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 Purification and properties of laccase from Ganoderma EIM-40

灵芝EIM-40漆酶的分离纯化及酶学性质



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ChineseJournalofBioprocessEngineering
Vol.10No.2
Mar.2012
doi:10.3969/j.issn.1672-3678.2012.02.008
NOPH
:2011-10-09
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:1672-3678(2012)02-0034-06
PurificationandpropertiesoflaccasefromGanodermaEIM40
HEGuobin,CAIShaoli,KEChongrong,HUANGPing,ZHANGMingliang,HUANGJianzhong
(KeylabofIndustrialMicrobiologyoftheMinistryofEducation,EngineeringResearchCenterofFujian
ModernFermentationTechnology,ColegeofLifeScience,FujianNormalUniversity,Fuzhou350108,China)
Abstract:AfterthecrudelaccasefromGanodermasp.EIM40waspurifiedusinglyophilization,fraction
alammoniumsulfateprecipitation,HiTrapphenyl(FF)hydrophobicchromatographyandQSepharose
FastFlowIonexchangechromatography,afinalyieldof53% andaspecificactivityof146foldwere
achieved.SDSPAGEanalysisbysilverstainingrevealedasinglebandwithmolecularweightof
653×104.TheoptimalreactionconditionsofthelaccasetowardGuaiacolandABTSassubstratein
clude55℃ and50℃ ofreactiontemperatureandpH48andpH45.Whenthetemperaturewasbelow
50℃ andthepHwasintherangeof40to50,thelaccaseexhibitedexcelentstabilityinthetwosub
strates.TheKmforoxidizingguaiacolandABTSwere6450and222μmol/L,respectively.Laccase
couldbeenhancedbyCu2+andinhibitedbyFe2+,Ca2+,andBa2+.
Keywords:laccase;Ganoderma;purification;enzymologicalproperties
  
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1min
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3
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30min,
-80℃
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6h

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20h


6
30%
(NH4)2SO4ŸE。
172 (NH4)2SO47~ü–
  
S
171
=>*7—­J
4℃
ª
4h


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08
mol/L(NH4)2SO47×@Ÿ/(20mmol/L,pH
56)
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173 HiTrapphenyl(FF)
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08mol/L(NH4)2SO47×@Ÿ/(20
mmol/L,pH56)
B$
1mL
7
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± ­ u &
1mL。
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(20mmol/L,pH 56)
Î G ¡ ª

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08mL/min,
Ö`
048~08mol/L
7
(NH4)2SO4Ÿ
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10min,
*`
0~048mol/L
7
(NH4)2SO4Ÿ/ÎGU]¡ª。`¸(5t‰X 
–}•v

g1>}•7¤œ

ÂD—­`
20mmol/L、pH56
7×@Ÿ/J
4℃

24h

K–E“–½A~‚u&
15×103),
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12h
#
öO:Ÿ/
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 ¡
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PEG6000
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h`

174 QSepharoseFastFlow
ªö‘–
  
u14mL
7
QSepharoseFastFlow
廚
”Z

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XK16/20
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7cm。
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Ÿ/
(20mmol/L、pH56)
B$
5~10`
˓@

±
­u&
20mL。

05mol/LNaCl
7×@Ÿ
/
(20mmol/L,pH56)
ÎGU]¡ª

¡ªÒ&
06mL/min。
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g1>
}•7¤œ

18 
j£‚£@`ß
181 
|}+À
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i
pH
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pH37~60
73+@
NaOH
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7×@
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(01mol/L)
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50
w
40℃
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6}•e+à…&
100%
Ö½A}•e

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pH37~60
73+@
NaOH
01/w
pH35~59
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30℃
=ª
2h,
~†k
50℃
=–”}•e

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&
100%
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35~70℃
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35~70℃
7D¦V
ª
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*

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pH
=
–”}•e

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4℃
}/}•e&
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Ö¯Ã
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183 
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pH
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(02~18mmol/L)
w
ABTS
‰X
(0005~10mmol/L)
–”}•e

»¼
Lin
eweaverBurk
%3Ë@}A‰X7
Kmª。
184 
͌Âiƒ„•v˜äÐ*}•7–”
  
S͌Â
(Zn2+、Mg2+、Fe2+、Cu2+、Ca2+、
Mn2+、Ba2+、K+)
iƒ„•v˜
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~†Ð³3|}

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30min,
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EDTA
Ñ\]¯3
1mmol/L)
7½q“úV

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Ñ\ ] &
5mmol/L,
ƒ „ • v ˜ Ñ \ ] &
1mmol/L,
k+˻]


pH
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äÐ͌Âiƒ„•v˜}/7}•e&
100%
Ö½A}•e

