免费文献传递   相关文献

Screening of xylanase-producing fungus and analysis of enzymatic properties

一株高产木聚糖酶真菌的筛选及酶学性质分析



全 文 :!11
"!

#
2013
$

%
&
 

 
(
 
)
 
*
 
+
ChineseJournalofBioprocessEngineering
Vol.11No.1
Jan.2013
doi:10.3969/j.issn.1672-3678.2013.01.007
!"#$
:2012-11-07
%&(

,-./01234567
(973
67

89
(2011CB710803)
)*+,
:¨  
©
(1983—),
L

’“ÚªK

«Q23&

23OP

k¬+ñ`&VT

ùÀÇ

TÁK
),
MN
,Email:jianhexu@ecust.edu.cn
;(œj<=í4>c56/74Ó1é>¢
%
 
o

pqr

#st

uvw

xyz

{|}2/0 56~€23Dk‚lmn

ƒ„
200237)
B
 
C

fghijñ¡

kl+,-.kl4mnox45p3&(564
/

õ¡
202
/pÄ"
96
s%+,)*„…ÑÐt

hi45

Ç·
96
sus%BEx

šy+,-.g/_GqrÿQECU2023,
Gzÿa
rDNA
aãW{|}SDEF€
w
(Aspergilusawamori)。
ÀÁ@-Ñõš‚‘‚ƒ
:pH60,
‘U|
4d,C
ŸF
30~40g/L
ƒ„…‹

GA
(NH4)2SO4†ŠüSuperdexG 75‡ˆA‰Š‹ˆ,õ¡ŒÍqrÿQÚ®<šô­opHF60,šô­o’“
F
50℃。
DEF

qrÿQ

L

L/+,
;96
sus%

ƒ„…‹
?G>HI
:Q946.5    
JKLMN
:A    
JOPI
:1672-3678(2013)01-0035-06
Screeningofxylanaseproducingfungusandanalysisof
enzymaticproperties
SUNJie,YUHuilei,LIChunxiu,KOUZhenfu,XUJianhe
(StateKeyLaboratoryofBioreactorEngineering,EastChinaUniversityofScienceandTechnology,Shanghai200237,China)
Abstract:Hundredsofvarioussoilsampleswerecolected.Firstly,564strainswithhaloformingzones
wereisolated.Then,thexylanaseactivitiesof202strainswerefurtherverifiedbya96deepwel
microplatescreeningassay.Theactivitiesfrom96deepwelmicroplateandhaloformingplateshowedthe
sametrend.ThebeststrainECU2023withthehighestxylanaseproductionwasidentifiedasAspergilus
awamoribyinternaltranscribedspacer(ITS)sequencing.Thecultureconditionswereoptimizedas
folows:30~40g/Lcorncobpowder,pH6.0,cultivationfor4d.Furthermore,themainxylanase
componentwascrudely waspurified by ammonium sulfate precipitation and Superdex G75
chromatograph.Thexylanaseactivitywasoptimalyperformedat50℃ andpH6.0.
Keywords:xylanase;fungi;strainscreening;96deepwelplate;corncob
  
!&x–áÿò,Œùi`&ºï

æÅ
üüŒÖ,hºï­â`´«®úcd

&
x‚^“”j=>õ–
,´
«!_Õ$Ú`
¯x¬+ñ–G#

æj•Îïfg)Œ¯•Ö

¨©

ÿ[@|¯x¬+ñ“”dÙ` ³

¯x¬+ñ`´«ù¯TŸ¬+ñ

k¬+ñ
q¯xñâR‚×

¬+ñZd{pβ 1,4 dl
ô_‹uŒ`bcd_‹

uk¬+ñgZÑ°
!Y`ù¯

U¬+ñcÕp«T@ôá‹

*h
¾í./Ö
15%~35%[1],
:ž@|k¬+ñ™ò
p±àÈÏ`¬+ñÔÕiÑ/

á:¯x¬+ñ
`@|žâ

k¬+ñ`ÿ[@|g‰«Ï{Ô
×`²¾Õi
,´
«TŸ´4` β 1,4 ¯dlô
³|ÔǍ4`Cõ0k³|Ô

¡_´µd¶j
dlÔâ
),
•–´4ÔTŸ¯_d0xÔ
(endo
1,4 β xylanase,EC3.2.1.8)ǯdlÔ(β D
xylosidase,EC32137)。
=>õ“”`k¬+ñ
Ô±±pÏԕ‹‘’“”
,´
«Îïjº%

