus、Rhodosporidium、Rhodotorula、
Streptomyces、Trichosporon
âÅ
,
|ÎïT§8ç
、
ç
、
- ç
、
¹ ô â
。
Agrobacterium
radiobacter AD1、 Aspergilus niger、 Rhodococcus
erythropolis、Solanumtuberosum、Rhodotorulaglutinis
âÎ
ï`
EHs
23_ÖEs
,
Cå]æs§.ù.ì
,
)
AspergilusnigerEH
æ2lÔ]À
。
2.2
%!f·ghTU
@
20
$Î
,
Kdæ!=>õïà#̧^w
EHs`
ó&
,
µZæj&%.ì`
EHs
±±%
hÕef`dà¯)òº¾¿Ï"Ú
¾àáR`
EHs,¨
uwÔ12Áq©ßà
áRÚ:â¼½
。
&%$swÔ}T«z
»¿
,
u)Ôs¯²T`*+
CôÔ1K
#
。
¨©
,
KdõCÉpP§¡G#:©ßà
áR`
EHs
Ç:w
EHs`
%$
。
<=&`a@
`45
、
0¨½`a`Ïpq0¨)+;<`
õ
,
"G#`
EHs
0¨æÖòº
。
ùÏÅ
h`
EHs
1æe·¸.ù.ì
,
¡
Aspergilus
niger、Agrobacteriumradiobacter、Rhodococcusglutinis
Ç
Solanumtuberosum
Îï`
EHs。
<=
EHs
½
r^`e(
,
½ºÐÁW§ÈEs`§
|
,Barth
â
[25]
¯¨§^
EHs
½Uá¿`Ð
Á
,
Ö!0¨rê,OPÈÏÚ¾STRx`
EHs
áf§01
。
.
1
ö§_@
5
$·¸.
ì`à¯
EHs
q0ÂSTRx
。
§
1
¹º_©ª§¨5
EHs
7p1é
Table1 CatalyticpropertiesofclonedEHs
Îï Ü ª2@zro¾Ô1 ©àáR 3
Rhodococcus opacus
ML 0004
Vmax=2.24mmol/(L·min·mg),
Km=45mmol/L
[26]
Mugilcephalus ND 99% e.e.(R),15.4%
w°
[27]
Novosphingobium
aromaticivorans
Vmax=40mmol/(L·min·mg),
Km=6mmol/L
E=1.4(R) [28]
Metagenome 42mU/mg E=5.9(R) [29]
Streptomyces
carzinostaticus
ATCC15944
kcat,R=133min
-1,
Km,R=0.5mmol/L
E=44(R) [30]
Streptomyces
globisporusSB1010
kcat,S=48min
-1,
Km,S=0.6mmol/L
E=37(S) [31]
Rhodosporidium
tortiloides
Vmax=10.5mmol/(L·min·mg),
Km=595mmol/L
100% e.e.(R),
18%
w°
[32]
18
!
1
# ºÛHâ
:
¶ôx|Ô`á¿01qÔÐ4
.
Îï Ü ª2@zro¾Ô1 ©àáR 3
Cupriavidus
metalidurans CH34
kcat,R=4.8s
-1,
Km,R=2.05mmol/L
E=2(R) [33]
Vignaradiata(mung
bean)
kcat=129min
-1,
Km=12mmol/L
99% e.e.p(R),
67%
w°
Vrê
Bacilus
megateriumECU1001 83U/mg E=56(R) [34]
2.3 EHs
>»2
efx)+µ*ÙPToRÍ6âO
òë!}*+Ô`1RÇàáR
,
u)òpÔ
`4iÜ./
,
µ*©ªÔohi/Î
EHs
ÕÖ&STÀ`./ÇRº
,
Ù/
&),«zi
。
<=¶ôTx|
ÔῺÐÁ`ÈEs|
,
áòijÈ:
¿À¥+ß
(
,
)`
¯Ä&@OÇ
EHs
1R`
:µ^å;<
,
EHs`
ÙPTURÍ6
Ö@ $
EHs
i~`23ä/
。
æ¦Ð&u/#§
EHs`
:µ^ïàO
[3,35],
T
:① UV/VIS¯ Éɳ,",µ
¹}M³Ú:
;② µ*NaIO4ôTYY,á¯ÉÉ
³o÷Éɳ
,
ÙwYY`^
;③ NBP(4
(pnitrobenzyl)pyridine)
,NBP
ºU¶ôTh
&̧¹b
,
ò
600nm
så
;④ Reetz
âÐ4§i
ESI MS
:µ^Ù
EHs
©àáR
`O
,
p
1∶1
`
(R) PGE(phenylglycidyl
ether)
Uøõ
(S) PGE
ÕÖÜ
,
hnæj
R
Ç
S
¿E`Üowx^¥
,¨
©xÝáâ
ò×ÙÜÇw`
e.e.
