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Identification of acetyl xylan esterase from Bacillus subtilis and effects of inducers on its production

枯草芽胞杆菌乙酰木聚糖酯酶的鉴定及诱导剂对酯酶活力的影响



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ChineseJournalofBioprocessEngineering
Vol.11No.1
Jan.2013
doi:10.3969/j.issn.1672-3678.2013.01.011
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:1672-3678(2013)01-0060-05
IdentificationofacetylxylanesterasefromBacilussubtilis
andeffectsofinducersonitsproduction
TIANQianqian,SONGPing,CHENChen,LINGMing,JIANGLing,LIShuang
(ColegeofBiotechnologyandPharmaceuticalEngineering,NanjingUniversityofTechnology,Nanjing210009,China)
Abstract:AnacetylxylanesterasefromBacilussubtilisCICC20034wasidentifiedandtheefectsof
inducersonitsproductionwereevaluated.TheresultsshowedthatBacilussubtilisCICC20034secreted
107×105ofhighmolecularweightesterase,theesterasewasidentifiedasanacetylxylanesterasevia
massspectrometry,andmolecularweightofthesubunitoftheesteraseis356×104。Addingethyl
acetateandxyloseasinducersintofermentationmediumextremelyincreasedtheproductionofacetyl
xylanesterase,however,otherinducers,suchastributyrin,xylan,corncobpowberandchitosanshowedhad
noefectsontheesteraseproduction.TheactivityofacetylxylanesteraseofBacilussubtilisCICC20034
reached062U/mLusingethylacetateasaninducer,thusitisthehighestactivitylevelamongwild
bacterialacetylxylanesteraseproductionstrains.
Keywords:acetylxylanesterase;Bacilussubtilis;inducer
  
N„¯_d-Ô
(acetylxylanesterase,

AXE)
Zd×òpz¯_doÁ_¯d–„0T`
Zj¯d˜„0T“…Ý0Û,`-Ô
,¯
×jâ
,-Ô-vⅦ,òpx|É4ÒÓ,q{„0T


—˜fg`ÜãR
[1]。
N„¯_d-
Ԕk¬+ñ¯_d`@|–-=.«Õi
[2];
“
)—˜òß`„0-Ô1R

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ø0%Ü
¡,Ç%Ü%ñ

¥„0‹Œ β 0„6×X&ñ


—˜“‚`)thiÓª
[3]。
N„¯_d-Ôw&%{ÏÖV%
,´
«˜
Volvarielavolvacea[4]、Trichoderma[5],Aspergilus[6],
Penicilium[7]、 Schizophylum[8]、 Rhodotorula[9]、
Fusarium[10]
Ç
Streptomyces[11];
º%w&%{´«Ö
Bacilus[12]、 Thermobifida[13]、 Firobacter[14]
Ç
Thermoanaerobacterium[15]
â

¦jV%

º%N
„¯_d-Ô{צB

)-Ô12Á
[16]。
pd{:12Ž)‘%N„¯_d-Ô
Ö23ã

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)%ÅN„¯_d-Ô

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–,)tó&%
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K0LMN,-

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dâT=
Sigma
VW

12 
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efg
10、
hiš
5、
NaCl10。
4hwÔ`a0
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`a0

FCÀ
œ(^
05%[12]。
13 
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a
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05%
`
ò{^òs
250mL
ó
50mL
4hwÔ`a0`R
€–
,37℃、200r/min
4h
70h。
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st饦
125% SDSPAGE
þ×Ç_+y„6]šþ×
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VW

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i
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Îl
100mL。
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,37℃、55
r/min,
¹§
15h[18]。
16 
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«|__+y„6þ×ÊË
Rousselot
â
[19]
`


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SDS,255%
SA
,005%
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þ×

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10min,
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TrisHCl(100mmol/L,pH75)
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[12]。1
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2.1 
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ð
1)。
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-Ô¹§
;2—
¥GSH¹§
G
1 
‹Œ†nc,4ž5=VŠ#PQ¥¦
Fig.1 NativePAGEanalysisoftheculturesupernatant
fromBacilussubtilis.
:i«|_+y„6]šþ×Ç-Ô¹§U
¥GSH¹§á‹¹§„×Ù-Ô¦¯Äx
^

J9`›œÇþ×O„¬Ë3­
[19]。
J9Š
ÅÁÈ

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þ×*+”1²–­s

pה-ԛj_‹…o

áâ(ð
2。
æð

ò
ó

-Ô¦¯Äx^Ö
107×105。
2.2 
‹Œ†nc234Ⅰ%‚pq5hi
z²ÙŽ)‘%
CICC20034
-Ô`þ×
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1—
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G
2 
‹Œ†nc234Ÿt>é™
Fig.2 Molecularweightofesterase1
fromBacilussubtilis
5qxD

ð
2,
×é

–¦h`-Ô5q
),
•­
sefxxݲÙ
(LCMS/MS)。
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i–sÁÂ
Mascot
–s#Ì`ef²Ùáâ¡.

*-

¹-ÔefUN„¯_d-Ô`ef¯r
Ö
655(
b‚j—ª
53),
Y^˜™°ìÌ
34%, 
       
ø0,½ŒU|ݚ)‘%
FZB42
`N„¯_
d-Ô_Öò@

.