ª]

:\Bª

&[øZÄ
Â7GH

Z[
Ca2+、Ba2+、K+
P7ÄÂ&
Cl-,
jÃ&
SO2-4 。
2 
yz%{)
21 
j£‚gYì¨
211 
°}/7\}
  
oJKK=0/º,óX‚
[12],
qM°}/÷


óFÞ«Û

wx`
30% ~80%
7
(NH4)2SO4
Ÿ/¯W3\}7Û7

`°*
,` 30%
7
(NH4)2SO4ŸE—­,æ8~7º,óX‚÷È´
ŸE

ám>ÙJÉʂ7ŸE

I6¯3\}
7Û7

S
400mL
°}/ì`°\}
、75%
(NH4)2SO4–+Ñ\}¶20mL,\}[20­。
`°\}7ˆ|¶&
578%,(NH4)2SO4–7ˆ
|¶&
385%。
212 HiTrapphenyl(FF)
ÂD‘–
  
05mL
—­±­ÂD‘–Ë

!O?(`
í
048~08mol/L(NH4)2SO47×@Ÿ/
(20mmol/L,pH56)
U]¡ª@‘O7

_>}
•v 7 S

! + ? ( ` í
0~048mol/L
(NH4)2SO47×@Ÿ/(20mmol/L,pH56)¡
ª

M3O`>}•v7S

g1>}•¤œ
1À®\}

MÞú3

s8

o3

I6º@

S

>|}•v

S

E|}•v
。SDSPAGE
¡
eú3

s8

f
1 

HiTrapphenyl(FF)
o¾pxf:
Fig.1 HiTrapphenyl(FF)hydrophobic
chromatographyoflaccase
M—
Öä²ÉÊ
;1—
`°\}}

2、3—
€F}‡v;U¼
R250
‹^
f
2 
ì¨j£
SDSPAGE
nf:
Fig.2 SDSPAGEpaternofpurifiedlaccase
  
o3



S

—­7
SDSPAGE
‡v;U¼
R250
‹^MÞé8?‘O7x?

6f~µÞ?D

213 QSepharoseFastFlow
ªö‘–
  
ÂD‘–*7—­ìK–ª

20mL
—­
±­ªöË
,`
í
05mol/LNaCl
7×@Ÿ
/
(20mmol/L,pH56)
ÎGU]¡ª

ÅMO`>
}•v7S

g1>}•¤œ1À®\}

MÞú
3

s8

3

V?¦§S>}•

214 
€FMÞ
  
z{
EIM 40
|}~€FMÞúƒ

s8





|}ì—`°™
、(NH4)2SO4U]
–
、HiTrapphenyl(FF)
ÂD‘–w
QSepharose
FastFlow
ªö‘–~€F*

€F­œ&
146,
ˆ|¶&
53%。
63
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!
10
"
 
f3 

QSepharoseFastFlow
YZîqpxf:
Fig.3 QSepharoseFastFlowionexchange
chromatographyoflaccase
22 
j£‚£@`
221 
|}7
SDSPAGE
  
ìÂDwªö‘–*

A|}ÎG
SDS
PAGE
½

MÞú3

s8

  
o3


:SDSPAGE
ы7MÞ&‘Ox
?

6f~—­€]?à

¼¾¿ÉÊ7½A¾
÷¶A½A~‚u%3

M|}7½A~Â
‚u&
653×104。
222 
|}7+À
pH
i
pH
²”v
  
~†6¸(5tw
ABTS
&‰X

–”|}7

pH
i
pH
²”v

MÞú3

s8

…
1 
hi
EIM 40
j£gYì¨yz
Table1 PurificationresultsoflaccasefromGanodermasp.EIM40
€FRÆ
å“@

mL
å•e


m(
åÉÊ
)/
mg
ñ•e

(U·mg-1)
ˆ|¶


€F
­œ
°}/
4000 5664 54912 1031 1000 10
—`°™
1070 3274 17277 1895 578 18
75%(NH4)2SO4– 200 2181 13765 1584 385 15
HiTrapphenyl(FF) 280 677 00627 10793 120 105
QSepharoseFastFlow 378 299 00198 15115 53 146
M—
Öä²ÉÊ
;1—
`°\}}
;2、3—
€F}ы
f
4 
ì¨j£‚
SDSPAGE
nf:
Fig.4 SDSPAGEpaternofpurifiedlaccase
  
