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Preparation of cellooligosaccharide by enzymatic method

酶法制备功能性纤维低聚糖的研究



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ChineseJournalofBioprocessEngineering
Vol.8No.1
Jan.2010
doi:10.3969/j.issn.1672-3678.2010.01.005
OPQI
:2009-04-23
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EGI、EGI
­
CBHI
ØZ±²³§Õµ³±²
Ù¶¡B[Ng·¸

å5¹º—˜±²Ù¶¡à>»‰¼½ã§¾¿Oì

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:1672-3678(2010)01-0023-05
Preparationofcelooligosaccharidebyenzymaticmethod
ZHUNianqing,XIAWenjing,YONGQiang
(KeyLaboratoryofForestGenetics&Biotechnology,MinistryofEducation,NanjingForestyUniversity,Nanjing210037,China)
Abstract:TwoendoglucanasesEGI,EGIandexoglucanaseCBHIwerepurifiedfromTrichodermareeseiRut
C30andthedegrademechanismonceluloseofEGI、EGIandCBHIwereinvestigated.Then,theprolifera
tionofBifidobacteriumadolescentisusingcelooligosaccharideswasstudied.Theoptimalpreparationcata
lyzedbyEGwas1U/gcelulosefor90minhydrolysisandtheratiosofcelobiose,celotrioseandcelote
traosewere438%,348% and79%,respectively.Withcelobioseandcelooligosaccharideascarbon
source,theproliferationmultipleofB.odolescentiswas214and284,respectively.
Keywords:celooligosaccharide;homogeneouscelulase;Bifidobacteriumadolescentis
  
lmnop
(celooligosaccharides,COS)
ëÀX
Ì
2~10
0•–pëž β-1,4-pxVPÛS’
3͚knop”‘y

€|ijknop\ˆ

CDijknop3Ðk

jL)×&‰N3ˆ
‰
[1]。
lmnop3gh Dj45
[2],
+lm’
H~sÈÓ¿Hfghlmnop3K¨ÒÝþ

If45Š‹B/
(Trichodermareesei)RutC30
lm
’H½=~sÓ¿.Œ•opH
EGI、EGI
ª§Œ
•opH
CBHI
Qlm’3R2xY=Olmno
pQÜi&‰N3ˆ‰B£

=eÍElmn
op3]úgh³;Yn¤Ç

ÛElmnop>
yÞkû3×st45Olmnop3q£³;
¤Ç

1 
†‡Lf
1.1 
HIMÜNOPf_žF
=Š‹B/
(Trichodermareesei)RutC30
N]

}~[íÞ>yYÉ45jkl
),
8¿÷3{
|Á›\/-ghlm’H
[3]。
6Wõ¿‚¤
F

éß

ßÀ¬öX

ÞÀ¬öX

Z[ž©

q*
ÈY3.Œ•opHÓ¿
EGI、EGI
ª§Œ•op
HÓ¿
CBHI[4]。
1.2 
HIJK}_žF
8ö÷ø=ŒŸ¿?3noBp>0;Žª
¿?3lm’H~sÓ¿

~|
50℃、80r/min
Ê
—¦§<©=H2s]ÚÖ

H2œóœ

¥Õ
”•
5min
HÔI

|
3000r/min
ÁÀž
10min,
´o¨›Õqlmnopš›

1.3 
QRSq_st
Üi&‰N
(Bifidobacterium adolescentis)
ATCC15703,
›ç‹ÌNm*G
,^
‘bk€

Ì
=Ùbc/-É[45[¯>yN]kl=ž
³;

‡î…†‡
[5]:
ƒ„…
5g/L,
’…
5g/L,
‘’
†‡
5g/L,
-.G
10g/L;
¿?zxß

8‡î…†‡=ŒŸ
3g/LC
P
,1g/LL

‰W“12WåÒ«Ô

Üi&‰N~|
37℃
‹Ìʗ…†“=…†

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1.4 
fwop
1.4.1 
.Œ•opHI­3¡]
[3]
=
10g/L
l·lm’”•›EAy

