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Cloning,purification and characterization of promising 2-deoxy-D-ribose 5-phosphate aldolase from Staphylococcus aureus N315

来自Staphylococcus aureus N315的2-脱氧-D-核糖5-磷酸醛缩酶的工程菌构建、表达纯化与性质鉴定



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ChineseJournalofBioprocessEngineering
Vol.11No.1
Jan.2013
doi:10.3969/j.issn.1672-3678.2013.01.009
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StaphylococcusaureusN315
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DERA(SaDERA),
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z{QG›XˆÀÁÑõrX3[

hi[

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±²Œ<
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ü
45℃;SaDERA
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T
pH110、25℃
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T
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¸
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:1672-3678(2013)01-0047-07
Cloning,purificationandcharacterizationofpromising2deoxyDribose
5phosphatealdolasefromStaphylococcusaureusN315
HANLei,CHENXi,FENGJinhui,WUQiaqing,ZHUDunming
(NationalEngineeringLaboratoryforIndustrialEnzymes,TianjinInstituteofIndustrialBiotechnology,
ChineseAcademyofSciences,Tianjin300308,China)
Abstract:ThegenomeminingwasusedtofindapromisingDERAsfromthegenomicdatabase.Afterthe
optimizationofthecodons,aDERAfromStaphylococcusaureusN315(SaDERA)wasoverexpressedinE.
coliBL21(DE3),purified,andcharacterized.ThepurifiedSaDERAwasobtainedafterasimpleonestep
simplepurification.Thehomodimericenzyme(57kDa)hastheoptimalactivityatpH77and45℃.It
showedquitehighstabilityatalkalinepH(pH11.0)and89% ofactivityremainedafterincubation(25℃
for24h).Itshowedaresistancetoacetaldehydeandmorethan70% ofactivityremainedafterexposure
to03mol/Lacetaldehydefor30minat25℃.Toconfirmitssyntheticpotential,thedoublealdol
productwassynthesized,purifiedandidentified.
Keywords:2deoxydribose5phosphatealdolase;genomemining;Staphylococcusaureus
  
Ô¨•:àáR

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hij-§T)&w*+–

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R}ST
C—C
ô`‘Œ

òÕÖQR¯Ä)t‹
Œ`.«)—
[1]。2
¥ô

£d

Ë,0Ô
(2deoxydribose5phosphatealdolase,DERA,EC
4124)
ZCå4/`?dd{NÒØE`


”&ß0
,DERA
ò@}ST•v>Ü
NÇ

SA

Ë,4&0hu&Œ

¥ô

£d

Ë,

ÊUÏ{ó&`
DNA
õ
ötu
[2]。
¹ÔÅj

ם0Ô

ډ«Û¨Ä

]
pd"”A`Øø,Ö1RÑ/
[3]。
DERA
—˜H`ÜÝ
[4]。
p§Nî

DERA
5ºðp+



Bõ+ûÕfß
[4]。
Ž
‘Œ`Þߖ—`¿Eÿ[æ
DERA
Ù“)µ
zZ

¿E
。DERA
Z?dd{ºSTN`=0
‹oǝ×xcÕ`0C0‹`0Ô

~<•±
yÐÑ

¨ÖfßUyß1Z

pNÕfß`0
‹wWòÕÖ­dõ0‹`yßÜ

!u”0
Üt¶sÏ"QR–—

YÉ0‹`wòp&Œ
ØÙ`k0u–ŧ­dõ0‹
[5]。
¹ã/<Ž
ŒÖ‹ŒÏ{„˜dÇdr&`˜2)—

Ԍ0‹wòpij‹ŒŸ¯×n
(statin)
`QR4åß
[6-9],
~dh*+pDÓ`

«Q
R`EbÖÜ

—˜fI`hiåJ

<=
DERA
hiÓª`æçÚ?(E

ÍÎ
ÍÏ`ab
DERA
`0¨
(deoC)
e·¸

™½Ç.
ì

~çæèé`
DERA
¯qÎïj
Aeropyrum
pernix(
FߏÖ
BAA81452,

)[10]、Escherichia
coliK12(CAA26974)[11]、Paenibacilussp.EA001
(ACT313388)[12]、Pyrobaculumaerophilum(AAL63
340)[13],ThermococcuskodakaraensisKOD1(BAC677
08)[14]、Thermotoga maritima(AAD36625)[13]、
Yersiniasp.EA015(ACN71238)[15]。
>u

