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Structure basis of EHs and development of novel EHs as biocatalyst

环氧水解酶的结构基础及新酶开发



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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
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:1672-3678(2013)01-0077-10
StructurebasisofEHsanddevelopmentofnovelEHsasbiocatalyst
KONGXudong1,2,YUHuilei1,ZHOUJiahai2,XUJianhe1
(1.StateKeyLaboratoryofBioreactorEngineering,EastChinaUniversityofScience&Technology,Shanghai200237,China;
2.StateKeyLaboratoryofBioorganicandNaturalProductsChemistry,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
  


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¾sѵâ ×"l{
[1-2]。
©î

QR¶
ôåßòij‹ŒXOÒn
(L
ê&
)、
XÓÔI
n

Õ}–Ö
)、
µé–×n

}µúØ
)、
ÙÚ`

(Frontalin)
ââ
[3]。
QR¶ôTU•Ð¶wUÑYYòµ*
Ï{&VTO„‹Œ

TŸ((ôÔo*ôT
ÔST`ywڍn¶ôT

¥§ÔST`¶ô®
¯

¶ôx|Ô
(epoxidehydrolases,
†n
EHs,EC3.
3.2.3)
STîˆý¶ôT`x|®¯o©Û
dRx|
。EHs
fg“”jÜmª

|

ÙÚÇ
ó&â A*˜&ß0

_y”
20´
µ
70
$
õ4/¶ôx|ÔZæj•”Ümªß0ç
R¶ô×x`õöº
[4]。
•”&ß0`´
«&œºTŸ

”Ümªß0@|çR¶ôT
`|睺

”|ÇÙږÊU}¶ô×`
¯Ä`õöUkœpqÊUó&¶ô×3ï`
õöâ
[5]。20´
µ
90
$õnó&Îï
EHs`
fg23|§
EHs
Îï`¼½

<Ž”QR¶ô
T&‹Œ–`hiŒÖ23ä/

ÕÖ&
STÀ
,EHs
—˜h¿Ú‰«AŖÄÇÛÔ

Ô
`Îïfg

©àáR:â}/

æefghi
jQR¶ôTqUÑYY`‹Œ

÷ø´«!
EHs`
á¿Uº

ŽE
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/

ŽÎïjÝݑ8B‘
%
(AgrobacteriumradiobacterAD1),
UÜmªÎï
`
sEH
—˜¾ïR
。ArEH
Åα/βx|Ô-v,•S
Tá¿æα/βx|ÔÞßá¿~Ç1"™Äá¿~
ùŒ

ð
1a),
Î=j α/βá¿~` Asp107、His275U
Asp246
*ùŒ`STRTßÑj~

"á¿~cÕ

Cå*4/`‚à¯
EHs
]Åj~d-v

¹bß
á¿`èéÕÉ×Ù§Ñj™Äá¿~–`


ø,
Tyr152/Tyr215
”ST*+–UÜá‹ÇÛ9¶
ôжÕi

 AUc¾¿
,Argiriadi
â
[7]
èé§Î
ïj~
(Musmusculus)` MssEH(PDBID:1CQZ)
b
ßá¿

Ž`

|—˜U
ArEH
×2`α/βá¿~U
™Äá¿~

8>™Äá¿~–α helix` ¿ã“”
dÙ¥q

µ•–`

"
Tyr`
ÑØ«zdô

­d
õþ§ŽdÊU§STh

¹á¿–1RÑ/
Ñj

"

‘µé–

UŽ*ST`È4ÒÓ,¶ô
T«zà‹

”Ž`

|5T}

"ڗ˜ST
º`á¿~


MssEH
Y_ß`‘Œ-.«Õi

!

" α/βx|Ô×` EHá¿ZÎïjÞ½ƒ
(Aspergilusniger)` AnEH[8],
8>ŽUÜmªó

EHs(mEH)
¾ïRÈ:

µcá™âÙá¿~

¨©Zd{ò{R`
EHs。AnEH
–×2j
mEH
`
hE

À|Ñã:~

!α/βx|Ôá¿~`Üà
б‘Œ "Ԍ`α helix¶›Ì™Äá¿~`
äà

!u”,™Äá¿~Uα/βx|Ôá¿~Èå
–`æ‹

!
2004
$l~çŒ÷˜

"α/βx|Ô
×
EH
bßá¿eèé
,¯
qZÎïjK
(Homo
sapiens)` HssEH(PDBID:1S8O)、
Îïj8ç
(Solanumtuberosum)` StEH(PDBID:2CJP)
ÇÎï
jᣑ%
(Mycobacteriumtuberculosis)` MtEHA
(PDBID:2BNG)。
•–
HssEH
U
mssEH
«z¦2