o3



6¸(5t&‰X

|}7+À
pH
&
48,
k
pH40~50
79:U}•e!†W
æ
;30℃
ª
2h
7œ¼é8

k
pH40~50
U

ÇÃ}•e¯3
90%

,pH37~53
79:U

ÇÃ}•e¯3
80%


ƒ]@÷D7²”v

6
ABTS
&‰X

|}7+À
pH
&
45;30℃
ª
2h
*7œ¼é8

k
pH40~50
U

ÇÃ}•e
¯3
80%


ƒ]@÷D7²”v
,pH
nJ
41
<æJ
51
ç

}•ÈŽ?µª

f
5 
j£3r
pH
‘
pH
sß`
Fig.5 OptimalpHandpHstabilityoflaccase
223 
|}7+Àª]iª]²”v
  
~†6¸(5tw
ABTS
&‰X

–”|}7
+Àª]iª]²”v

MÞú3

s8

  
o3



6¸(5t&‰X

+˻]&
55℃,
k
40~65℃
U½A}•eæJ
90%;
kWQ
ª]=ª
1h
7œ¼é8

ª]mJ
50℃,¯
Ã}
•¯
95%


ª]mJ
60℃
çüŽ
60%
6±}
•

 xÃ78~z{|}½ñ
[13-14],
ª]²”v?
D

àJ
55℃
ç}•eÉÊ=l
,70℃
ç‰.>


LM

kª]mJ
50℃
çIÐ?ಔv

6
ABTS
&‰X

+˻]&
50℃,
k
50~60℃

73 
!

# ÄÜfP

z{
EIM 40
|}7~€Fi}v‚
f6 
j£3rŽŒqŽŒsß`
Fig.6 Optimaltemperatureandthermal
stabilityoflaccase
A}•e.FWæ

kWQª]ª
1h
œ¼é8

ª]mJ
50℃
ç
,¯
Ã}•¯
80%


àJ
50℃
ç}•eÉÊ=l
,60℃
ç‰.>}•

LM

k
ª]mJ
50℃
çIÐ?ಔv

224 
’bӜ
(Km)ª
  
6¸(5tw
ABTS
&‰X

k
50℃、pH45
xy=–”WQ‰X\]=7½qÒ¶

»¼
Lin
eweaverBurk
Ë%3

MÞú3

w3

s8

f
7 

LineweaverBurk
°±

tuv

Fig.7 LineweaverBurkplotsoflaccase(guaiacol)
f
8 

LineweaverBurk
°±
(ABTS)
Fig.8 LineweaverBurkplotsoflaccase(ABTS)
  
o3
7、
3

IM|}ŒF¸(5t‰X7
Km=6450μmol/L,ŒF ABTS‰X7 Km =222
μmol/L。
225 
͌Âiƒ„•v˜A}•7GH
  
‰m͌Â
(5mmo/L)
wƒ„•v˜
(1mmo/L)
A|}•v7GHúƒ

s8




:Cu2+
A}>g•%`
,Zn2+、Mn2+、Mg2+
A
}•e7GHWæ
,K+、EDTA、SDS、Tris
A}_>
WQ³]7_g%`

jV
Fe2+、Ca2+、Ba2+
+û_
g[}7•v

…
2 
QdYZ‘…wk`›j£k`‚ƒ„
Table2 Efectsofmetalionsandsurfactants
onlaccaseactivity
͌Âi
ƒ„•v˜
½A}
•e
/%
͌Âi
ƒ„•v˜
½A}
•e
/%

100  Mn2+ 98±11
Zn2+ 98±10 Ba2+ 37±03
Mg2+ 99±15 K+ 63±07
Fe2+ 31±50 EDTA 74±16
Cu2+ 114±25 Tris 73±07
Ca2+ 36±10 SDS 63±02
3 
y
 
)
  
¬``°\}
、(NH4)2SO4–、HiTrapphenyl
(FF)
ÂD‘–w
QSepharoseFastFlow
ªö‘
–Az{
EIM 40
-./ÎG~€F

ÅM€F
|}

€F­œ&
146,
ˆ|¶&
53%。SDS
PAGE
ы7MÞ&‘Ox?

½A~‚u³&
653×104。
6¸(5tw
ABTS
&ŒF‰XÎG
}v‚89


pH
~†&
48
w
45,
+˻
]~†&
55
w
50℃,2
m‰Xk
pH40~50
9
:U

ª]mJ
50℃
ç

}7²”v1÷D


(5t&‰Xç
Km=6450μmol/L,6 ABTS&‰

Km=222μmol/L。Cu
2+
A}Æg•%`

Fe2+、Ca2+、Ba2+
I+û_g}7•v

L˜6j

[1] 
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