µz1“
”ò{.ì^Á

ØÙR¥pq12Á`c/

¨©

#Ìd{:.ì^

:ØÙR`

:12`
DERA
pwÖ.«

Â23.ì

²Td{Î=
StaphylococcusaureusN315
`
DERA(SaDERA),
“
23•¦ÐRx

1 
QRSTU
11 
QR
111 
xS

%$q´«JÀ
pET 28a(+)
xSˆßÇ
E.coliBL21(DE3)
£
yoº)T=
Novagen
VW
。FastDigestë°R0
xÔT=
Fermentas
VW

efgÇhiáC]
Î=
BD
VW
。Bradford
efœ³JÀWT=#´
µVW

¥GSH
R250
T=
Solarbio
VW

NADH
T=
Codexis
VW
。d 2
¥ô£d

Ë
,
(DR5P)、
Ë,+d¿ÔÇSAË,¥HԐ
‹~
(GPD/TPI,
Î=IEêùû

T=
Sigma
Aldrich
VW
。40%
Nx{~T=,n;k

•
ÚJÀ]ևÀ¯¨²

112 
´«ä¬Í±
ZHWY 200D
Œ`a¦«¬

t­JŠV
W
);SW CJ 1FD
!K)Õ]

®KMLVW
);
APV2000
:/‡åÉÊä

I,
APV
VW
);RC6+
:ƒ8$–—â
(Thermo
VW
)。
ef²Tq[Ô¦
¯Äx^™Ù]”
GE
VW`
AKTAPurifier10
e
f¯–²TÁÂt­s
。SpectraMax M2eԗä
T=
MolecularDevices
VW
;microTOF QI
ExÝ
(MS)¯
¨ä

rê:;M6
microTOFcontrol
qrê
›œM6
DataAnalysis
]T=
Bruker
VW

12 
%!gh
121 
rê,OPq&`a@¯¨
”U,,Þ&;<`a–—
(NCBI)`
efr
ê,
(htp://www.ncbi.nlm.nih.gov/protein)
–

p
“2
deoxydribose5phosphatealdolase”
o
“deoxyribose
phosphatealdolase”
Öås_

“p
“bacteria”
ÖëÙ

×
Ù¹à`
DERA。
µ*”
ExPASy
`
ProtParam

(htp://www.ncbi.nlm.nih.gov/protein)
t6®¹àÔ
`œ#œTãR

zp}kNø,"rÏoZOr
`

òº€ò{R

Çϙò{RÁ`

µ*
BioEdit¯
¨M6


7.0.9)` ClusterW
ϽŒ¾

,¯
¨¹à`
DERA
`”A½Œ

_›×Ùpd{Î
=j
StaphylococcusaureusN315` DERA
Ö23ã

*ÚÖ
SaDERA),
•ø0,ʽŒ`Fߏ
Ö
BAB43223。
122 
–bÄ}TÇ0¨‹Œ
µ*”Ë`
jcat
–bÄ}T+½
(htp://www.
jcat.de)

SaDERA
`ñ0¨½Œ­s§¬”
E.coli
–.ì`–bÄ}T

>n”

|(t§
His6—3。”×Ù§0¨”|`ë°ÔçqÑcn
æt­Ûg&?;45˜ëVW‹Œ

13 
f·c5Êý7(545§¨Gp
131 
)+%`¿À
xS`áC

Ôx

VT

£,]šþ×

0
¨`FC.ìq
SDS PAGE
âìÓ~Õ]¬Ë

[16]
­s

C`0¨¿Àl
pET 28a(+)
xSˆß`
NcoI
Ç
EcoRI
”ÔxÑ/cÕ

V
Tl
E.coliBL21(DE3)
!^£yoº%–

132 
C`Ô`.ì²T
z)+%òs
800mL
}›œ³Ö
100μg/mL
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LB
~ß`a0–
,37℃
‚`a
(200r/min)
l
OD600Ö 10¿,œ(›œ³Ö 1
mmol/L` IPTG
­sFC.ì
(4h、37℃)。
–—
(4℃、6000r/min、10min)
³%