æ

|`èTË,Ôá¿~Ç

|`¶ôx|Ôá
¿~ùŒ

=
20´
µ
70

EHs
e4/n
,EHs`
ST
âœpy̧fgEs`23
[9]。
Ì
1993
$
Lacourciere
â
[10]
™Ù§”
H182O¶·DóSß EHs
(ÉVTnw–ÝÝRžÏ

Ÿd¾¿á‹§í
¿_Ž4.`¥§Ôbßá¿ÇSTâ°pq•
U
mEH
”½Œt`¦2R

á0`/”*‰éòy
`STâœ

ð
1(d))。
•–
Asp
”STh`!
dõ–ÕÖj£JÀ­¯¶ô3ÃÄ

UܑŒ
-–Õß

•Ú

"$0
His
U
Asp
µ*êCx¯
Ė`xÄ

w&
OH-
j£­¯-–Õß4&x
|

…ÝUÑYYÿŒSTh`!Yõ

x|*
+–-–Õß`³0ô*‘Œ`ôÀ–ÄUsÕ
tU@`

"´4ø0ùŒ`ôë

!u‘ŒØo
á¿

Ì
1999
$
ArEH
bßá¿`èé­dõ×
æ§~dâœ

¾¿4/STh5^q

"Î=
ìÄá¿~`
Tyr,
µ*U¶ôôÃđŒHôe
(•þÀR

-=ÁÙÜá‹ÑØÇÛ9¶ôÐ
¶`Õi

1.2 
t•V
A4ùú4
!d"«α/βx|Ô×EHs` bßá¿(Îïj
K`
LTA4
x|Ô

j
2001

Thunnissen
â
[11]



ZCåèé`?dd"z¶ôx|nw&«U
ÑYY`
EH。
•á¿Åjα α!üýÞß,æN|
á¿~

STá¿~Ç

|á¿~Rà¯ùŒ

ð
1b)。
STá¿~—˜aäefÔá¿ãš

T}d"
Zn2+
á‹Ñ/

¾¿—˜fRy
A4x|ÔÇø0íÔ
1R

á‹D‰UÙ/¥+ìÓáâòp×ٕST
âœ

ð
1e),
h4&¿ìÑá‹j
His295、His299
U
Glu318
`
Zn2+
ÕÖ
Lwies
,*C¶ôж

w&`
C63
87
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Ÿ–ÄæfRy–`¥îRy^l
C12,375
1T`x¯Ä–êC­0ÿŒx|h

G
1 EHs
‘pԋ
Fig.1 Catalyticmechanismofepoxidehydrolases
1.3 
´µV
1,2 EHs
2003
$
,Arand
â
[12]
|¨§Îïj¦û¦\%
(Rhodococcuserythropolis)
`ÏÐy
1,2 EHs
(limonene1,2epoxidehydrolase,LEH)` 3D
á¿
97 
!

# ºÛHâ

¶ôx|Ô`á¿01qŽÔÐ4
(PDBID:1HS6),
ŽÅjd׎`
EHs,
‚‘7
149
"ø0,

Åj~d×
EHs`
bßá¿5TŸá
£‘%
(Mycobacteriumtuberculosis)` MtEHA(PDB
ID:2BNG)
ǔ4ƒ%
(Streptomyceslasaliensis)
–Ê
U_q‹Œ`
lsd19(PDBID:3RGA)。
Žd`á¿
ãšZ—˜



" α üýÇ6":³ï½`
βÞß*ùŒα/βð…á¿,ð`0‘Œ1"E
sef0à`Üዢ_

•ST$0TŸÑj
Üà`”"
Asp
UÑj”øcÕ`
Arg,
¢_•Ú
௴«æûxR$0*ùŒ

ð
1c)。
U!d×
EHs
¦¾
,LEH
STâ°7T}dõh

ð
1f)。
”
Tyr53
U
Asn55
`Û9D

æ
Asp132
êCx–`

"


w&`
OH
j£­¯¶ô

U©¾¿
Asp101
z
xÄ^ñ¶ôôÃÄ

há:n
Asp132
›zx
ĵ*
Arg99
^Y
Asp101,
<ÔYÌy±…o

æj
LEH
µ*(õhÿŒx|

Ú^qÜUÔ`–
Õ߅o
,¨
©
LEH
×
EHs
˜òºòyxpî`j
£JÀ¡
N3
-、NO2
-、CN-、Cl-、Br-、I-、OCN-、
SCN-、HCOO-
âì/¶ôж