¡]a
ÜE
550nm。
]žs0wHI­~˜Eû¿–>
’
1μmol•–pjô3H?,U/mL。
1.4.2 
§Œ•opHI­3¡]
[6]
=Qš‡—‡ β D lmÐpx (pNPC)E
Ay

¡]aÜE
410nm。
]žs0wHI­~˜
Eû¿–>’
1μmolQš‡—tjô3H?,
U/mL。
1.4.3 
lmnop3¿«
8
Agilent1100
E£›¤™©¬o

£
BioRad
AminexHPX-42A(300mm×78mm)
®×n¿«

•–¤E±È<

•™
06mL/min,
®—
85℃。
¿
ьg•–p

lmÐp3´Êš›

¢£§´f
¡]¥¦=3lmnopû? ˜

•–p

lm
Ðp3û? ˜œ´ÊùŸ¡]

lmöp

lm
ñpû? ˜¾¹lmÐpùŸ¡]

1.4.4 
&‰NNÄû? ˜3¡]
&‰NNÄû? ˜¢£wµf¡]

˜
gN›¼¨˜

ÒNÄû? ˜ ρ(g/L)3´Ê®
$

…†›6
3000r/min
Àž
30min,
NÄ6zN
<Ü=

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·¸ù¶œ=zN
8
620nm
¡¼¨˜
A620,ÒÇ´Ê®$ùŸæ½NÄû?
 ˜

2 
xy%z)
2.1 
HIMÜNOPfTUHIM_Vš
Ay
50g/L
3noBp>0;Ž

lm’û?
¿»
8573%,

),
ûalm’
EGI、EGI
ª
CB
HI
H£?E
5U,
|
pH48、50℃
Á¿ÑH2
4h,
uv0yO
HPLC
ÿ©òÿ



e
1 
-WÇK}Ü
EGI、EGII
~0WÇK}Ü
CBHI
TU\™_
HPLC
e
Fig.1 HPLCanalysisofsuagrproductshydrolyzedbypurifiedEGI,EGIIandCBHI
  EGI
R20y=èDlmÐpª•–p
,Karls
son
:
[7]
ëžgh/á
EGI
Í=]™R2lmö
p

de0y=ÝÏáo‘˜
(Dp)>2
3nop

EGI
E™­3.Œ­•opH

$xŒ4•opu
ý3β 1,4 pxV,>’lmnop,+X0y=
ð?D
Dp5~8
3lmnop
(Dp>7
3lmno
pãš<
)。Medve
:
[8]
¢£ì
Novo
lm’H=È
Y3
EGI
R2šW›œ3lm’0y=Ù¦?D
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Dp>4
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。CBHI
X™­3§Œ­•o
pH

*ò„#3lmÐpEuvR20y


? ˜E
193g/L,
Çp3
9011%,
•–pªl
möp>?Jî

¿ÑÇp3
643%
ª
346%。
Nidetzky
:
[9]
¢£
CBHI
R2©ž

lm’
),
q*
3œK¤sÖ

0y=lmÐp

•–pªlmö
p¿Ñ
92%、5%
ª
3%。CBHI
G§×Ÿlm’
Üý

=lmÐpEuv0y

YR2×n*%Á
s0Ì

0•–pӒ3lmÖpÚ
,CBHI
R2A
y3œKX0>s0lmÐpªlmöp

œb
CBHI
Qlmöp3 ªB£JR

+
CBHI
Qlm
öp`ÜDs]3R2B£

Y
CBHI
¡QB£|
lmöpÚ

R0yWElmÐpª•–p

d
e

lm’6
CBHI
B£œ

0yӒÎÏlmÐ
p

•–pªlmöp

{]&qxY3Íjk
Vrsanska
:
[10]
8
CBHI
3œ„ªÛL&qxg=D
jEn

If345×st/á

.ΥopHR2l
m’è0>
Dp
E
2~4
3lmnop

ÍQlm’
H<2xYB×st3ò¢ª–:

2.2 EGII
ÜpžFHIJK}
Ì.Œ•opH
EGI、EGI
ª§Œ•opH
CBHI
Qlm’R2xY345 Ø
:EGI
R20y
E•–pªlmÐp

ZÇp=•–p3waÛ%
sw

ã¿lmnop3gh
;EGI
ª
CBHI
R2l
m’30yuvElmnop

de×st45
EGI
H£?ªH2ÚÖQghlmnop3ST

2.2.1 EGI
H2ÚÖQlmnopÓ¿>?3
ST
8
1g
lm’
EGI
H£?E
5U
Ú
,pH48、
50℃、
Ayû? ˜
50g/L
{|ÁR2ghlm
nop

H2žŸ=o]pû? ˜Owa$ÚÖ
3ýYòÿ
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ÿ



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2、
ÿ

Í!
:0~15min
.lmÐpªl
möpEuvH20y
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3lmnopÝ
Ïá
。0~60min
.

lmöp>?£8o(

60min
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。0~30min
.

lmñ
p>?>™o(

1œ$ÚÖ3"Ü

>?£8Á
R

8
100min
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720min
.

•–p>?D®23ˆŒ

1œ$ÚÖ
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EGI
B£|
Üýlm’0>lmnop

H2žŸ#Û0>
Dp
e
2 EGII
ÜU&æÂHIJK}"ä$%_ÃÄ
Fig.2 Efectsofhydrolysistimeonthe
concentrationofsugarproducts
e
3 EGII
ÜU&æÂHIJK}XY_ÃÄ
Fig.3 Efectsofhydrolysistimeonthe
proportionofsuagrproducts
1~4
3•–pªlmnop

ã0>
Dp>4
3lm
nop
。EGI
ˤן͚3lmnopý3¥

ìSRnlmöpªlmñp3>?

deBæ
90min


eÚlmÐp

lmöpªlmñp
3>?¿ÑÇp3
498%、248%
ª
52%。
2.2.2 EGI
H£?QlmnopÓ¿>?3ST
H£?QH&q™˜STJí

8Y¦§\Á
3H&q=

òKAy ˜—Lí

3H&q™˜
ÒH£?’*w

8
EGI
ãÙH£?
、pH48、
50℃、50g/L
Ay3{|ÁR2
90min
ghlm
nop

H2žŸ=o]pû? ˜Owa$H£
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ÿ



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4、
ÿ

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y=op3waªû? ˜Ù¦ýYJí

•–p
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jX$%H£?3ˆŒ

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ä’H2™rÁR

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܍äÂHIJK}$%_ÃÄ
Fig.4 Efectsofenzymedosageonthe
concentrationofsugarproducts
e
5 EGII
܍äÂHIJK}XY_ÃÄ
Fig.5 Efectsofenzymedosageonthe
proportionofsugarproducts
1g
lm’ŒHì
1U
ˆŒ*
2U
Ú

