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Induction of β-glucuronidase in Penicillium purpurogenum Li-3

产紫青霉中β-葡萄糖醛酸苷酶的诱导表达



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ChineseJournalofBioprocessEngineering
Vol.10No.3
May2012
doi:10.3969/j.issn.1672-3678.2012.03.006
OPQI
:2011-10-31
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β¥¦Å§«¨Ç¬Ð°±²ÀÁ³j。ù¥¦KLb(¬N´_,DŒžµ¬¨Ç±²J:¥¦Å¬éRñ
¸òó
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(10g/LGL+12% Tween80)
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¹r

[±²!

±²
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SŒºšT
40℃
qr¥¦
48h。
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½¯

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ÏÐu
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:1672-3678(2012)03-0028-05
InductionofβglucuronidaseinPeniciliumpurpurogenumLi3
GUOXiaoxiao1,WANGXiaoyan2,FENGShijiang2,LIChun1,2
(1.SchoolofLifeScienceandTechnology,BeijingInstituteofTechnology,Beijing100081,China;
2.SchoolofChemistryandChemicalEngineering,ShiheziUniversity,Shihezi832003,China)
Abstract:CarboncataboliterepressionwasfoundwhenPeniciliumpurpurogenumLi3grewinmedium
containingothercarbonsourcesthanglycyrhizin.Theinductionstrategyofβglucuronidasewasstudied
andanewtechnologyfortheenzymeproducinginductionwasestablished,whichbasedontheoptimiza
tionoffermentationconditions.Theinducerwasaddedafterobtainingalargemyceliumbiomassinnutri
entmediumwithglucoseasthesolecarbonsource.Whenglucosewascompletelyconsumedinthenutri
entmediumcontaining5g/Lofglucose,20% inducer(10% glycyrhizin+12% Tween80)wasadded
every24h.Thentheculturetemperaturewasmovedto40℃ beforefermentationlastedfor48h.The
phenomenonof"secondproduction"ofP.purpurogenumLi3wasfoundaftertheprocedure.Theenzyme
activitywasenhancedfrom64799U/mLto2356U/mL,thusitraisedaboutthreefold.
Keywords:carboncatabolite;βglucuronidase;induction;Peniciliumpururogenum
  β tuKv=wV(βglucuronidase,EC
32131)
íH!=
(glycyrhizin,GL)
M¶å¯Ë
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9;M¶)u>βtuKv=wV
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GL
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:①GL→
GA(
H!b=
);②GL→GAMG(tuKv=)H
!b=
)→GA;③GL→GAMG;④GAMG→GA[1-5]。
Akao
ø
[2]
8ù਩Ÿ s>β tuKv=V;
<‰~‹á①、②、④¤WXЇ,OV>°¶‡A
¯9þ
。Lu
ø
[6]
Sª©s5Ø>β tuKv=w
V£çYZ
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alZeÍû

9È*“pXM
:
。Kuramoto
ø
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i†G9y4
(Cryptococus
magnusMG 27)
~2ˆ4
(StreptococcusLJ 22)
Â
Ã
GL
؏1A>
GAMG,
«¬­ø
[9]
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UV 48
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GL
؏
GAMG,
.V®Í‹û‰~ï
Ї
。Feng
ø
[11]
Ïæi>}>4B
Penicilium
purpurogenumLi3
µz{> β tuKv=wV,
àQÍû>Ї°¶}~¯~WX®¯

E£ç³
X
GL
MZ
GAMG
>íšòû{
8845%。
  
Ö

Ÿ4µz{>βtuKv=wVó ²
V

ÍG*WX
GL
MZ
GAMG
>d4µ:β tu
Kv=wV1©æ* ‰V

Í+§àk¯DE
ˆ•>:pqr
P.purpurogenumLi 3
µ:OVó
 ²xyV

û~89:VËÌs‰~ Z[T°
ù±

¿FÿH!=†ó²A

uÎxy}—4B
M—–:V

Ö¯V>YZíšÍ‹

«V®ˆ‹

  
QRÿβtuKv=wVûíz{óÖ¯,0
P.purpurogenumLi3
>89:VËÌÈæÆÇ

~
89‘’ŠX>)ً

v/À>:Vxy“”