ª
,
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JK
:
[1] ZelaszczykD,KiecKononowiczK.Biocatalyticapproachesto
opticalyactivebetablockers[J].CurMedChem,2007,14:
5365.
[2] AgustianJ,KamaruddinAH,BhatiaS.Singleenantiomericbeta
blockers:theexistingtechnologies[J].ProcessBiochem,2010,
45:15871604.
[3] ChoiW J,ChoiCY.Productionofchiralepoxides:Epoxide
hydrolasecatalyzedenantioselectivehydrolysis[J].Biotechnol
BioprocEng,2005,10:167179.
[4] OeschF.Mammalian epoxide hydrases:inducible enzymes
catalysing the inactivation of carcinogenic and cytotoxic
metabolitesderivedfromaromaticandolefiniccompounds[J].
Xenobiotica,1973,3:305340.
[5] HammockBD,GilSS,StamoudisV,etal.Solublemammalian
epoxidehydratase:actiononjuvenilehormoneandotherterpenoid
epoxides[J].CompBiochemPhysiolB,1976,53:263265.
[6] NardiniM,RidderIS,RozeboomHJ,etal.TheXraystructure
ofepoxidehydrolasefrom Agrobacterium radiobacterAD1:an
enzymetodetoxifyharmfulepoxides[J].JBiolChem,1999,
274:1457914586.
[7] ArgiriadiMA,MorisseauC,HammockBD,etal.Detoxification
of environmental mutagens and carcinogens: structure,
mechanism,andevolutionofliverepoxidehydrolase[J].Proc
NatlAcadSciUSA,1999,96:1063710642.
[8] ZouJ,HalbergBM,BergforsT,etal.StructureofAspergilus
nigerepoxidehydrolaseat1.8resolution:implicationsforthe
structureandfunctionofthemammalianmicrosomalclassof
epoxidehydrolases[J].Structure,2000,8:111122.
[9] HanzlikR P,EdelmanM,MichaelyW J,etal.Enzymatic
hydrationof[18O]epoxides:roleofnucleophilicmechanisms
[J].JAmChemSoc,1976,98:19521955.
[10] LacourciereGM,ArmstrongRN.Thecatalyticmechanismof
microsomalepoxidehydrolaseinvolvesanesterintermediate[J].
JAmChemSoc,1993,115:1046610467.
[11] ThunnissenM M GM,NordlundP,Haeggstrom JZ.Crystal
structureofhumanleukotrieneA4hydrolase,abifunctional
enzymeininflammation[J].NatStructMolBiol,2001,8:
131135.
[12] ArandM,HalbergBM,ZouJY,etal.StractureofRhodococcus
erythropolislimonene1,2epoxidehydrolaserevealsanovelactive
site[J].2003,22:25832592.
[13] MischitzM,FaberK.Asymmetricopeningofanepoxidebyazide
catalyzed by and immobilized enzyme preparation from
Rhodococcussp.[J].TetrahedronLet,1994,35(1):8184.
[14] PedragosamoreauS,ArchelasA,FurstossR.Microbiological
transformations28:enantiocomplementaryepoxidehydrolyzesasa
preparativeaccesstobothenantiomersofstyreneoxide[J].J
OrgChem,1993,58(20):55335536.
48
&
(
)
*
+
!
11
"
[15] ElfstromLT,WiderstenM.Catalysisofpotatoepoxidehydrolase,
StEH1[J].BiochemJ,2005,390:633640.
[16] XuW,XuJH,PanJ,etal.Enantioconvergenthydrolysisof
styreneepoxidesbynewlydiscoveredepoxidehydrolasesinmung
bean[J].OrgLet,2006,8:17371740.