“i
4℃
Ï8`
ÍÎ~
A(20mmol/LTrisHCl、pH80、500mmol/L
NaCl)
ÅÆ%ß
,-20℃
”“

p
50mL
ÍÎ~

.Ë%ßn

­s:/‡å
É%

–—
(12000r/min、4℃、15min)
p˜º)Ê
»

t™~

ƚÔ~

†n
CFE)
”i
022μm`

(Milipore)
*onµ*
HisTrapTM FastFlow
…
¨Ï
(GEHealthcare,5mL)
­sdõ²T

i
10%
ÍÎ~
B(
}
02mol/L
,-`ÍÎ~
A)
ûÖÏ
Ä

t9ni
10%
`ÍÎ~

ÎÅ

IÌîå™0
/0Ë¿

i
10% ~100%
`ÍÎ~

­sËRÅ
¥
(6
"ߞ
)。
³;‹“C`efn

µ*!o’
­sœ0¥Ì“œ(›ßž¯rÖ
10%
`SA

*™¯ón

j
-80℃
”“

14 
4Ó1é½¾
141 
1R¯¨O„
DERA
`1R™Ùµ*
GPD/TPI
ºT`O„”
ԗät­s
。DR5P
”
DERA
STD³|w&`
G3P
ºe
GPD/TPI
5Ãuˆ»
NADH,
¶-
340nm
ÇÈD`ÆɳD@
(ε=622mmol-1·cm-1)。æ
j
GPD/TPI
*^

©D@ƒ°U
G3P
`&Œƒ°d
ô

µz

~"ºTh”
25℃
D­s

hßÁ

100mmol/LTris HCl
ÍÎ~
(pH77)、1mmol/L`
DR5P、2U
`
GPD/TPI
Ç
035mmol/L
`
NADH
p
q4^`
SaDERA。
Æɳ`+TeԌå™
5min
p×Ùy±hƒ°
。1
"Ô12(Ñ
(U)
eÙÒÖ
”tuå™56D

Ó¯Ô³|
1μmol` DR5Pu

1μmol` G3P*‰`Ô^。
142 
¦¯Äx^`™Ù
SaDERA
`¦¯Äx^q•”{~–`_;
…oµ*
SDS PAGE
pq¯ÄïÎ×Ù

*i¯Ä
ïÖ
Superdex20010/300GL(GE
VW
)。
Ъ¦Ö

150mmol/LNaCl` 20mmol/L
Ë,ÍÎ~
(pH
74),
ЃÖ
04mL/min。
ef`¦¯Äx^æ
—Õef`”Ñߞ­s6®#Ì

—Õef¯q
Ö
Ovalbumin(44×104)、Conalbumin(75×104)、
Conalbumin(158×105)、Feritin(440×105)、
Thyroglobulin(669×105)。
143 
_4h56
_4h56¬³|Ç0‹`1R¯¨O„
ΙÙ

j_4h
pH,
*iÍÎßÁ
(100
mmol/L)
Ö
NaAc HAc(pH40~60)、Na2HPO4
NaH2PO4(pH60~70)、Tris HCl(pH70~95)
Ç
Na2CO3 NaHCO3(pH95~110)。j_4
h³

h”
25~65℃
D­s

144 
ª2@Êr
ª2@Êr¬³|Ç0‹`1R¯¨O„Î
™Ù

%Z
DR5P
àCÚ¾œ³
(04~8mmol/L)。
.ª2@Êr
(KmÇ Vmax)q•P¥æ SigmaPlot
v12.2`
ËR‰‹º#Ì

15 
Zi1>¢
151 pH
ØÙR
j
pH
dyR

*iÍÎßÁU_4
pH
™Ù
¿¦¾

“”jÚ¾
pH
ÍÎ~–`Ô{~
(05
mg/mL)`
Ԕ
25℃
D²
24h
n

™Ù•$“
12

152 
äØÙR
j³dyR

“”j
100mmol/LTris HCl
ÍÎ~
(pH77)
–`Ô{~
(05mg/mL)
”Ú¾
³D
(4~65℃)
²
10min
n™Ù•$“12

֙Ù
SaDERA
”_4h³
(45℃)
D`12
k$#

‘ÁÔ~
(20μL)”Ú¾`¿Õ/C0“
™Ù•$Ú12

153 
NdyR
Öå™
SaDERA
N`dyR


100
mmol/LTris HCl
ÍÎ~
(pH77)
Ç
300mmol/L
N`Ô{~
(05mg/mL)
”
25℃
D²

”Ú
¾`¿ÕC0‘ÁÔ~
(20μL)“µ*óE!o’
(AmiconUltra05mL,Ultracel10k)
p˜N
[13]。
”i
100mmol/LTris HCl(pH77)
„…ÌÃߞ
cn