ËÆÇ4–M—~ β tuKv=wVYZ>m
5ËÌ

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î

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@

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ÿ
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ó β tuKv=wV>“pXM:X£


1 
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QRµ~rstÏæ>}؏!ˆ•

  PDA
wx)

G*
P.purpurogenumLi 3

®X

wx~ˆ•

$µ¶


)300g/L,
tuK
20g/L、
AT
17g/L(
ób¢–wx)
)。
*
121℃
²4
20min。
  
:Vwx)

å#Þ>¸Dwx)
[15],
ì@‡
ˆÚ‰ŠX
[16],
ÿ
28g/L
>H!="ƒZ¹
IK
,NaNO33g/L,K2HPO408g/L,!ÆÊ012%
>
Tween80,
ÏÐ
pH;250mL
KT®i“
80mL,
121℃
²4
20min。
  
³xwx)

ÿ9;…U>IK

ò>ˆz

>
?@

ò¡¯¢z~tuKø

îYG

uZ¹:
Vwx)s>H!="ƒ

  
xy}
:10g/L
H!="ƒ¡

Es¼Q
—˜iì
12% Tween80。
  
wx‘’
[17]:
‡
4℃
ˆ‰>4•
PDA
=ˆwx)‹
,30℃
wx
3d
Èæ4B®X


î$’A$®X>4î

‘’
PDA
–wx)

30℃、180r/min
º»wx
3d
5Øî-

‡î-
ÿ
5%
>‘’:Vwx)s
,30℃、
180r/min,
89
120h。
12 GL
Æ
GAMG
r…€
[11,17]
  
89„Ø>89ì
8000r/min
°
10min,
’
05mL
‹®G4P£ •
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ìŸ]ó
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20μL,™1mL/min,¼ŒÚ25℃。
13 
r˜Di9:
[17]
  
’

®
(80mL)
89„Ø>
Ppurpurogenum
Li3,
°ýÁ4–

G
pH45
>
NaAcHAc
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G
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‹áÓ+×Ù4–


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ŸåRš
400W),
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ދ®¸ó¨V

14 β deÁphq˜˜™¨’[11,17]
  
°üY’
200μL¨VÊ’800μL¼Q
2g/LGL、pH45
>
NaAcHAc
Ó+¡s
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Ї
1h
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’
05mL
Ї
G4P£ •
10mL,¨
GûíS¹º»¢£
GAMG
>¼˜

  βtuKv=wV®£±ó~‹á‘’",¹
1min
WXMZ
1μmol>GAMGµ¶>V˜óA]
V®g
(U)。
15 
`•y¨’
  
4–M¶˜££·G¡—˜‰

’A®
(80mL)
89ÈæÂë

GžŸÂHI4–

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ëÃaÄ;
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105℃
sv¡•¨—˜

*—iÉAœðžü+
˜

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(DCW)。
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†‡%ˆ)
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sz{tuvwi v
  
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>ÜîYÄ

92 
!

# £¤¤ø

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),
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Ϊ‘ÿH!="ƒ
ó²A

uÈæ,¿§s

‘îÞ­®wx

~
24h
~
120h
i†’¨££ β tuKv=wV®
n–89s
GAMG
>¼˜

ÂÃÄz
1。
 
1 C
Ó\e˜ijk
Table1 Efectsofcarbonsourcesonproducingenzyme

u
ρ(Cu)/
(g·L-1)
89þV®

(U·mL-1)
ρ(GAMG)/
(g·L-1)
24h 120h 24h 120h
H!=
3 1557 8713 029 213
IK
5 0 6376 0 152
tuK
5 0 8407 0 205
>?@
5 0 7418 0 189
򡯢z
5 0 5011 0 126
ò>ˆz
5 0 3528 0 087
 