[17] PedragosaMoreauS,MorisseauC,BaratiJ,etal.Microbiological
transformations37:Anenantioconvergentsynthesisofthebeta
blocker(R)Nifenalol usingacombinedchemoenzymatic
approach[J].Tetrahedron,1997,53:97079714.
[18] MischitzM,KroutilW,WandelU,etal.Asymmetricmicrobial
hydrolysisofepoxides[J].Tetrahedron:Asymmetry,1995,6
(6):12611272.
[19] SimeoY,FaberK.Selectivityenhancementofenantioandstereo
complementary epoxide hydrolases and chemoenzymatic
deracemizationof(+/-)2methylglycidylbenzylether[J].
Tetrahedron:Asymmetry,2006,17:402409.
[20] ZocherF,EnzelbergerM M,BornscheuerUT,etal.Epoxide
hydrolaseactivityofStreptomycesstrains[J].JBiotechnol,
2000,77:287292.
[21] TangYF,XuJH,YeQ,etal.Biocatalyticpreparationof(S)
phenylglycidyletherusingnewlyisolatedBacilusmegaterium
ECU1001[J].JMolCatalB:Enzymatic,2001,13:6168.
[22] PanJ,XuJH.Markedenhancementofepoxidehydrolase
productionfrom TrichosporonloubieriECU1040bysubstrate
inductionandfedbatchfermentation[J].EnzymeMicrobTech,
2003,33:527533.
[23] LiCF,LiuQ,SongX,etal.Epoxidehydrolasecatalyzed
resolutionofethyl3phenylglycidate using whole celsof
Pseudomonassp[J].BiotechnolLet,2003,25:21132116.
[24] LiuZ,LiY,PingL,etal.Isolationandidentificationofanovel
Rhodococcussp ML0004 producingepoxidehydrolaseand
optimizationofenzymeproduction[J].ProcessBiochem,2007,
42:889894.
[25] BarthS,FischerM,SchmidRD,etal.Sequenceandstructureof
epoxidehydrolases:Asystematicanalysis[J].ProteinsStructure
FunctionandBioinformatics,2004,55:846855.
[26] LiuZQ,LiY,XuYY,etal.Cloning,sequencing,andexpression
ofanovelepoxidehydrolasegenefromRhodococcusopacusin
Escherichiacoliandcharacterizationofenzyme[J].Appl
MicrobiolBiotechnol,2007,74:99106.
[27] LeeSJ,Kim H S,Kim SJ,etal.Cloning,expressionand
enantioselectivehydrolyticcatalysisofamicrosomalepoxide
hydrolasefromamarinefish,Mugilcephalus[J].Biotechnol
Let,2007,29:237246.
[28] WooJH,KangJH,KangS,etal.Cloningandcharacterizationof
anepoxidehydrolasefromNovosphingobiumaromaticivorans[J].
ApplMicrobiolBiotechnol,2009,82:873881.
[29] KotikM,StepanekV,MaresovaH,etal.EnvironmentalDNAasa
sourceofanovelepoxidehydrolasereactingwithaliphatic
terminalepoxides[J].JMolCatalB:Enzymatic,2009,56:
288293.
[30] LinSJ,HorsmanGP,ShenB.Characterizationoftheepoxide
hydrolaseNcsF2from theneocarzinostatinbiosyntheticgene
cluster[J].OrgLet,2010,12:38163819.
[31] LinSJ,HorsmanGP,ChenYH,etal.Characterizationofthe
SgcF epoxide hydrolase supporting an (R)vicinaldiol
intermediateforenediyneantitumorantibioticC1027biosynthesis
[J].JAmChemSoc,2009,131:1641016417.
[32] LiuZQ,ZhangLP,ChengF,etal.Characterizationofanewly
synthesizedepoxidehydrolaseanditsapplicationinracemic
resolutionof(R,S)epichlorohydrin[J].CataCommun,2011,
16:133139.
[33] KumarR,WaniS I,Chauhan N S,etal.Cloningand
characterization ofan epoxide hydrolase from Cupriavidus
metaliduransCH34[J].ProteinExprPurif,2011,79:4959.
[34] ZhaoJ,ChuYY,LiAT,etal.Anunusual(R)selectiveepoxide
hydrolasewithhighactivityforfacilepreparationofenantiopure
glycidylethers[J].AdvSynthCatal,2011,353:15101518.