G#pYYpî`
•Ÿw
[13]。
1.4 EHs
5-./01
jα/βx|ÔÞßEHsÇLEH×EHs,”x
|îˆý¶ôT*+–¾¿“”©àáRÇ
:~àáR¼½

ãÙ`
EH
òº}jST

Eo

EÜ`x|

“”©àáR

òijˆýÜ
­s®¯

ð
2)。
¾¿j(``



í
EH
­¯˜Cõ` α3ÃÄ¿,w¿E4&òV
w&

EYY

uí­¯ƒCõ`β3ÃÄ¿,w
¿E”Ñ&Œ

EYY

¨©

íST”{¿E`
Üp¦`ÑØàáR­sx|¿

òì/ˆ
ý¶ôT`©ÛdRx|G#(d¿E`Y
Yw

~d*+òµ*EHs
ojn:iԄÇT@„ST¶ôx|`O„
ì/

¡ùi
Aspergilusniger
Ç
Bacilussufurescens
%$òôTLNy­s©ÛdRx|G#
89% e.e.
Ç
92%
`³°
[14]。
u|Îï`8ç
StEH[15]
Ç-ç
mbEH[16]
¯qò(`STôTLN
yǍK0ôTLNy`©ÛdRx|

Furstoss
â
[17]
ùi
Aspergilusniger
šÔU,x|`
O„iˆý`K0ôTLNy°±§¦h`

EYY

“_›p
99% e.e.
Ç
58%
³°‹Œ§

Nifénalol。
G
2 EHs
¶·/01
Fig.2 Regioselectivityofepoxidehydrolases
2 
®
EHs
˜~‘p+5¯E
2.1 
¸M6/TU
=
20´
µ
90
$õ-Kdó&Îï`
EHs
­s§Es`23

4/§‚ÓºðàáRx|¶
ôT`º%

V%%$

|§
EHs`
Ôï¼½

_y`ïà´«!攔`%$04噕
EHs
1R

}jàáºð˜ž@|yw`%$

ºx|
æ©w&`¶ô–Õß

oºdy¶ôTçR`
%$

up¶ôÜÕÖ?d

ï8BJ9­
s¢;`aïàoûõá:ܜ³­sóTï
à

ò¬ãÙÜ!=>õ–fgïà
EHs
w&
%$

=
1993
$-
,Mischitz
â
[18-19]
ïàÌdӗ˜
¶ôTx|Ô1R`º%%$

“.§dÁŒè
é

TŸ‚^%$`ÜݲÙ

ùi
EHs
­s°
±‰D`QR‹Œ
、EHs
ÁÙT
、EHs
ST`©Û
dRx|â

Ù¯þ§º%
EHs
”˜â‹Œ–`
hid2

„,`
Zocher
â
[20]
W¬w
EHs`
V
%%$­s§¦h`)Õ

•–
Aspergilusniger
.
ì`
EHs
©neEs23“æì/§2lT

Zocher
â
[20]
™Ù§
120
$4ƒ%ôTLNy`
1RqàáR

!–ïàÌ
Streptomycesantibioticus
Tü4
©àáR

ªì
31,
“)U‚à¯cåèé
`%$—˜lÍ`©àáR

,0_Ææ¼H
œ)‚@ùÀǑ½ù!8B–ïà#Ì

$º%
Bacilus megaterium ECU1001
Ç

$ V %
TrichosporonloubieriECU1040,
ŽdL00xSA
q—˜lÍ`©àáR
,E
ª¯qìÌ
478
Ç
658[21-22]。
GH‚@
Li
â
[23]
ùi
Pseudomonas
sp.
žº)®¯

