免费文献传递   相关文献

Optimization of tannase production by solid state fermentation from Aspergillus niger

黑曲霉固态发酵生产单宁酶的条件优化



全 文 :!11
"!

#
2013
$

%
&
 

 
(
 
)
 
*
 
+
ChineseJournalofBioprocessEngineering
Vol.11No.3
May2013
doi:10.3969/j.issn.1672-3678.2013.03.002
!"#$
:2012-03-22
%&(

¢–ÃŽÄ7Å9:)+56
(KSCX2 YW G 050)
)*+,

V
 
Æ
(1988—),
Ç

ÈɕÊ1

ËE78&

78FG

)&H&

°";

PQ1
),
78Ì
,Email:zmzheng@ipp.ac.cn
„€…†‡3456ˆ‰Š;[\‹Œ
G
 
H

*IJ

K
 
L

K
 
M

N@O

PQR

.@S5< TUV672;BCDEF

WX
230031)
>
 
?

78t+FuvBUwxy)z{/012L|}D#$VW

u+
PlacketBurman
~P€‚ƒE
FM„

…†„‡na
、(NH4)2SO4ˆd‰Š_`(„a,J‹Œ~PŽJFu‘’。u+ BoxBehnkenF
uv~PC‚ƒEFM„“”•Uw

–sJ—#$VW

˜
250mL
™š›œž
10g
…†„‡
、44g
Ÿ b
05g
¡¢

£z¤
(mL/g)2∶1
¥¦$§¨£©ª«
(NH4)2SO421g/L、MgSO4·7H2O1g/L、NaCl1g/L,#$¬pH
­®

_`
57×107
¯(„°e
30℃
cdg#$
4d。
efVWg

L|}2a±
40U/g
²%s
114U/g,3
³‚
´Pµ¶~P·¸¹«
115U/g,
Pµ9º»D¼½¶

@AB

L|}

z{/0

xy)

FuvB
CDEFG
:TQ925    
HIJKL
:A    
HMNG
:1672-3678(2013)03-0007-06
Optimizationoftannaseproductionbysolidstate
fermentationfromAspergilusniger
JINWei,NIEGuangjun,WANGLi,WANGPeng,GONGGuohong,ZHENGZhiming
(KeyLaboratoryofIonBeamBioengineering,ChineseAcademyofSciences,Hefei230031,China)
Abstract:Responsesurfacemethodology(RSM)wasusedtooptimizecultureconditionsfortannase
productionbysolidstatefermentationwithAspergilusniger.PlacketBurmandesign(PBD)experiment
screenedoutthreesignificantfactors:galnutpowdercontent,(NH4)2SO4 concentrationandspores
numberofinoculum.Thesteepestascentexperimentapproachedthegreatestresponsespace.Box
Behnkendesign(BBD)experimentwasusedtofurtheroptimizethesignificantfactors.Theoptimum
cultureconditionsfortheproductionoftannaseareasfolows:each250mLflaskloaded10ggalnut
powder,44gricehuskand05gwheatbran,liquidsolidratioofthemediumwas2∶1andnutrient
solutionwasconstitutedby(NH4)2SO421g/L,MgSO4·7H2O1g/LandNaCl1g/L,withanaturalpH
value,themediumwasinoculatedwith57×107sporesandculturedunder30℃ for4days.Underthose
optimizedcultureconditions,theproductionoftannaseincreasedfrom40U/gto114U/g.Thereliability
ofthemodelwasverifiedthroughtriplicateexperimentswithanaverageyieldof115U/g.
Keywords:tannase;solidstatefermentation;Aspergilusniger;responsesurfacemethodology
  
CÍÎ
(tannase,EC31120)
bcdtÏÎ

?²‘H&×&

átÏÐa&LM×CÍ
W

ÑÏd¢háÿOÒÓWÿ

&“Ô¡XWOd
:ˆ•
[1]。
CÍΒՍ

ˆ‰

†‡

Œ{v&
Rij±²‚ÌO

‚ï¡€
[2-5]。
Á‘Y6
ïCÍÎ&×[Öά^3

&×tus

ñW׈
|}{àž

ÊËíCÍÎhŸ ˆ‚
[6]。
  
¹ØIJbn’ÔjÙÂj˜–’‘thô
õû

H&&[’¹eôæÚÛ½

×&ÎÙd
:LM×h&.*+
[7]。
K´µ&×CÍ
Îh¿ØIJF%÷g

¹ØIJF%&×CÍÎ
hèÜå53’