™Ù•$Ú12

16 
ijeç¼@7j~hi
161 
¯¨O„
W……¨
(TLC)
56

5…ÀÖN,N-
(EA);
ë§ÀÖQaë§À
(V(
Qa
)∶V(
œ
H2SO4)∶
V(H2O)=5∶10∶85);RtÈSë§。Ԍ0‹w
2,4,6
R¥ô d`¾¤ªÂÖ
05,
ëT-§

MS
56

þUVþ–ÄD’
(ESI+),
àáh/·
(SRM)
WXD’

UVþ/
45kV,
VTzÐ^
6L/min,
–Ä^Y躒³
180℃,
Z[zÖ
N2,WX\°10Hz。
162 
Ԍ0‹hqw²Ù
h{~
(10mL):100mmol/L
Ë,ÌÍÎ~
94 
!

#
 
ð
 


Î=
StaphylococcusaureusN315` 2
¥ô

£d

Ë,0Ô`)+%¿À

.ì²TURx²Ù
(pH=74),03mol/L

,8mg
²Ô

h56

25℃

,100r/min
¦

p
TLC
/·h­+
,3d
ná:h

(

†ßž`+û›Åhn

1tÄ
Ø
15min,
–—p˜ef
。40℃
D(/ý°h~l
1mL,
i°±
TLC
­s²T

ÉD`wiN,N-
(EA)
.{n

*o

°Ç

C
025mg
{j
1mL
mili Q


­9Ì
MS
p噕¦¯Äx^

2 
XYSZ[
21 
˜~klÓ>¢
SaDERA(
Fߏ
BAB43223)
æ
220
"ø0,
$0ùŒ

æ
ExPASy
eft`
ProtParam
ºó

SaDERA
`œ#¦¯Äx^Ö
2334×105,
œ#
pI
Ö
471。
ø0,ϽŒ¾

ð
1)
áâë-

SaDERA
U Î ï j
Paenibacilus sp.EA001
(PaeDERA)[12],ThermococcuskodakaraensisKOD1
(TkDERA)[14]、Yersiniasp.EA015(YerDERA)[15]、
Thermotoga maritima(TmDERA)[13]、Pyrobaculum
aerophilum (PaDERA)[13]、 Aeropyrum pernix
(ApDERA)[10]
Ç
EscherichiacoliK12(EcDERA)[11]
`
DERAs
½ŒdôR¯qÖ
640%、517%、
515%、473%、290%、285%
Ç
274%。
Þ§]Jõ.¦[”A`ø0,$0

T§]Jõ.¦2`$0
;▲õ.Ë,0á‹:;õ.1RÑ/
G
1 SaDERA
Smn5
DERAs
5opq—t
Fig.1 MultiplesequencealignmentforSaDERAandtheotherreportedDERAs
05
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  EcDERA
–^q‘Œ
Schif
&k2­0‹
h`Ðô$0
(Asp102、Lys167
Ç
Lys201)
”
SaDERA
–ÿ[”A
(¯
qh
Asp89、Lys151
Ç
Lys180)。
”
EcDERA
–
,Lys167
UܑŒ
Schif
&

u”ÞßsՖ
Lys167
ò@`
Asp102、Lys201
^qfߝ`ÞßàáR`xÄTV¤*+
[17]。
¨©

c?
SaDERA
òº—˜STº2

Desantis
â
[18]
èé§
EcDERA
`Ë,ዢ_
æ
Gly171、Lys172、Gly204、Gly205、Val206、Arg207、
Ser238
Ç
Ser239
ùŒ


SaDERA
–

h`$0
Ò É Z
Gly155、Phe156、Gly183、Gly184、Val185、
Arg186、Thr200
Ç
Arg201。156(
¬
SaDERA


