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ChineseJournalofBioprocessEngineering
Vol.11No.1
Jan.2013
doi:10.3969/j.issn.1672-3678.2013.01.011
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JOPI
:1672-3678(2013)01-0060-05
IdentificationofacetylxylanesterasefromBacilussubtilis
andeffectsofinducersonitsproduction
TIANQianqian,SONGPing,CHENChen,LINGMing,JIANGLing,LIShuang
(ColegeofBiotechnologyandPharmaceuticalEngineering,NanjingUniversityofTechnology,Nanjing210009,China)
Abstract:AnacetylxylanesterasefromBacilussubtilisCICC20034wasidentifiedandtheefectsof
inducersonitsproductionwereevaluated.TheresultsshowedthatBacilussubtilisCICC20034secreted
107×105ofhighmolecularweightesterase,theesterasewasidentifiedasanacetylxylanesterasevia
massspectrometry,andmolecularweightofthesubunitoftheesteraseis356×104。Addingethyl
acetateandxyloseasinducersintofermentationmediumextremelyincreasedtheproductionofacetyl
xylanesterase,however,otherinducers,suchastributyrin,xylan,corncobpowberandchitosanshowedhad
noefectsontheesteraseproduction.TheactivityofacetylxylanesteraseofBacilussubtilisCICC20034
reached062U/mLusingethylacetateasaninducer,thusitisthehighestactivitylevelamongwild
bacterialacetylxylanesteraseproductionstrains.
Keywords:acetylxylanesterase;Bacilussubtilis;inducer
N¯_d-Ô
(acetylxylanesterase,
0
AXE)
Zd×òpz¯_doÁ_¯d0T`
Zj¯d0T
Ý0Û,`-Ô
,¯
×jâ
,-Ô-vⅦ,òpx|É4ÒÓ,q{0T
T
,
fg`ÜãR
[1]。
N¯_d-
Ôk¬+ñ¯_d`@|-=.«Õi
[2];
)òß`0-Ô1R
,
òpST
7
ø0%Ü
¡,Ç%Ü%ñ
C
¥0 β 06×X&ñ
i£
,
`)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{צB
,
)-Ô12Á
[16]。
pd{:12)%N¯_d-Ô
Ö23ã
,
4/¦¯Äx^Ú¾jæèé`
)%ÅN¯_d-Ô
,
É`4h¿
Õ
。
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,
p
#àád{`N¯_d-Ô1CÀÎá:Ô
12
。
1
QRSTU
11
cd
)%
CICC20034(
,)tó&%
{
)。
K0LMN,-
、
Þ¯_d
、¯
dâT=
Sigma
VW
。
12
%
{Ä`a0
(g/L):
efg
10、
hi
5、
NaCl10。
4hwÔ`a0
(g/L):M9CA
`a0
,
FCÀ
(^
05%[12]。
13
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=»C
1
¶%òsó
50mL
{Ä`a0`
R
,37℃、200r/min`
a
6h。
p
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250mL
ó
50mL
4hwÔ`a0`R
,37℃、200r/min
4h
70h。
14
st饦
125% SDSPAGE
þ×Ç_+y6]þ×
ÊË3
[17]。
Á¦¯Äx^ÕefTj
Takara
VW
。
15
|4
Õ×nC
100mgα Û,-Ç100mgβ Û
,-
,
2mL
+ûÿ[{|
,
(s
100mg
ÁH
B,
]
。
i
01mol/LpH70
Ë,ÍÎ~Ù
Îl
100mL。
zØjtͪ
,37℃、55
r/min,
¹§
15h[18]。
16
ú=5=V#PQ¥¦
«|__+y6þ×ÊË
Rousselot
â
[19]
`
O
。
t9ÍÎ
(pH68):016mol/LTris,795%
SDS,255%
SA
,005%
5MH
。
ß`J9
40℃
x²
5min,80/120VSDSPAGE
þ×
,
þ×n
xÅ
10min,
Øj߯r
05% TritonX100
`
TrisHCl(100mmol/L,pH75)
ÍÎ~*ÃR
,
-Ô¾)Ô¹§
,
#Ì-Ô¹§5q
,
xÅns
¥GSH¹§
。
17
<=í|4uv5i
pK0LMÛ,-ÖÜ
,410nm
Då
Ôx|Üw&`K0LM`Æɪ
,
ÙO
ÊË3
[12]。1
"(ÑÔ1ÙÒÖÕ5
6D
,
Ó¯Ôx|w&
1μmolK0LM*`
Ô^
。
2
XYSZ[
2.1
nc|4¯f4
:i_+y6]þ×q-Ô¾)Ô¹§
4hn`t~sþׯ¨
,
.þ)
%ò¯Ùd{-Ô
(
ð
1)。
1—
-Ô¹§
;2—
¥GSH¹§
G
1
nc,45=V#PQ¥¦
Fig.1 NativePAGEanalysisoftheculturesupernatant
fromBacilussubtilis.