V%

ÝË%â
[2]。
æèé`Ô1:`%$´«
; – ” ¯ ƒ Å
(Trichoderma sp.)
Ç ½ ƒ Å
(Aspergilussp.)
–

Â23´«Zùid{"ƒï
à`Q¼!=>õ–ïà:wk¬+ñÔ`Ž%
$

“•wÔRº­syõ23

^Â`%$ïà)Չ«»·‚^`¿ÕÇ
Û2

—˜¸#È

.•R¥âc/
,¨
©ÀÞd{
:µ^ïàO„Z("ïà­+`Ðô¨ñ

¬
+ñÔ:µ^ïà`O„æeÏ"‘½ùè
é
[3-5],
uz©û]hijk¬+ñԖ>Š(è
é

”yõ¯–`¹à%$•ï*+–

÷øùi
96
ºüÁ­s%$`a

Ô~¯–pqÔ1™Ù
`"ƒïà*+

¾¿v>k¬+ñÜ`›
œpqœ³w¯_dÔ`€Çžâ

p#Ö~
n­dõ`hi237Ù01

1 
QRSTU
1.1 
?@ABS}~CD
*i89:=[,}

10
cm
`89

p¶·Ï9R֗Õ

³;Ì`89i
ƒ%x»³„…n

­sûüïàÇ~ßïà

ïàÜã会

½pq‘Þ¾ješÊ
Œ¿S

Å*
015mm
¯9ï#Ì*‰‚‘`š
À

›¯qz
10g
šÀØj
500mL
R€–

(s
4% NaOH300mL,121℃
(ä°Á
20min,
89n
i‰*op˜Áß

o~
4℃
”“*Ã
,5mol/L
HCl
–Çl–R
,4℃
ÄØ*Ã

4/˜3Ý&
Œ
,8000r/min
–—
15min,
˜t™

3ݛi
95%
NYÅÆ

É

_n`’…3ÝÅÇoÃÄn

ÕÖ
ó&ïà`k¬+ñÜ

1.2 
c(b6
yï`a0
(g/L):
k¬+ñš
100,
hij
45,NH4NO330,NaCl50,K2HPO420,MgSO4·
7H2O02,¼Ò 150;pH70(~ß`a0Œ¯¦
¾

Ú(¼Ò
)。
z89„…nˆ‰Ìyï`a0
ûü
,`
a
2~3d
n

‹C(%Š­s~ß²`a

›ò{̎``aûüt

™Ùk¬+ñÔ1Rý
þÿ`‚‘
[6]。
1.3 
c(e6
z’…k¬+ñ3Ý]‡ûŔ`aÆt
(3~5mm
dz
),
ÄØ
1~2h,65℃
Ço*Ã

#
Ì`]dÜW»

iº¬xŒ5»…

ÕÖ
96
ºEºüÔ4h`Ü

Ó"º–(s
1mL

`a0

Ú}ã会

Ç

»tuÏ°`ÜW
»

Øj
30℃、500r/min
`¦«¬
(HTCBiotech
Co.)
t`a
2d,4000r/min
–—
5min,
™Ùt™
~Ô1

™ÙßÁÖ
(96
ºEºü
)100μL¯_d
{~
(1mg/mLbirchwoodxylan,Sigma
VW
)、180
μL50mmol/LpH5.0Û,ÜÍÎ~、20μLÔ~。
50℃
x²”
1h
n

(s
120μL3,5 YK0x
D,
(DNS),
ÁÈ
5min,
I89n(s
160μL˜–
Äx

‡n¤C
200μLÌԗü,iԗä™Ù
550nm
DÆɪ

1.4 
<=í4uv5ši
”J’–¯q(s
1mL¯
_d{~
(1mg/mL)
Ç
18mLpH50
Û,ÜÍÎ~

›(s
02mL
„…

†`šÔ~
,50℃
h
15min,
(s
2mLDNS,
Á
È
10min,
ÙÎl
10mL。
i¯Éɳ6™Ù
550nm
ÇÈ`Æɳ
(OD550),™Ù¿pÍÎ~ÕÖsf
Ë