[35] SmitM S,LabuschagneM.Diversityofepoxidehydrolase
biocatalysts[J].CurOrgChem,2006,10:11451161.
[36] vanLooB,SpelbergJH,KingmaJ,etal.Directedevolutionof
epoxide hydrolase from A. radiobacter toward higher
enantioselectivitybyerorpronePCRandDNAshufling[J].
ChemBiol,2004,11:981990.
[37] ReetzM T,Tore C,EipperA,etal.Enhancing the
enantioselectivityofanepoxidehydrolasebydirectedevolution
[J].OrgLet,2004,6:177180.
[38] NardiniM,RickR B,JanssenD B,etal.Structureand
mechanism ofthe epoxide hydrolase from Agrobacterium
radiobacterAD1[J].JMolCatalB:Enzymatic,2001,11:
10351042.
[39] ThomaeusA,NaworytaA,MowbraySL,etal.Removalofdistal
proteinwaterhydrogenbondsinaplantepoxidehydrolase
increasescatalyticturnoverbutdecreasesthermostability[J].
ProteinSci,2008,17:12751284.
[40] ReetzMT,BocolaM,WangLW,etal.Directedevolutionofan
enantioselectiveepoxidehydrolase:uncoveringthesourceof
enantioselectivityateachevolutionarystage[J].JAmChem
Soc,2009,131:73347343.
[41] KotikM,ArchelasA,FamerovaV,etal.Laboratoryevolutionof
anepoxidehydrolasetowardsanenantioconvergentbiocatalyst
[J].JBiotechnol,2011,156:110.
[42] XuY,XuJH,PanJ,etal.Biocatalyticresolutionofglycidylaryl
ethersbyTrichosporonloubieri:cel/substrateratioinfluencesthe
opticalpurityof(R)epoxides[J].BiotechnolLet,2004,26:
12171221.
[43] XuW,XuJH,PanJ,etal.Enantioconvergenthydrolysisof
styreneepoxidesbynewlydiscoveredepoxidehydrolasesinmung
bean[J].OrgLet,2006,8:17371740.
[44] HeWH,HuaY,FanLQ.IsolationoftotalRNAandcloningof
epoxidehydrolasegenefromVignaradiata[J].FoodDrug,
2011,13:100103.
58
!
1
# ºÛHâ
:
¶ôx|Ô`á¿01qÔÐ4
[45] JiaT,XuJ,YangS.ImmobilizationofBacilusmegaterium
epoxidehydrolaseanditscatalyticperformance[J].ChinJ
Catal,2008,29:4751.
[46] JuX,PanJ,XuJH.Enantioconvergenthydrolysisofp
nitrostyreneoxidecatalyzedbymungbeanepoxidehydrolase
[J].ChinJCatal,2008,29:696700.
[47] ChoiWJ,LeeEY,YoonSJ,etal.Biocatalyticproductionof
chiralepichlorohydrininorganicsolvents[J].JBiosciBioeng,
1999,88:339341.
[48] BaldasciniH,GanzeveldKJ,JanssenDB,etal.Efectofmass
transferlimitationsontheenzymatickineticresolutionofepoxides
inatwoliquidphasesystem[J].BiotechnolBioeng,2001,73:
4454.
[49] LeeEY,YooSS,KimHS,etal.Productionof(S)styrene
oxidebyrecombinantPichiapastoriscontainingepoxidehydrolase
fromRhodotorulaglutinis[J].EnzymeMicrobTech,2004,35:
624631.
[50] YeatesCA,KriegH M,BreytenbachJC.Hydroxypropylβ
cyclodextrininducedcomplexationforthebiocatalyticresolution
ofapoorlysolubleepoxide[J].EnzymeMicrobTech,2007,40:
228235.
[51] GenzelY,ArchelasA,BroxtermanQB,etal.Microbiological
transformations.47.Asteptowardagreenchemistrypreparation
ofenantiopure(S)2,3,and4pyridyloxiraneviaanepoxide
hydrolasecatalyzedkineticresolution[J].JOrgChem,2001,
66:538543.
[52] ChoiWJ,ChoiCY,DeBontJAM,etal.Resolutionof1,2
epoxyhexanebyRhodotorulaglutinisusingatwophasemembrane
bioreactor[J].ApplMicrobiolBiotechnol,1999,53:
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