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Purification chitosanase from Streptomyces hygroscopious and its catalytic properties

链霉菌壳聚糖酶的纯化及其酶学性质



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ChineseJournalofBioprocessEngineering
Vol.11No.3
May2013
doi:10.3969/j.issn.1672-3678.2013.03.010
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:30℃

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°}
˜«Z[}˜D
333%,40℃

1h
°}˜«Z[}˜D
222%。ChA
D}¤ãuZÏo«
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(mL·min),Vmax«0318μmol/(mL·min),Km«1×10
-2mg/mL,
¥CæçG€RCД¶
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ChA
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Mn2+
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Pb2+
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。Mn2+
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:1672-3678(2013)03-0052-07
PurificationchitosanasefromStreptomyceshygroscopious
anditscatalyticproperties
YANGLihong,CHENGShiwei,FENGZhibin,ZHOUNannan,KONGYunhong,MingYongfei
(InstituteofMicrobialEngineering,ColegeofLifeSciences,LudongUniversity,Yantai264025,China)
Abstract:ThechitosanasepurifiedfromStreptomyceshygroscopiousisolatedfromtheofshoreinYantai,
andthenpropertiesofchitosanasewereinvestigatedtoprovideatheoreticalbasisforitsapplication.The
crudeenzymeofchitosanasewasextractedbyammoniumsulfatefractionprecipitation,andtwotypesof
enzyme,(ChAandChB),wereobtainedbygelfiltractionchromatographywithSephadexG100.The
catalyticpropertiesofChAwerefurtherstudied.ThemolecularweightofChAis416kDa,whichwas
monounitprotein,andthemaximumUVabsorbanceswereat220nmand280nm.TheoptimumpHof
ChAwas5055whenchitosanwasusedassubstrateandtheoptimumtemperaturewas55℃.Thermal
stabilityexperimentshowedthat333% ofactivitywasretainedafterheatingat30℃ for1h,222% of
activitywasretainedafterheatingat40℃ for1h.TheinitialreactionrateofChAwas62×10-3
μmol/(mL·min),themaximumratewas0318μmol/(mL·min),andtheMichaelisconstantKm was
10×10-2mg/mL.Furthermore,ChAshowedrelativesubstratespecificity.K+,Na+,Li+,Mg2+,Ca2+,
Ba2+,Zn2+,Cu2+,andCo2+hadlabileefectsontheenzyme,whileMn2+couldactivatetheenzyme.The
heavymetalionsHg2+,Pb2+,Ag+,andCd2+ inhibitedtheenzymeactivity.Mn2+couldmakeamixed
activationefectontheenzyme,butZn2+displayedacompetitiveinhibitionontheenzyme
Keywords:catalyticproperty;chitosanase;bioseparation;Streptomyceshygroscopious
  
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Table1 PurificationofchitosanasefromStreptomyceshygroscopiousSC1
׈–B
>e_

mL
m(
>Àf
)/
mg
>ά

(μmol·min-1)
gά

(μmol·min-1·mg-1)
×ˆæŠ •*
/%
ÞÎ
1000 127716 79070 00619 1 100
30%(NH)2SO4GH 1000 542963 51827 00955 154 6554
80%(NH)2SO4GH 4 5224 2066 0396 639 261
SephadexG 100
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(ChA) 8 299 356 119 192 045
  
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d…T
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SephadexG100column
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ÓÔ1Š;Ôf9ghia,-
Fig.2 Polyacrylamidegelelectrophoresisofchitosanase
22 
ÓÔ1Š
ChA
÷cE“¿w;j+
C
SephadexG 75
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dÐÑÀf
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SephadexG 75
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mL,
‘6

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C
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ÐÑÞ#46

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5。ChA
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•÷§…‡*Å
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‘6

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4)
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(43000);
3—
ˆ¥FÀfÎà
(25000);4—
r[Î
(14400)
D
3 
e;#$Q÷cE“¿w;@G
Fig.3 Relationshipbetweenelutionvolum
andrelativemolecularmass
\1[
SC1
àø÷Î
ChA
õ¦ô±Óôøh
ρ3@46
6,
‘6

á7

:Β
220
O
280nm
Kij‚0ô±ÓO

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23 
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ChA
;ŠN¿<=
231 
‚†
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pH
§\1[
SC1
àø÷ÎhST3@46
7,
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pH
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50~55
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50
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55
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d¬åðÍrs

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4 ChA
;
SDS
Ôf9ghia,-D
Fig.4 SDSpolyacrylamidegelelectrophoresis
ofchitosanaseChA
D
5 
k÷cE“¿wJ@lm€
Fig.5 Standardcurveoflowmolecularweight
D
6 
d…T
SC1
ÓÔ1Š
ChA
;nKo!pD
Fig.6 UVabsorptionspectrumofChAfrom
StreptomyceshygroscopiousSC1
D
7 pH
cŠª~;de
Fig.7 EfectsofpHontheactivityofChA
55 
!