ÙÒÓ¯ÔSTw&
1μmol¯ d`Ô^Ö1"
12(Ñ
(U)。
1.5 
EFj4©ª«p
4h`a0
(g/L):
ã会
100,KH2PO4
30,
efg
10,K2HPO420,MgSO4·7H2O05,
NaNO320,CaCl2·2H2O01,Mandels1%(V/V,pH
50)。
•–
Mandels
{~Œ¯Ö
MnSO4016%,
ZnSO4014%,FeSO405%,CoCl202%
[7]。
4h}
T”ó˜
50mL`
a0`
250mL
̑€–­s
,¯
qk2`a¿Õ
、pH、C
ï{×Ç

­s


`aá:nz`a~”
10000r/min
–—
20
min,
Ct™~™Ù)îÔ`12

1.6 
cdhi
”
15mL
–—’–(s
100μLƒ%x,ÉC
3~4
¶Ž´`a`%Êßj–—’–

¦«]‡n

8000r/min
–—
2min,
Çt™~

”
15mL
–—’
–(s
150μL³|~(“¹‚@–,:Ë)tó&
8ïU`a–—áf
),
¦«]‡n
,85℃
h
25
min
#Ì0¨ù
DNA
šá~

ùi
ITS
½Œµi¶
63
&
 

 
(
 
)
 
*
 
+
  
!
11
"
 


:5′TCCGTAGGTGAACCTGCGG3′;

:5′
TCCTCCGCTTATTGATATGC3′)
­s
PCR
‹e

²T
q™½

_nz™½áâ­s
BLAST
¾

1.7 
45Gp7bH§I
³;4ht™~

1²–­s
(NH4)2SO4¯^


Ó(s
20%
Ðdz`ºÄ
(NH4)2SO4šÀ
n

ÄØ
05h,8000r/min
–—
10min。
3Ý`
"¯^efi
50mmol/L、pH60
Û,ÍÍÎ~{
|

ý¨*Ã

“™Ù¯_dÔ12

ài
40% ~
60%
efù¯­s!o
,4℃、8000r/min
–—
30
min,
Cœ0n`Ô~
4℃、10000r/min
–—
10
min,
Ct™~
500μL­s SuperdexG 75]š*
o

:i
AKTAExplorer
…¨ÁÂ
,50mmol/LpH
6.0
Û,ÍÍÎ~Å¥

ЃÖ
0.5mL/min,
=ª³
;Ӓ
500μLù¯,¯ q™ÙÔ1,SDS PAGE„
™Ù¦¯Äx^


pH
Ô12`€

z
Ô~ØÎÌÚ¾
pH
ÍÎ~
(pH30~60
ÏÐ,Ì
ÍÎ~
,pH55~80
Ë,ÌÍÎ~
,pH80~100
?¾ÑÜÌ,ÍÎ~

–

\ÝØ
5h,

05
h,
™Ù$Ú¯_dÔ12

h³Ô1
2`€
:¯
q”Ú¾³D

™Ù¯_dÔ12
Î×Ù_4³

2 
XYSZ[
2.1 
c(6/7hi
2.1.1 
%$y^ïàáâ
!=>õ–ïà¯_dÔæ˜èé
[8-10]。
÷
ø:i

{Ú¾`_ÕÒ¾ÕÖïàÜ

¾¿
Ü­sϛœ

˜p¬+ñ╟Œ¯


•–`k¬+ñŒ¯

pïàºwk¬+ñÔ`%
$

t|Á:;Ì`89

ÅÁß`a0¯–

~
ß²`an

™Ù1Rýþÿ‚‘

áâ(ð
1。
ÔêÜÇ89`Ú¾

¥˜
11
"ÓÉ
,D
ª
.-ýþÿ`ñu
,d
ª.-%Šñu
,D/d
‚.
-%$`wÔº2é

æð

òó

¥˜
564
"
(%Š./0§k¬+ñ@|Ô1R

•–
362
$
%$`ýþÿ
D/d
”
0~5

,123
$%$`ýþ
ÿ
D/d
”
5~8


ýþÿ
D/d
‚j
8`
¥˜
79
$

ª#Ñv`Z

‚Ïrq˜%Ê`%$`
D/d
1‚j
8。
¾¿4/px{Rß`‘Þ¾ÕÖÜ
¿

Î…ïà#́:Ô1`%$

­dõ
ïà#Ì`
D/d
‚j
5` 202
$%$­s§¯_d
Ô12`•ï

G
1 
JA_`aUb6j4c(5KLMN
Fig.1 Primaryscreeningofmicrobialstrains
throughhaloformingmethod
2.1.2 
%$•ïáâ