D
¾

Ñ`ø0,$0æ&R`
Lys
ØÎÖûx`
Phe,
e(§Ë,ዢ_`ûxR
;238
Ñ`ø0,$0
æ
Ser
ØÎÖ4ÈÈ`
Thr,
ue‚§Ë,ዢ_
`sÕі
;201
Ñ`ø0,$0æ–R`
Ser
ØÎ
Ö&R`
Arg,
*+§Ë,ዢ_`Äþ¶·

~
ç*+z<#
SaDERA
U«Ë,Cõ`×T‹
`ዺ2eé

!u˜9jԌ0‹h`­s

22 
…†
SaDERA
5§¨Gp
SaDERA
—˜òß`ò{R
,37℃
FCÆò.
ì0‚^ò{ef

æð

òó

æj
SaDERA
q
§
His6—3,µ*dõ†(`ÿϲTÆò#Ìþ
ײ`C`Ô

ð
2,
×é
PE)。
NI—
ŠFC`[º)
;W—
FC`[º)
;S—
FC`º)
ÉÊ~t™
;PE—
Å
HistrapTM
jDž¨cn`²Ô

M—
ef¦¯Äx^—˜
G
2 SaDERA
5
SDS PAGE

Fig.2 SDSPAGEanalysisofSaDERA
SaDERA
²Ô`¾Ô1Z
1189U/mg(
.
1),
Z¦¾56DÎ=
PaeDERA
`¾Ô1
(62U/mg)
`

†
[12]。
23 SaDERA
r%K
µ*]š*o…¨
(Superdex20010/300GL),
SaDERA
`”ÑߞÖ
14.1mL,¨
©
SaDERA

`¦¯Äx^ÂÖ
557×104。
æ
SDSPAGE
ó

•(ߦ¯Äx^Ö
255×104,
*p¹Ô”{
~–p¾ïY_ߑ’“”

©áâUæèé`
DERA
`[0rdô

§
1 SaDERA
5stGp¶·
Table1 Purificationoftargetenzyme
²Tõö
ߞ

mL
Õef

mg
Õ12
a/

¾Ô1

(U·mg-1)



²T
†r
šÔ~
b 50 170 8986 52.9 100 1.0
ÿυ¨
32 64 7611 118.9 85 2.2
 
Ñ
:a
Öd"Ô12(Ñ
(U),`
”
25℃
DÓ¯Ô³|
1μmol
DR5P
*‰`Ô^
;b
֚Ô~

Z
3.1g
F%
(800mL
%~
)`
É%t™

24 
%u4Ó1é
SaDERA
`Ô@Rx¯¨¡ð

*-

æð

(a)
Çð
3(b)
òó
:SaDERA
_4h56Ö
pH
77。
Žº”d"H`
pH
./
(65~95)

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u)—˜¦í:`&
dyR

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pH110
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–
25

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24h
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93%
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—
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40℃
²
10min
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ò
ó

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,30min
0”,§
:j
70%
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2h
n

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6h
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SaDERA
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EcNAL
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G
3 SaDERA
54Ó1é>¢
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G
4 SaDERA
5›dvw1
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áâ¡
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L/(mmol·s),
~U23_Ù¯`:12`
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¦2
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uUæèé`
PaeDERA[12]
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2 SaDERA
S8x
DERAs
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Table2 ApparentkineticsparametersofSaDERA
andtheotherDERAs
Ô
Km/
(mmol·L-1)
kcat/s
-1 kcat∶Km/
(mmol-1·L·s-1)
SaDERA 1.13±0.07 198±2 175
PaeDERA[12] 145 320 0.24
EcDERA[18] 0.64±0.01 68±1 106
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20h
n
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u
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9 Aå™ÚÌw
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[12]。
æð
5(b)
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2,4,6

ô d
(C6H12O3),ÆNÔŒ”É=0‹`w,
(d¾Ññ`œ#xX¾
(m/z)
Ö
1320786,
œ#
(ÜÎÖ
1550684,
ì™Ö
1550640。
¯–w°
395%。
áâ.þ
SaDERA
—˜STԌ­h
`º2

u)þë}j23_Ù¯`
EcDERA。
25
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eçj~hi
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ƒ„JK

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35 
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