?²×©¦CÍÎ

%Y׈
),
Îh&×}*i3

Îijih
pH
݁åO¦
݁å{èé
[8-9]。
žŸ

SICÍιØIJ&
×F%bp3CÍÎ&×tu

rs&דøhj


  
yzQ+’‘#“ü”•h

Ö3×CÍ
ÎhlÞ1[Ö5Å&×[Ö

Qßà

á¼{â
&ãäŹØôæO

·

QÌÍsþØÂCÍ
WhåæXk5Åçp

‚TFèˆ¹
ØIJôõ

78)5j%YrsCÍÎh&×
“øOklCÍÎh&ׁ‚

1 
OPQRS
11 
Ty
  
CÍÎ3×hlÞ1[Ö

‘yz+’‘ñ
W“ü

12 
ŽQ
  
á¼Kßà ÔÕ½£è•ž

åæXkéÅ
åæXêë¡C
025mm
“X*§•ž

Ô¡XW
ìá

ñí×

O×CÍÎˏ

î–
Sigma
ïð
);
àä

ñòÍ
,(NH4)2SO4,MgSO4·7H2O,NaClJÅ
ñí×

–‡ÔˆŽ¤jÊïð
);TU 1901
óô—õ¦á4ñôô\;

ö÷í-‚ùøj
Êùúïð
)。
13 
]^RS
131 
òóô
  
ºòóô
:PDA
òóô

ûüÄ`òóô
),
‚Y[d„™

lÞ1¹ØIJ×CÍÎôLò
óô

á¼
44g、
CÍW
06g、V(
œ`Fr¿
)∶
m(
¹Ø
)=16∶1;
œ`Fr¿ª“
(g/L):
(NH4)2SO410、MgSO4·7H2O1、NaCl1。mª““
ñª
250mL
12â¢ýþJì

132 
òóF
  
òóôC3ˆÅ[¡

Md
1mL
lÞ1ŒXÿ
¿
(364×107
!ŒX
),
‚MdÛëñýþ>òó
ôKŒXߕJì

"ªY
30℃
òów¢òó
4d。
133 
Þοhp¤
  
òó#“¡

‰
10∶1(mL/g)
h¿¹g(ã
pH
50、005mol/L
hGHW GHW$%&r¿

ýþ
J졪´µ½
180r/min
p
15h,
‚¡‚‘Í
å§(*§Ç•Þο

134 
CÍÎάhρF
  
6ï–)½*?µcCÍάnhρF

mdCÍάnρF^2

}Q÷~gB

Æ
ùMQCÍWÅ+hρF,-B0
[8]。
’
Sharma
{
[10]
78hôL½§CÍάnhρF
.ílc–‚l

Qp3CÍάnhρEOwMQ•žQCÍWÅ+hCÍάn

  
‚
pH50、005mol/L
hGHW GHW$%
&r¿/ËÔ¡XWìár¿
(001mol/L),
Þ
οNO

æ

¤

0¤ðJ(ã
025mL
Ô¡X
Wìár¿

ñ­Ð1Å2fð

ϤðO§3ð

¤
025mL
½/GHW GHW$%&r¿(ã2
fð¢
,025mL
NOhÞο(ãϤ𢡝

4¤ðJ"ã
30℃
tå5¢„â
5min。
¢¡ñ’
+j¤ð¢(ã
03mL
ñòÍàä
(005mol/L)
r¿

’
30℃
tå5¢6ã„â
5min。
‚¡’

4¤ðñ­(ã
02mLKOH(05mol/L)
r¿

§
3ð¢C7(ã½/NOhÞο
025mL,
’
30℃
tå5¢„â
5min。
.#“¡¥4¤ðR
‚
4mL
×ØtNO
,15min
¡’8[
520nm

.ß•h±ô\O

‰%
(1)
0æ±ô\O
ƒˆ
(Δ520):
Δ520 =(A
Ϥ
-A
2f
)-(A
§3
-A
2f
) (1)
  
‚×CÍÎˏO
pH50、005mol/L
hGH
W GHW$%&r¿/Ëij^2άnC9h
CÍÎr¿
(U/mL),
‚:Îr¿‰3½/ρF
Ë•Îg¬nK±ô\OƒˆhÐÑÞ#
,Y
Å
±ô\Oƒˆ
(Δ520),XÅCÍÎg¬n(U/mL)。
C9CÍάnxÅ¥ñ;rÏ
1μmolCÍW
áÿ+ghΖ