202
$%$`¯_dÔ12­s§•ï

À
Þ§d"0j
96
ºEºü`%$`a

Ô~¯–p
qÔ1™Ù`û]

“§¹O„`~ÕòsR
UrêòÔR

Õj

k¬+ñ’…3Ýe()Œ
]d5»…

Öק‚^Úò{Ü`ØÙn^*
+

•É

zà¯%$`yï
D/d
Uºü™#`¯
_dÔ12¾

4/”øðñ0Âdô

ð

(a))。
zzÚå
13`
%$Ô1UTÛ`

$””
%$

t­)tó&*

­s§¾

ð
2(b))。
TÛ`%$¯qÖæÜûÝÃè%
(Phanerochaete
chrysosporium)、
Þ½ƒ
(Aspergilusniger)、
-§¯ƒ
(Trichodermaviride)
Ç ¡ < ¯ ƒ
(Trichoderma
reesei),
4/•–Þ½ƒ`¯_dÔ12”TÛ`
%$–Z_:`

uïà#Ì`
PAM111、UV4、
PDA23、H29、RS14、RS88、RS91、RS97、WS40、
WS191、M1、ECU2023、WDF8`
Ô11¦:

•
–
ECU2023`
Ô1_:

ÂZ
A.niger
Ô12`
1.3
†

%$
H29、M1
Ç
RS88
W1ë-0§:`¯
_dÔ12

—˜:12`%$]w&þë`
%ÊßoÜÄ

2.1.3 
%$²Ùáâ
ïàÌ`
ECU2023
%$­s²Ù

ùiʅ
V%

qhi
)`
£dß
rDNA
0àVßÕÖ:
(internaltranscribed spacer,ITS)
` O „


ECU2023
½Œ­s
BLAST
¾

²ÙÖà[½ƒ
(Aspergilusawamori)。
2.2 ECU2023
c(EF©ª5«p
2.2.1 
`a¿Õ`€
ð

Ö4h`a¿Õ¯_dÔ12`€

æð

òó
:`
al!

v¿¯_dÔ12ìÌ_
73 
!

# ¨
 
©â

d$:w¯_dÔV%`ïàqÔ@Rx¯¨
G2 
e6c(5<=í4uv—O
Fig.2 Xylanaseactivityassayinsecondround
screeningofstrains
:

Ô1!‚Ì‘ÒÉÖ
A.awamoriECU2023、
RS88、A.niger、M1、H29,
~U
96
ºüïà`Ô1á
âZdô`

ò{
24h
n

Ì˜B^%ÊoÜ
đŒ

µZ¯_dÔ12Á

`a¿Õ!*
48h
n

˜‚^%ÊoÜÄw&

u)¯_dÔ12
øƒe(

”!

v¿ìÌ_:ª

%ß`a¿Õ
!*
4d
n

Ô`121˜*@Á

G
3 
“”¸´t<=í4uv5wx
Fig.3 Efectsofculturetimeonxylanaseactivity
2.2.2 pH
`€
µ*yï

•ïp_n×ÙÔ1_:`
ECU2023
ÖàÙ%$

•`a0
pH
­s

4
/
ECU2023` pH
4h./f

”
pH40~90
./0Ô1¥Ú!*
7%,
”
pH60
¿Ô1ìÌ
_:

~U3­
[11]
èé`¯_dÔ12_*
pH
¦á‹

2.2.3 C
ï`€
¥§
13
{

ï
(20g/L),
TŸdç(d

bcd
、¯

)、
D’Ü

ób¬+ñ
(α
celulose)、
âZ0¬+ñÜ
(CMC)
â

Çv>Ü

ãä¼

½

‘Þ¾šâ
),
wÔáâ¡ð

*
-



òpî0

ípv>ÜÖ

ï¿
,¯
_dÔ12«þë:j•ŸÜ

•–pã会
Ǒ޾š`žâ_ß

Ô1¯qòìÌ
367
Ç
289U/mL。
´«Ã¨òºZv>Ü8>f˜Å
*ϛœ

µZ¢}¯_d

òpFCw&¯_d
Ô

Ucåèé`ãä

½ÇSÜâ`FCžâ
¦h
[12-15]。
­dõ:iã会Ǒ޾šÕ
ÖÜ



w¯_dÔ`€

G
4 
“”%

¬«pXY
Fig.4 Optimizationofcarbonsourcesin
fermentationculture
2.2.4 C
ïx^œ³`€

`áâ(ð
5。
æð

òó

ECU2023
ùiÚ¾0x¿

•Üdy³W˜¥
q

µ1˜d"_4œ³

ã会Ö
40g/L,
‘Þ
¾šÖ
50g/L。
v>ܜ³*Áo*:1€
¯Ù¯_dÔ

p‘Þ¾šÕÖÜ¿

Áj
40g/L
oø:j
50g/L
1‚‚@Á¯_dÔ12

u
pã会ÕÖÜ¿

”
10~70g/L
1ºØÙ}
¯Ù0¯_dÔ

ܜ³*Á¿ÚùjFC%
ßwÔ

œ³*:¿‘ŒÇ`Ë}…

uÚº
Ù¯‡!u€^x

4h*+–

4/”`a
y#Ü`Áß¿S3”€Üà

í`ad¼¿
Õn

%ßæÅzÁß¿STåoˆT

4h~Ü
83
&
 

 
(
 
)
 
*
 
+
  
!
11
"
 
àƒ¿S3Ý

èˆ#

%ßï/\…

~òºm
ƒ%ùik¬+ñ`O’˜Ð

!Åƞæ;

pv>xÖÜ

Wò‚‚@ÁŒÂ

:i
30~
40g/L`
ã会_֋4

G
5 
“”%

¬5陿s«p
Fig.5 Optimizationofcarbonsourceconcentration
infermentationofxylanase
2.3 
<=í45Gp74Ó1é5bH§I
Å
40% ~60%(NH4)2SO4¯^3Ýn,t
9l
SuperdexG 75
­s]š*o

ð
6(a)
օ
¨Å¥½Ë

Ô1Î0/”
20
’õö

¾Ô1ì
Ì
36.6U/mg,
²T†rÖ
27.7
†

³°Ö
093%,
~Õ*+–K#Ï
。SDS PAGE
á
â(ð
6(b),
4/þëef5q`¦¯Ä
x^Ö
2.2×104。
G
6 
<=í4
SuperdexG 75
PQ¶R
ST¹º’
SDS PAGE
¥¦G
Fig.6 GelfiltrationonSuperdexG75columnof
xylanasexylanaseactivityand
SDSPAGEofpurifiedxylanase
  
š²Tn`¯_dÔ_4
pH
Ç_4³¯q
Ö
60
Ç
50℃(
ð
7),
¾¿
,pH
Ö
50~80,
³
Ö
30~50℃,¯
_dÔ121¦ØÙ

G
7 pH
’rst<=í4uv5wx
Fig.7 EfectsofpHandtemperatureonxylanaseactivity
3 
X
 
[
yõïà̺wýþÿ`
564
$ó&%$

ùi0j
96
ºEºü`ïàO„•–
202
$%
$­s§"ƒ•ï

¥#Ì
13
$:w¯_dÔ`%
$

•–pa
ECU2023
`Ô1_:

²ÙÖà[
½ƒ

•`a56­s§}T

×Ù`a56

pH
Ö
60,`
a¿ÕÖ
4d,
_*

ïÖã会
,C
Ö
30~40g/L。
yï%$ýþÿ1RU
96
ºEºü™#`
¯_dÔ12­s¾

4/Ô12`+TðñZ
0Âdô`

:Ô1`%$i”{O„™Ù`1
93 
!