lmöpû? 
˜®2³E*
039g/L,
Çp3
298%,
1œ$H
£?3ˆŒ

>?3Rn

Yûalm’ŒH
1U
Ú

lmñpû? ˜E
009g/L,
Çp3
79%,
1œ$H£?3ˆŒS]™Rn

ûalm’ŒH


Ú

lmñpì0y=&P

Yûalm’ŒH
10

Ú

0y=lmöp3waßE
158%。
IœKÒ
Nidetzky
:
[9]
45
EGI
H£?Qlm’R20y3
ýY3œKsÖ

Y&qĽ=H£?ˆŒ

>À3
EGI
¾ÒF
lmñpªlmöp3R2

BæH£?Eûa
lm’ŒH
1U


=~sÈÓ¿
EGI
£?E
ûalm’ŒH
1U,
H2ÚÖ
90min,
R20y=
lmÐp

lmöpªlmñpÇp3wa¿Ñ
E
438%、348%
ª
79%。
ìlmnop3Ӓ
œ„ªwaÎ

s|ȝ
[11]
gh3lmnop

2.3 
HIZ}

HIJK}ÂQRSq[\_]^
#$
  
¿Ñ=lmÐpªHfgh3lmnopE

P

8‹Ì{|Á…†Üi&‰N

…†žŸ=
o¾»3ýY©ªòÿ
6、
ÿ



e
6 
HIZ}Â_`QRSq[\_ÃÄ
Fig.6 GrowthofB.adolescentisusingcelobiose
assolecarbonsource
e
7 
HIJK}Â_`QRSq[\_ÃÄ
Fig.7 GrowthofB.adolescentisusingCOS
assolecarbonsource
Ìÿ
6、
ÿ

Í!

lmÐpE

Pj]™ˆ‰
&‰N
,6h
Ú&‰N ˜)*TíÉ

NÄû
? ˜Ì…†6#3
021g/L
ˆŒ*
045g/L,
ˆ
‰F
214
º

…†
6h,
ÇÒ«p3û? ˜ì6
73
3g/L
ÁR*
247g/L,
p¢£rßE
1767%。
=Hfgh3lmnopE

P

Ù¦8
6h,

û? ˜)*TíÉ
054g/L,
ˆ‰F
284
º

&
‰NQlmnop3¢£s|lmÐp

…†
36h,
Çpû? ˜ì673
300g/L

200g/L,
p¢£rE
3300%。
6t45 Ø

l
mnopQ&‰NDs]3ˆ‰B£

lmno
pãjEôÄ&Y½¾¼µZóoÔÄ.3D€
N¢£

deñnop¿÷BE&‰d¬#ۈ‰
ôÄ.3Üi&‰N

ëžlm’H~sÓ¿R2noBp;Ž`
qE«ŒÉ3lmnop

RCDJï3q£H

+ìÉ[Y÷˜Ï/

sùr
,`
qÈH’I
óE

Csùr

~sÓ¿
EGI
ª
CBHI
R2lm
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s,ƒ„d¬1Ö3¬ÙB£

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£Jn

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st45

3 
x
 
)
45FŠ‹B/
(Trichodermareesei)RutC30
l
m’H½]uv3

]HÓ¿
EGI、EGI
ª
CBHI
R2lm’3xY

ì0y=lmnop3Ӓ
ªwa÷˜Î
,2
]H­RX
EGI
¿‘|lmno
p3gh

×stQ~sÓ¿
EGI
3H2ÚÖªH
£?×nFsY

=
1g
lm’H£?
1U,
H2ÚÖ
90min
ETò

ghq*3lmnop=lmÐp

lmöpªlmñpÇp3wa¿ÑE
438%、
348%
ª
79%。
¿Ñ=lmÐp

lmnopE

Pˆ‰Üi&‰N

NÄû? ˜ˆ‰º»¿Ñ
E
214
ª
284。
M“6i

[1] 
®d¯

ijknop
[M].
‰~

YÞÉ[ÏLZ

2004:395.
[2] 
°±²



ZM

:

ÎW<2lm’g´lmnop34
5
[J].
0YÞÒÉ[
,2008,28(1):7982.
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´¾µ

¶·~

üýþ

ë¸?QŠ‹B/0lm’H3S
T
[J].
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,2008,28(2):5557.
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[4] 
ï$Ü

Š‹B/lm’H3¿ÀÈYOq£345
[D].
}~

}~[íÞ
,2008.
[5] 
…M

èW

noBpQÜi&‰N3ˆ‰
[J].
bcÝÞ

2001,22(7):1517.
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Res,1992,227:1925.
[11]
ȝ

Bûlm«CHfghlm¹p345
[D].
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