 9•ž;0%
(2)。
Y=34309X+00679,R2 =09865 (2)
  
abÐÑÞ#fáQ;0æÞοhCÍÎ
¬n
(U/mL),
l9áQ;0æ¹ØIJ¥<Ò
æôæ+×CÍÎάn
(U/g)。
135 PlacketBurman
¤=;
  PlacketBurman
¤bôY‡#>u?@àL
hÁtu¤=;

ƒQB˜haŠ“üæ?
}žXh¤F
[11]。

&
 

 
(
 
)
 
*
 
+
  
!
11
"
 
136 
‚ABC¤
  
TD•F+0’|}hE@FGH2ƒ
ëñ.I…Ž

²œJ@‚0TFG2ƒ¼
Kj}hTD•F+

‚ABC¤Fƒ
‘Í

j}QJ@‚0TFG
[12]。
137 BoxBehnken
¤=;
  
ab‚ABC¤•žh¢šé

‚
Box
Behknen
¤=;

lc–§
PlacketBurman
¤
“üæh±²STžXlv1ž!1tuhT
èˆ=;
[13-14]。
2 
_`Qab
21 
deˆ‰Š56;‘?de’“”•
  
Cž!78h*+¢I3

åæXkìLC
ÍW5Åçp

Q‹’òóô¢Ž(†–ßà

JƒkllÞ1¹ØIJ&×CÍÎ

ÅíiE€
QèˆCÍÎ&×ôõ

 M]hSTžX¢Nü
ßà–
(X1),἖(X2),åæXk–(X3),
(NH4)2SO4‡\(X4),¿¹g(X5)Q‹MdŒX
–
(X6)5Å|}ž!,‚PlacketBurman¤=
;lc–“ü±²STžX

mž!¤3
(+1)、
s
(-1)2
!tu

TOÅ¥<Òæôæ+×CÍ
Îάn
(U/g),
¤=;‹3@45
1。
  
‚
Minitab
Oõ§ž!h±²åSTlvñ
í

3@45
2。
ž!§TOhSTPÿÅ
X3、
X4、X6、X5、X1、X2,ÖÆ X3、X4‹ X6{ž!háQ’
75%


žŸ~üÅ
Æe

!
ž!§CÍÎ&×Jj
f
1 PlacketBurman
^–—˜_`
Table1 PlacketBurmanexperimentaldesignandresultsoftannaseproduction
RS
X1
m(
ßà
)/

X2
m(
á¼
)/

X3
m(
çp
)/

X4
ρ((NH4)2SO4)/
(g·L-1)
X5
¿¹g

(mL·g-1)
X6
MdŒX–

(107
!

CÍÎgά

(U·g-1)
1 -1(33) -1(0) -1(0) -1(5) -1(16∶1) -1(182) 472
2  1(55) -1 -1 -1  1(25∶1)  1(730) 363
3  1 -1  1(108) -1 -1 -1 2403
4 -1  1(10) -1 -1 -1  1 526
5 -1 -1  1  1(20)  1 -1 6965
6 -1 -1 -1  1  1  1 748
7  1  1  1 -1  1  1 4047
8  1  1 -1  1  1 -1 1147
9 -1  1  1 -1  1 -1 4058
10 -1  1  1  1 -1  1 9209
11  1 -1  1  1 -1  1 9101
12  1  1 -1  1 -1 -1 924
f
2 PlacketBurman
^_`™šE›
Table2 Estimatedregressioncoeficientsof
PlacketBurmanexperiment
ƒ– QŠ QŠÐÑ,-
T P

33303 4248 784 0001
X1 -3328 4248 -078 0469
X2 -0118 4248 -003 0979
X3 26336 4248 620 0002
X4 13521 4248 318 0024
X5 -4423 4248 -104 0346
X6 6688 4248 157 0176
 
T
:S=147158,R-Sq=913%,R-Sq(Adj)=809%。
22 
œžŸ^
  
‘
PlacketBurman
¤3@ñíá7
,3
!
±²ž!åæXk–
、(NH4)2SO4‡\OMd
ŒX–J§×Îj
R¤u(F
G

Öab
PlacketBurman
¤=;üý•†h
tu5ÅBC¤„…é

5

¤=;‹3@

‘5

á7

PåæXk
163
g,(NH4)2SO440g/L,MdŒX–Å 617×10

!P

dšž!Å
PlacketBurman
¤=;h¢
šé t u P

l Þ 1 × C Í Î ¬ n ‚ 3
(1038U/g)。
9 
!