# ¨
 
©â

d$:w¯_dÔV%`ïàqÔ@Rx¯¨
21Z¦:`

òpæÖ~""ƒïàû]Z
ò``

¾¿
,DNS
„™5Ãd±±‰«Ï"õ
ö

j‚^J9`›œ»·÷Ï¿Õ

ùi
96
ºEºüò‚‚á:¹O„`ž°

zÎWòz
©û]hijïà•ŸÔ×

”

ï¿

4/

ï`àápq

ï`œ³1€¯_dÔ`¯
Ù

u‚Ïr¯_dÔ1e¯ÙÌ)î

¨©

:
i§v>_ÕÒ¾ÕÖÜ

•–ã会¢}
k¬+ñ

4/Õ

ï¿e(÷Ï

0xœ³ÍÁoÍ:“«˜
¦¾`žâ

ܜ³Í:òºw&‚^˜çR
`–Õw

–èÔ`‹Œ

>u

ܜ³*Á÷
چpFC¯_dÔ`w&

yõ²T#Ì`¯_dÔ12”
pH50~80
./01¦ØÙ

—˜d”`2thiÓª

¦
`µ*­dõ`¯Ä*÷

òij÷é

kl

ºï
â)t

ùiÂ3èé`ïàû]4PÈώ`
¯_dÔ

˜ùj­¯_dÔ`wtThi

w
•Zòpµ*á:U¬+ñÔ`²¾ÕiÎ|
«zêx`ºïU¶·âò,Œ45¼½

ƒ„JK

[1] SahaBC.Hemicelulosebioconversion[J].JIndMicrobiolBiot,
2003,30:279291.
[2] 
â´M



KŽÃ

â
.¯
_dÔ0¨23­5
[J].
ó&
@ÖC
,2006,26:6167.
[3] ChandrasekaranA,BharadwajR,ParkJI,etal.Amicroscale
platformforintegratedcelfreeexpressionandactivityscreening
ofcelulases[J].JProteomeRes,2010,9(11):56775683.
[4] ChundawatSP,BalanV,DaleBE.Highthroughputmicroplate
techniqueforenzymatichydrolysisoflignocelulosicbiomass[J].
BiotechnolBioeng,2008,99:12811294.
[5] KingBC,DonnelyMK,BergstromGC,etal.Anoptimized
microplateassaysystemforquantitativeevaluationofplantcel
waldegradingenzymeactivityoffungalcultureextracts[J].
BiotechnolBioeng,2009,102:10331044.
[6] ChenW P,AndersonAW,HanYW.Productionofglucose
isomeraseby Streptomycesflavogriseus[J].ApplEnviron
Microbiol,1979,37:324331.
[7] MandelsM,WeberJ.Theproductionofcelulases[J].JAm
ChemSoc,1969,95:391414.
[8] 
Tì¦

K†¡

èíW

â

d&R¯_dÔ:w%$`ï
à

wÔ56}Tq•”ޏå&îf–`hi
[J].
_t
&;<@è
,2005,13:235240.
[9] 
ïÇð

ñM¹

DOŒ

â
.¯
_dÔ&w%$`ïàqw
Ô56`}T
[J].
–,&)+ÖC
,2007,27:4144.
[10] 
ùŸO

ò3ó
.BaciluspumilusWL 11¯
_dÔ

`²T

²Ùq•Ü@|O’
[J].
&)+@è
,2005,21:
407413.
[11] BegQK,KapoorM,MahajanL,etal.Microbialxylanasesand
theirindustrialapplications:areview[J].ApplMicrobial
Biotechnol,2001,56:326338.
[12] BegQ K,Bhushan B,KapoorM,etal.Production and
characterizationofthermostablexylanaseandpectinasefrom
Streptomycessp.QG113[J].JIndMicrobiolBiotech,2000,
24:396402.
[13] GuptaS,KuhadR C,BhushanB,etal.Improvedxylanase
productionfrom ahaloalkalophilicStraphylococcussp.SG13
usinginexpensiveagriculturalresidues[J].WorldJMicrobBio
tech,2001,17:58.
[14] KuhadRC,ManchandaM,SinghA.Optimizationofxylanase
productionbyahyperxylanolyticmutantstrainofFusarium
oxysporum[J].ProcessBiochem,1998,33:641647.
[15] PuchartV,KatapodisP,BielyP,etal.Productionofxylanases,
mannanases,and pectinases by the thermophilic fungus
Thermomyceslanuginosus[J].EnzymeMicrobTechnol,1999,24:
355361.
04
&
 

 
(
 
)
 
*
 
+
  
!
11
"