  
/z

òÅ

89
24h
<

¿QÿH!="
ƒó²A


P.purpurogenumLi 3
M—<

î
Fxy:M β tuKv=wV!WX GLMZ
GAMG。
Ë89<Ê՗

³xwx)s>IK

t
uKø™í

uíÆ

†óxy}>H!="ƒ
îJ¾8×Rí

xy
P.purpurogenumLi 3

:V!³X
GL
:M
GAMG。
øÞ
P.purpurogenum
Li3
4Bµ:> β tuKv=wVíxyV,~
V>YZËÌs‰~Ì Z[T°ù±

QE7™
í

u‰~<
,¸
HÆÊH!="ƒ†óxy
}

O4Bî9Fz{Ö>V

¿QÈwx)s>
™í

uíÆÞ

4–îJ¾FG
GL
!:Mβ t
uKv=wV

  
/*üM¶~FGIK

>?@

򡯢z

ò>ˆz<

 ‡EÂÃótuKÎÃKøK
Þ«†ó³x¶—ÊÿFG

,^ÿtuKó

u
ÆÇ
PpurpurogenumLi 3
4z{ β tuKv=
wV> Z[T°ù±

22 
deÁ\`•y`Æe˜ijk
  
ÿ¼
25g/L
tuK>³xwx)Èæ
PpurpurogenumLi3
4Bwx

¹
12h
’¨££
89stuK>¼˜~4–M¶˜

ÂÃÄY
1。
  
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Å

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u<

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ìÇA]™ç>Õy#
,12h
<4–¸J¾M
—
;20~50h
ó.ìM—#
;50~110h
4–M—©
q
1 
deÁ\`•y`ijk
Fig.1 Efectsofglucoseoncelgrowth
*˜£67

4–M¶˜Î1ðó
499g/L。
/*
‰~ Z[T°

tuK‰~‘’"4B9Fz{
Ö¯V

89‹®–4–7ó°Þ>‹®s
Ï¢£9β tuKv=wV®¯。4–È’˜
£#<89stuK>—˜…Uó
85g/L,
óí
tuKó
165g/L。
23 
¾xh\`•y`Æe˜ijk
  
~ÿH!="ƒó²A

u>:Vwx)s
Èæ
P.purpurogenumLi3
89wx

¹
12h
’¨£
£4–M¶˜~89s βtuKv=wV>V®
n

!ižO4BZ[
GL
>ïð

ÂÃÄY
2。
q
2 
¾xh\`•y`%e˜ijk
Fig.2 EfectsofGLoncelgrowthand
enzymeproduction
  
/Y

òÅ
:P.purpurogenumLi3
4–:Vó
M—‡‚Á

M¶˜>PÊý

u>óíôeS
N

4B>M—ìËA£<Ê>ȇ#

36~60h
<Ȓ.ìM—#
;60~140h
4–M—
©*˜£67

M¶˜Î1ðó
051g/L;140h
Þ
ó2}#

4–J¾Ï¡

Ȓ˜£#Þ>
60~
130h
1˜YZ βtuKv=wV,84h<þV®
{Î1ð
534U/mL。
  
ڋµá
,P.purpurogenumLi3
4BÿtuK
ó²A

u<

4–M¶˜-™PÊ

ûM¶˜Î
03
M
 

 
Ê
 
“
 
Ë
 
Ì
  
!
10
"
 

(499g/L)
ÍÿH!=ó²A

u<
(051g/L)
‘û1(
10
é

0* ²VÍÈ


òÉ>M¶˜îí‘ûV®>Aîæ‰

Í:V
ËÌs‰~Ì/tuKÎE7ñZ[

u>Z>
iÃZ[¶T°í‡

,^çý~5ØÍ1M¶˜
>;<êÑîT°

Zóûí:V>NO

  P.purpurogenumLi3
µ: β tuKv=wV
æ*xyV
,GL
uí4–M—>

u°¶Êí:V
xy}

Aæˆ4–¶·A£>
GL
†ó

uÿË
ÌÏßM—

€Aæˆ
GL
†óxy}

E…UËû
òF0:V9F

íÚ~‹á­®89)ًk
Ê
GL,
ÆÇxy¶…U0:V>?@

ÿkl˜

k
l<Ê~89<Êó,¿Èæ
L9(34)
eÙ§s

Â
Öæ‰ižÄz

~z
3。
/z

~z

òÅ

H
!=>kʘ0xy:V>?@Î1

Ebí89<
Ê

æÍ

‘’"V®nðÎû

ýæÍ

þÍ

‹
V®nfQ‘û

.89<ÊPÊ1
12h。
Vÿ8
9<Ê0xy:V?@Íòÿ–al~c>FG
š

qóæÍ

óÎّ’
,¸
89
36h
ÞkÊ
004%
>
GL,
«89<Êó
72h,^
<ÎûþV®{
64799U/mL,
V>M:þUó
897U/(mL·h)。
 