# V
 
Æ{

lÞ1¹ØIJ&×CÍÎhôõèˆ
f3 
œžŸ^–—˜_`
Table3 Experimentaldesignandresultsofpathof
steepestascent
R
S
m(
åæX
)/

ρ((NH4)2SO4)/
(g·L-1)
ŒX–

(107
!

CÍÎgÎ
¬
/(U·g-1)
1 054 10 182 543
2 086 20 327 778
3 123 30 472 926
4 163 40 617 1038
5 210 50 762 998
6 264 60 907 743
7 327 70 1050 409
23 
e ¡‹Œ–—˜_`E›
  
ab‚ABC


BoxBehknen
€<
oSTž!

d=;‹¤3@45
4。
f
4 BoxBehknen
^–—˜_`
Table4 BoxBehknenexperimentaldesignand
results
¤
RS

m(
åæX
)/


ρ((NH4)2SO4)/
(g·L-1)

ŒX–

(107
!

CÍÎ
gά

(U·g-1)
1 -1(086) -1(20)  0(617) 1064
2  1(264) -1  0 538
3 -1  1(60)  0 1024
4  1  1  0 865
5 -1  0(40) -1(327) 1008
6  1  0 -1 670
7 -1  0  1(917) 1060
8  1  0  1 576
9  0(163) -1 -1 1036
10  0  1 -1 982
11  0 -1  1 993
12  0  1  1 958
13  0  0  0 1049
14  0  0  0 1074
15  0  0  0 1124
  
UVF+áQBçQ/mž!KTO"
wh…°Q

%‚: ˜áQ€‚ƒIJ3@
‹IJôõ

mž!lvUVDO¡á•žUV
F+

£‚
Minitab
Oõ§5

TOKmž!l
vUVD•¡

•žCÍάn§åæXk–
(A),(NH4)2SO4文\(B),MdŒX–(C)R
WOhÁa]5UVF+

Y=10823-1851A+272B-136C+
868AB-365AC+047BC-1899A2-
262B2-639C2 (3)
  
‚
Minitab
Oõ§½/UVF+Öϕ

åæ
Xk
10g,(NH4)2SO421g/L,MdlÞ1ŒX–
57×107
!

CÍÎgάJÏO=
114U/g。
§
:èˆôõlv

a±jX夐

ϕuJO
Å
115U/g,
)3@KLYJÏOZñM@

X
í ˜h݁å

  
‚
Minitab
OõOõ§¤3@lvF-ñ
í

3@M5

+N

UVF+¢mƒ–TOST
h
‘

[¶\

]*

hOÜß


ƒ–h
 ˜
P<005,
UV ˜

á^å‡A3

’øhtu()”hž!Åca5

Q‹Áa5
A2,C2,
_~÷J^

‘6
1(a)
á4

méh™-uJñ’cô
ù#½

áQù`Qaæ: ˜D•+\bç

J
ÏOOÏO"wij3\h÷°å

á‚Y‚ƒ
CÍÎ&×ôõhLYJÏ

-*
DesignExpert
Oõ§ ˜F++5h1
‹T6§h{3#6

6
1(b)、
6
1(c)
O6
1(d))
ƒgBù`QÏOmƒ–Oƒ–"w§T
OhST

%‚:6

!ƒ–¢h

!ƒ–¹
’¢štuO

9§c¦

!ƒ–_~STCÍÎ
h&×lvñíKdÌ

{3#he„áQù`
Qñíæ_~5‚hvf

ghe5N

!ž!_
~5‚
9hedK"÷.

‘6

á7

åæ
Xk–O
(NH4)2SO4‡\h_~5‚§CÍÎ
h&×STBv

åæXk–OMdŒX–a
"

9
(NH4)2SO4‡\OMdŒX–"wd”•Ô
j_~5‚

01
&
 

 
(
 
)
 
*
 
+
  
!
11
"
 
f5 BoxBehknen
^_`R¢E›
Table5 VarianceanalysisforBoxBehknenexperimentalresults
¶· ’‘SeqSS AdjSS AdjMS F P
UV
9 468129 468129 52014 1139 0008

3 289025 289025 96342 2109 0003
A 1 282790 282790 282790 6191 0001
B 1 4753 4753 4753 104 0354
C 1 1482 1482 1482 032 0594
uF
3 139452 139452 46484 1018 0014
A2 1 121761 129157 129157 2827 0003
B2 1 1226 2005 2025 044 0537
C2 1 16466 16466 16466 360 0116
_~5‚
3 39652 39652 13217 289 0141
AB 1 34225 34225 34225 749 0041
AC 1 5336 5336 5336 117 0329
BC 1 090 090 090 002 0894
™-,-
5 22840 22840 4568
iD
3 19923 19923 6641 455 0185
×,-
2 2917 2917 1485
•;
14 490969
D
1 
e ¡£¤R¥;¦¢E§¨˜ˆ‰Š©Šªe ¡«¬DE›­‘?’®¯°;±²).
Fig.1 Interactionsbetweensignificantvariablesofresidualsformodelequationofresponsesurface
andresponsesurfacecontourlineplots
11 
!