L0¶ôN¡Z,N-ì̧
95% e.e.sÇ25%`³°。’“)t‚@RS,‘
½ù¯–#Ì`
Rhodococcussp.ML 0004
%$ò
    
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ijSTx|²’ ¶ôŸ ,&w

z{,
[24]。
l~ÖÅ

昂^ó&%$Ç|eïà̗˜
¶ôx|Ô1R

ó&´«Î=j
Agrobacterium、
Aspergilus、 Bacilus、 Beauveria、 Corynebacterium、
Pseudomonas、Rhodococcus、Rhodosporidium、Rhodotorula、
Streptomyces、Trichosporon
âÅ

|ÎïTŸ§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
æ˜2lÔ]À

2.2 
%!f·ghTU
@
20


Kdæ!=>õïà#̧‚^w
EHs`
ó&

µZæjš&%.ì`
EHs
±±%
h•Õef`d‘௓)òº¾¿“”Ï"Ú
¾àáR`
EHs,¨
u“”wÔ12Áq©ßà
áRÚ:â¼½

š&%$­swÔ}T«z
»¿

u)Ô­s¯–²T`*+…CôÔ1K
#

¨©

KdõCÉpP§¡žG#:©ßà
áR`
EHs
Ç:w
EHs`
%$

<=&`a@
`45

0¨½Œ`a`žÏpq0¨)+;<`
­õ

"ƒG#Ž`
EHs
0¨æŒÖòº

ùÏÅ
h`
EHs
1æe·¸“.ù.ì

¡
Aspergilus
niger、Agrobacteriumradiobacter、Rhodococcusglutinis
Ç
Solanumtuberosum
Îï`
EHs。
<=
EHs
™½
r^`e(

•½ŒºÐÁW˜§ÈEs`§
|
,Barth
â
[25]
¯¨§‚^
EHs
½ŒU•á¿`Ð
Á

Ö!0¨rê,–OPÈÏÚ¾STRx`
EHs
áf§01

.

Œö§_@

$Œ·¸.
ì`à¯
EHs
q•0ÂSTRx

§
1 
¹º_©ª§¨5
EHs
7‘p1é
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%

[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%

[32]
18 
!

# ºÛHâ

¶ôx|Ô`á¿01qŽÔÐ4
Œ.
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%

ŠV‰rê
Bacilus
megateriumECU1001 83U/mg E=56(R) [34]
 
2.3 EHs
>»2
efx)+µ*ÙP­ToœRÍ6âO„
òë!}*+Ô`1RÇàáR

u)òp‹‚Ô
`4iÜ./

µ*©ªŽÔohiŽ/΋
‚
EHs
ÕÖ&STÀ`./ÇRº

‹Ù/˜
&‹Œ)—,«z˜i

<=¶ôTx|
Ô῝ºÐÁ`ÈEsœ|

á‹òijÈ:ž
¿À¥+ß

,
)`
¯Ä&@O„Ç
EHs
1R`
:µ^å™;<


EHs`
ÙP­TUœRÍ6Œ
Ö@ $
EHs
i~`23ä/

昦Ð&u/#§
EHs`
:µ^ïàO
„
[3,35],

:① UV/VIS¯ Éɳ„,†™"ƒ,µ
¹}M³Ú:
;② µ*NaIO4ôTYY,ዯÉÉ
³„o÷Éɳ„

™ÙwYY`^
;③ NBP(4
(pnitrobenzyl)pyridine)
„
,NBP
ºU¶ôTh
&ŒÌ§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

Ç

¿E`Üow“”x^¥
,¨
©•xÝáâ
ò×ÙÜÇw`
e.e.
ª

!u×ٍ©àáR

⑤ Looâ[35]pù¦OÖ`-À,”}1,2 ¶ôO
¡`¼ÒûütÙP­T`%$`
EHs
12­
sïà

µ*%Šx§‹à1R%$

Reetz
â
[36]
_j¬
AnEH
`©àáR­s
§ÙP­T

µ*dú…­
PCR
“i
ESI MS
­s
:µ^ïà

#̧¥+ß
A217V/K332E/A390E,
U
WT(Wildtype)
¦¾L00xSAq`àáR
(E
ª

æ
4.5
á:̧
10.8。
_@Ÿd÷ùip
LacZαÕÖè&ef`H§%ŠïàO„z AnEH
`.잰á:§@
50
†
。vanLoo
â
[37]
i…­
PCR
Ǿï.ù`O„
ArEH
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ƒ„JK

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