:i«|_+y6]þ×Ç-Ô¹§U
¥GSH¹§á¹§×Ù-Ô¦¯Äx
^
,
J9`Çþ×O¬Ë3
[19]。
J9
ÅÁÈ
,
(s%0NY
,
þ×*+1²s
,
p×-Ôj_
o
,
áâ(ð
2。
æð
2
ò
ó
,
-Ô¦¯Äx^Ö
107×105。
2.2
nc234Ⅰ%pq5hi
z²Ù)%
CICC20034
-Ô`þ×
16
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1
# 11â
:
)%N¯_d-Ô`²ÙqFCÀ-Ô12`
M—
ef¦¯Äx^Õ
;
1—
)%4hÔ~
(
-Ô¹§
)
G
2
nc234t>é
Fig.2 Molecularweightofesterase1
fromBacilussubtilis
5qxD
(
ð
2,
×é
2
¦h`-Ô5q
),
sefxxݲÙ
(LCMS/MS)。
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isÁÂ
Mascot
s#Ì`ef²Ùáâ¡.
1
*-
。
¹-ÔefUN¯_d-Ô`ef¯r
Ö
655(
bjª
53),
Y^°ìÌ
34%,
ø0,½U|Ý)%
FZB42
`N¯_
d-Ô_Öò@
(
.
1
Çð
3)。
N¯_d-Ô1
2`ÙÏpÛ,-
(
K0LMÛ,-
、
Û,
-
)
ÖhÜ
,
Â23-ÔÛ,-`:1
R
(
ð
1)
Uef²Ùáâ¦dô
。
|Ý)%
FZB42
`(ߦ¯Äx^
Ö
356×104,
uv>
oD)%-Ô`
¦¯Äx^Ö
107×105,
.þ¹-Ô=>
o
D÷òºÖÏ_ß
。
/~
,
|Ý)%
FZB42(
)%[{
916
q)%[{
5B6)
`N¯_d-Ô.