# V
 
Æ{

lÞ1¹ØIJ&×CÍÎhôõèˆ
3 
_
 
b
  
‚líCÍάnhρF

•žíwMQ
QCÍWÅ+hCÍάnρF

£‚T
èˆílÞ1¹ØIJ&×CÍÎh)h

-*1ž!1tuhèˆ

•ž‚ƒ&×CÍÎh
¹ØIJòóôOòóôõ

¥
250mL
12âÂß
à
44g、
á¼
05g
OåæXk
10g,
¿¹g
(mL/g)2∶1,
MdlÞ1ŒX–
57×107
!


Fr¿ª“
(g/L)
Å
(NH4)2SO421,MgSO4·7H2O
1,NaCl1;
òóô
pH
’¢

òóˆ30℃,
Pw

d。
©¦CÍÎh&× 
40U/g
p3¯
114U/g,
b
?èˆïh
29
æ
。3
a±jX夐uJOÅ
115U/g,
Xí ˜há^å

B"„…òóô

舡hòóô?²¹e“ñÅßà

á¼Oçp
åæXk

¶·Yâ&ãäOÞ()hj&×
Š

“øsþ

9CÍÎhזè08\p3

  
78†g6㒕†h&.ølv"0
¤&×

klCÍιØIJ&׏3)&ˆ

=žrsCÍÎh&דø

×&bçCk}Óh
6h

‚ƒHI

[1] LekhaP,LonsaneB.Productionandapplicationoftanninacyl
hydrolase:stateoftheart[J].AdvApplMicrobiol,1997,44:
215260.
[2] AguilarCN,RodríguezR,GutiérezSnchezG,etal.Microbial
tannases:advancesand perspectives[J].ApplMicrobiol
Biotechnol,2007,76(1):4759.
[3] ChávezGonzálezM,RodríguezDuránLV,BalagurusamyN,et
al.Biotechnologicaladvancesandchalengesoftannase:an
overview[J].FoodBioprocTechnol,2012,5(2):445459.
[4] 
l³m

n³o

pqo

CÍW΋d‚78lä
[J].
¡
Š)&Ü
,2008,29(2):317319.
[5] 
Ùrs

lÞ1×CÍÎh78Oj`÷¢¹ˆlÞ1¨
©&•“Ô¡XWìáh„¢
[D].
tu

v,0Ž
,2002.
[6] 
-Áí

wx

yíz

CÍÎh78lä
[J].
{|-)

2004,26(1):1619.
[7] PandeyA.Solidstatefermentation[J].BiochemEngJ,2003,13
(2/3):8184.
[8] 
+}~

³€

„hš

{

CÍÎIJ&×h78lä
[J].
¢–Â{
,2008(6):16.
[9] RanaNK,BhatTK.Efectoffermentationsystem onthe
productionandpropertiesoftannaseofAspergilusnigervan
TieghemMTCC2425[J].JGenApplMicrobiol2005,51(4):
203212.
[10] SharmaS,BhatTK,DawraRK.Aspectrophotometricmethodfor
assayoftannaseusingrhodanine[J].AnalBiochem,2000,279
(1):8589.
[11] MilerA,SiterR.Usingthefoldedover12runplacketburman
designtoconsiderinteractions[J].Technometrics,2001,43(1):
4454.
[12] MontgomeryDC.Designandanalysisofexperiments[M].3rd
ed.NewYork:JohnWiley&Sons,1991:430.
[13] 
y,

w*

‚ƒL

µ;舜’H&IJ¢h‚
[J].
‹sâ&0ŽŽ)

’¢ÃŽ„
,2009,40(3):465468.
[14] FereiraSLC,BrunsRE,FereiraHS,etal.BoxBehnken
design:analternativefortheoptimizationofanalyticalmethods
[J].AnalChimActa,2007,597(2):179186.
21
&
 

 
(
 
)
 
*
 
+
  
!
11
"