Çð
3)。
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2`™ÙÏpÛ,-

K0LMÛ,-

Û,•
-

֝hÜ

Â23–-ԍÛ,•-`:1
R

ð
1)
Uef²Ùáâ¦dô

|ݚ)‘%
FZB42
`(ߦ¯Äx^
Ö
356×104,
uv>…oDŽ)‘%-Ô`
¦¯Äx^Ö
107×105,
.þ¹-Ô=>…o
D÷òºÖÏ_ß

/~

|ݚ)‘%
FZB42(
)‘%[{
916
q)‘%[{
5B6)
`N„¯_d-ԓŠ.š
。Bacilussubtilis168
(GenBank:NP_3882001 )、Bacilussubtilis
ATCC6633(GenBank:ZP_06875135.1)、Bacilus
pumilus(GenBank:2XLC_A)`
N„¯_d-Ԕ=
>…oD¯q֛_ß



›_ß

¦¯Äx^
¯qÖ
220×105、45×105
Ç
190×105[12,20-21]。
Ž
)‘%
CICC20034
N„¯_d-ԗ˜¦¯
Äx^
107×105。
Uæèé`-Ԙë!`¥

ª#­dõ.šÇEs23

§
1 
‹Œ†nc
CICC20034
|4Ⅰ5sté¡hi
Table1 IdentificationofesteraseⅠ fromBacilussubtilisCICC20034viaproteinmassspectrometry
ef %$ Y^˜™°
/%
ef¯r
¦¯Äx^
/103
|ݚ)‘%
FZB42 34 655 35.6
N„¯_d-Ô
(Cah)
)‘%[{
916
)‘%[{
5B6
Ž)‘%
TUB10 9 81 35.7
    
Ñ

t.áâæefxÝöi
Mascot
–s¶œ–s#Ì

ef¯r—ªÖ
53;¯
r‚j
53、
Y^˜™°‚j
9%
¿


C—efU–sef—˜ë!`¾ïR

Ñ

¦§à¯ÖxݲÙ`ø0,½ŒU|ݚ)‘%
(Bacilusamyloliquefaciens)
FZB42¯
d„0-Ô
Cah
ø0,»¼Y^½Œ`ìà¯
G
3 
‹Œ†nc
CICC20034
|4Ⅰ5sté¡hi
Fig.3 IdentificationofesteraseⅠ fromBacilussubtilisCICC20034byproteinmassspectrometry
26
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01+t‹Œ†nc|4u15wx
N„¯_d-ÔÖ¯_d@|ÔÁ–`dü

v
>¯_dq•@|

¡¯_d

_d

ã会

¯dâ
[11-12]
òpFCN„¯_d-Ô`‹Œ

z(
FCÀqÉ4ÒÓ,-
———
N,N-
(C2∶0)、
RO
,SA-
(C4∶0)
Ž)‘%
CICC20034
w)î
-Ô12`Õi

áâ¡ð

*-

æð

òó

Ú
œ(ߞFCÀ¿

Ž)‘%
CICC20034
òù
ŒE.ìdÙ^`-Ô

¾Ô1Ö
023U/mL;
œ(
¯_d

_d

ã会ÇRO,SA-4hw
Ô AƒFCÕi

N,N-Ö_*FCÀ

ߞÔ

062U/mL;¯
dW˜ë!`FCÕi

-Ôß
žÔ1Ö
046U/mL,
¾Úœ(FCÀÔ12¯qá

169%
Ç
100%。
æèé`š&º%wN„¯_
d-Ô`º2¡.

*-

Â23–Ž)‘%ß
žÔ1
062U/mL,CICC20034
wN„¯_d-ÔÖ
/~èé`š&º%N„¯_d-ÔìÌ_:1R

pnòµ*¿À)+%έdõá:12

§
2 
.c›Š<=í|45EFuv
Table2 Productionofacetylxylanesterasesfrombacteria
%{
š&%{wN„¯_d-Ô
ߞÔ1

(U·mL-1)
¾Ô1

(U·mg-1)

Ž)‘%
CICC20034 0.62 4.7
Â23
ɑ)‘%
— 0.66 [12]
Ž)‘%
SHS0133 0.2 — [22]
Ÿ ,ʅ‘%
— 0792 [14]
¡§:ú¢%
02156 05 [13]
G
4 
01+t‹Œ†nc
CICC20034
|4Ⅰu15wx
Fig.4 EfectsofinducersonactivityofesteraseⅠ
fromBacilussubtilisCICC20034
  
Ž)‘%
CICC20034
`4hwԽˡ
ð

*-

æð

òó

4hßÁ–ƒFCÀ“”
¿

4hå#
(0~20h)
wÔ÷ÚØÙ

n#wԉ
«4h¿ÕÈ
(>50h)。
pN,N-ǯdÖF
CÀn

-ÔòpØًŒ

wÔ¿Õáål
30~
50h。
u
Degrassi
â
[12]
èé`š&ɑ)‘%N
„¯_d-Ô4hwÔ¿Õ;–j
40~72h。
G
5 
‹Œ†nc
CICC20034
EFj4¹º
Fig.5 TimecourseofesteraseⅠ productionby
BacilussubtilisCICC20034
3 
X
 
[
Ž)‘%
CICC20034
ò¯Ùd{:¦
¯Äx^`-Ô

=>…oD¦¯Äx^Ö
107×105,
ÅefxxݲÙÖN„¯_d-Ô

(ߦ¯Äx^Ö
356×104,
N,N-֎
)‘%
CICC20034
wN„¯_d-Ô`_*F


4h~–-ÔߞÔ1Ö
062U/mL,
Öæó
èé`š&º%N„¯_d-Ô`_:Ô1

Ž
)‘%
CICC20034
N„¯_d-ԗ˜´(
36 
!

# ‘11â

Ž)‘%N„¯_d-Ô`²ÙqFCÀ-Ô12`€
èé`¦¯Äx^ãšÇ:-Ô1R

—˜fI
`hiÇ23åJ

ƒ„JK

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