。Bacilussubtilis168
(GenBank:NP_3882001 )、Bacilussubtilis
ATCC6633(GenBank:ZP_06875135.1)、Bacilus
pumilus(GenBank:2XLC_A)`
N¯_d-Ô=
>
oD¯qÖ_ß
、
(ß
、
_ß
,
¦¯Äx^
¯qÖ
220×105、45×105
Ç
190×105[12,20-21]。
)%
CICC20034
N¯_d-Ô¦¯
Äx^
107×105。
Uæèé`-Ôë!`¥
,
ª#dõ.ÇEs23
。
§
1
nc
CICC20034
|4Ⅰ5sté¡hi
Table1 IdentificationofesteraseⅠ fromBacilussubtilisCICC20034viaproteinmassspectrometry
ef %$ Y^°
/%
ef¯r
¦¯Äx^
/103
|Ý)%
FZB42 34 655 35.6
N¯_d-Ô
(Cah)
)%[{
916
)%[{
5B6
)%
TUB10 9 81 35.7
Ñ
:
t.áâæefxÝöi
Mascot
s¶s#Ì
,
ef¯rªÖ
53;¯
rj
53、
Y^°j
9%
¿
,
.þ
CefUsefë!`¾ïR
。
Ñ
:
¦§à¯ÖxݲÙ`ø0,½U|Ý)%
(Bacilusamyloliquefaciens)
FZB42¯
d0-Ô
Cah
ø0,»¼Y^½`ìà¯
G
3
nc
CICC20034
|4Ⅰ5sté¡hi
Fig.3 IdentificationofesteraseⅠ fromBacilussubtilisCICC20034byproteinmassspectrometry
26
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2.3
01+tnc|4u15wx
N¯_d-ÔÖ¯_d@|ÔÁ`dü
,
v
>¯_dq@|
,
¡¯_d
、
_d
、
ãä¼
、
¯dâ
[11-12]
òpFCN¯_d-Ô`
。
z(
FCÀqÉ4ÒÓ,-
———
N,N-
(C2∶0)、
RO
,SA-
(C4∶0)
)%
CICC20034
w)î
-Ô12`Õi
,
áâ¡ð
4
*-
。
æð
4
òó
:
Ú
(ßFCÀ¿
,
)%
CICC20034
òù
E.ìdÙ^`-Ô
,
¾Ô1Ö
023U/mL;
(
¯_d
、
_d
、
ãä¼ÇRO,SA-4hw
Ô AFCÕi
;
N,N-Ö_*FCÀ
,
ßÔ
1Ö
062U/mL;¯
dWë!`FCÕi
,
-Ôß
Ô1Ö
046U/mL,
¾Ú(FCÀÔ12¯qá
:§
169%
Ç
100%。
æèé`&º%wN¯_
d-Ô`º2¡.
2
*-
,
Â23)%ß
Ô1
062U/mL,CICC20034
wN¯_d-ÔÖ
/~èé`&º%N¯_d-ÔìÌ_:1R
,
pnòµ*¿À)+%Îdõá:12
。
§
2
.c<=í|45EFuv
Table2 Productionofacetylxylanesterasesfrombacteria
%{
&%{wN¯_d-Ô
ßÔ1
/
(U·mL-1)
¾Ô1
/
(U·mg-1)
3
)%
CICC20034 0.62 4.7
Â23
É)%
— 0.66 [12]
)%
SHS0133 0.2 — [22]
,Ê
%
— 0792 [14]
¡§:ú¢%
02156 05 [13]
G
4
01+tnc
CICC20034
|4Ⅰu15wx
Fig.4 EfectsofinducersonactivityofesteraseⅠ
fromBacilussubtilisCICC20034
)%
CICC20034
`4hwԽˡ
ð
5
*-
。
æð
5
òó
:
4hßÁFCÀ
¿
,
4hå#
(0~20h)
wÔ÷ÚØÙ
,
n#wÔ
«4h¿ÕÈ
(>50h)。
pN,N-ǯdÖF
CÀn
,
-ÔòpØÙ
,
wÔ¿Õáål
30~
50h。
u
Degrassi
â
[12]
èé`&É)%N
¯_d-Ô4hwÔ¿Õ;j
40~72h。
G
5
nc
CICC20034
EFj4¹º
Fig.5 TimecourseofesteraseⅠ productionby
BacilussubtilisCICC20034
3
X
[
)%
CICC20034
ò¯Ùd{:¦
¯Äx^`-Ô
,
=>
oD¦¯Äx^Ö
107×105,
ÅefxxݲÙÖN¯_d-Ô
,
(ߦ¯Äx^Ö
356×104,
N,N-Ö
)%
CICC20034
wN¯_d-Ô`_*F
CÀ
,
4h~-ÔßÔ1Ö
062U/mL,
Öæó
èé`&º%N¯_d-Ô`_:Ô1
。
)%
CICC20034
N¯_d-Ô´(
36
!
1
# 11â
:
)%N¯_d-Ô`²ÙqFCÀ-Ô12`
èé`¦¯Äx^ãÇ:-Ô1R
,
fI
`hiÇ23åJ
。
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46
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