2 
X›y\e˜ijk
Table2 Efectsofaddingsubstrateonenzyme
productionbyorthogonaldesign

w(GL)/

kl<Ê


89<Ê


þV®

(U·mL-1)
1 002 36 60 54466
2 002 48 72 56390
3 002 60 84 63251
4 004 36 72 64799
5 004 48 84 66273
6 004 60 60 58364
7 006 36 84 58411
8 006 48 60 62205
9 006 60 72 56128
 
 
3 
X›y\e˜jki€zr…
Table3 Rangeanalysisonenzymeproductionfrom
orthogonalexperimentresults
Ήu
w(GL)/

kl<Ê


89<Ê


k1 57250 58619 58123
k2 63594 62162 58688
k3 58125 58650 61875
R 5844 2700 3854
24 
RSe˜{|i}~
  
Ï0d4Ÿ {„…z{˜
=P1>ù±
[19],
Ú1FGtuKŒÈ4B#
M—ÿ5ØÍ1M¶˜

ÈtuKíÆ
,¸
4–©
*ÐÑà5

M—>„…à5

<ʒxy}

û·
’~ûŒ"

PÊxy¶…U~zˆ®¯}…UÈ
æ©bxy:V>ef

~wx)sÿ
30℃、
180r/min
wx4–

~tuKíÆ<

ÿ
20%

ʘʒxy}

û¹
24h
ÆÊ

b

xy


³’
40℃、180r/min
Èæ:Vwx
48h。
  
ÂY
22
>ÂÃižòÅ

4B~³xwx)
sȒ˜£#<óítuKúó
165g/L,
óH4
–È’˜£#Þ©*

uÐÑà5

ÚtuK—
˜…Ui†ó
3、5、7、9、11
~
13g/L,
ÆÇtuK½
¾…U0xyÞ4–M¶˜~xyÞV®¯>
?@

ÿä¼Q
3g/LGL
>:V)Ùwx)ó0
`

ÂÃÄY
3。
  
/Y

òÅ

xy
PpurpurogenumLi 3
4
–M¶˜ýtuK½¾…UôeSN

xyÞ4–
åù1


ù±

ûÿ½¾—˜…Uó
5g/L
tuK<4–M¶˜Î1


39g/L。
Í
~^tuK…U"

‡5Ø>
PpurpurogenumLi3
4–Èæ:VxyÞ

Eµ:M>βtuKv=w
þV®ò{
2356U/mL,
þ:Vwx)sz{OV
®¯‘û1(

é

ÚYVÿü£³xwx)st
uKÎٗ˜…Uó
5g/L。
q
3 
“”ŽdeÁœ\`•y–e˜ijk
Fig.3 Efectsofinitialglucoseconcentrationonbiomass
ofP.purpurogenumLi3andenzymeproduction
3 
†
 
)
  
_·ÆÇ1
PeniciliumpurpurogenumLi 3
4
Bz{βtuKv=wVËÌs CZ[T°ù±
>êef

·G5–‰Èæ:Vxy

~³x
13 
!

# £¤¤ø

:pqrsβtuKv=wV>xyz{
wx)sÈæ4–Á

«kÊxy}

v/1
PpurpurogenumLi3
4Bûíz{ βtuKv=
wV>xyef

4B ‘î*¼
5g/L
tuK
>³xwx)s

ÿ
30℃、180r/min
­Swx

~
tuKíÆ<ʒ
20%
xy}
(10g/LGL+12%
Tween80)
Èæxy

¹
24h
ÆÊ

b

xy
72h
Þ
/¸³
40℃
Èæ­Swx
48h。
xyÞ8ùO
4Båù1


ù±

!1˜z{ β tu
Kv=wV

þV®S)Ùwx)s>
64799
U/mL
‘û•
2356U/mL,
‘û1‡(

é

ÑA
À>:V“”

u‘û14–M¶˜

ÊH4Bû
í:V

‘û1g89–?~g<ʲV>Y


Qíê1 Z[T°

ú‹1Ze

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XM:àQ.yo±

M©6u

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