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SC1
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hST3@
46
8。
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á7

àø÷Î
ChA
‚†ˆ\Å
55℃,
Pˆ\3Y
55℃
PZ‘Yƒåi¬pqÎ
¬å‰Šrs

D
8 
zscŠª~;de
Fig.8 EfectsoftemperatureontheactivityofChA
àø÷Î
ChA
h¦Ýå3@46
9。
‘
6

á7

’
30℃
ˆå
1h
¡Î¬Å‚0άh
333%,40℃
ˆå
1h
¡Î¬Å‚0άh
222%,50℃
ˆå
1h
¡Î¬#>‹i
,60℃
ˆå
45min
¡Î¬#>‹i

D
9 
ÓÔ1Š;qQ+N
Fig.9 EfectsoftemperatureonthestabilityofChA
232 
Îk.„Í*

iÝAŠÏ
àø÷ÎhÎk.„Í\‹iÝAŠÏ
3@

ñ­46
10
O6
11。
D
10 
ŠrÀ nss;¼+
Fig.10 InitialvelocityofChAforhydrolysisofchitosan
D
11 
ŠrÀ tuvï;¼+
Fig.11 Km ofChAforhydrolysisofchitosan
‘6
10
áQ;0•æàø÷Î
ChA
hÎk.
„Ív(
Ǻ*

Å
64×10-3μmol/(mL·min)。‘
6
11
á•:ÎhiÝAŠ
(Km)Å1×10
-2 mg/mL,
‚
0.Í(Vmax)Å0318μmol/(mL·min)。
233 
Îh+jcåρ
5

ÅÎh+jcå783@

f
2 
d…T
SC1
ÓÔ1Š
ChA
;½¾wIN
Table2 SubstratespecificityofchitosanaseChA
fromS.hygroscopiousSC1
+ ֤Œ
/%
95%
³é¥\àø÷
100
85%
³é¥\àø÷
875
65%
³é¥\àø÷
551
‚àôp‹!
(CMC) 53
õø÷
(40,000) 37
‘5

á7

àø÷Î
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§³é¥\Å
95%
hàø÷tσn‚v

T³é¥\hrs

ÎtÏ
ƒn@f

X!1Kàø÷Î

ŒƒtÏ
GlcN—GlcN
÷þÿ

ãƒtÏ
G1cNAc—GlcN
÷þÿ
)[15]。
Ÿ¦
àø÷Î
ChA
†ƒÔHtςàôp‹!
(β 1,4
÷þÿ

Oõø÷
(β 1,3÷þÿ),d÷§¬åñ­
Å
53%
O
37%。

ChA
§+53Å÷§jc
å

§ÿhK˜‹ÿY.hôJjchüýå

Á^bRÍüý

234 
VXWX§Î¬nhST
ícÌ/VXWX
(Li+、Na+
O
K+)、
/ûV
XWX
(Mg2+、Ca2+
O
Ba2+)、
*VXWX
(Co2+、
Mn2+、Zn2+
O
Cu2+)
O±VXWX
(Hg2+、Ag+、
Cd2+
O
Pb2+)
§Î¬nhST3@ñ­46
12、
6
13、
6
14
O6
15。
65
&
 

 
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+
  
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11
"
 
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ÁIx2&Rü“cŠª~;de
Fig.12 Efectsofmonovalentmetalions
onactivityofChA
‘6
12
á7
:K+、Na+
O
Li+
§ÎJjcË5
‚
。K+
O
Na+
‡\’
015mmol/L
PcË*ñ­
Å
2076%
O
2083%,
9
Li+
‡\’
015mmol/L
PcË*=ž
5122%。
D
13 
2y&Rü“cŠª~;de
Fig.13 Efectsofalkalineearthmetalions
onactivityofChA
‘6
13
á7
:Ca2+、Mg2+、Ba2+3
d/ûVX
WX§ÎJjcË5‚


Ba2+
h‡\=ž
0075mmol/L
PcË*á=ž
5998%,
PWX‡
\=ž
005mmol/L
P
Mg2+
O
Ca2+
hcË*ñ­
Å
426%
O
1562%,
TVXWX‡\ñu(cË
+\JóY݁

‘6
14
á7
:Mn2+
§:Îj#¬5‚

TU
Mn2+
‡\u(#¬5‚^Vu(

P‡\=ž
0125mmol/L
¡#¬}óY݁

dàžáƒb
Mn2+
‰K¬å¢šhʼ
[16]。
K"÷.h
,Zn2+、
Cu2+
O
Co2+
§ÎJjcË5‚


Cu2+
§Îh
cË+\Bv

P
Cu2+
WX‡\=ž
01mmol/L
PÎi¬

‘6
15
á7
:Hg2+、Ag+、Cd2+
O
Pb2+
§ÎJ
jcË5‚
,Pb2+
O
Hg2+
hcË}@BÅ;<

P
Pb2+
O
Hg2+
‡\=ž
01mmol/L
P>Îi¬

D
14 
Ãz&Rü“cŠª~;de
Fig.14 Efectsoftransitionmetalions
onactivityofChA
Cd2+
‡\=ž
0125mmol/L
PÎi¬
,Ag+
‡\=
ž
015μmol/LPÎi¬。
D
15 
‘&Rü“c
ChA
Šª~;de
Fig.15 EfectsofheavymetalionsontheactivityofChA
235 
VXWX§Î#¬KcË5‚h¸nŽ
Mn2+
§àø÷Î
ChA
STh¸nŽÏ3@
46
16。
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16
á7
:Mn2+
>Îk.h
Vmaxu
0
,KmO@ß,d}Åߕ˜#¬5‚。Mn
2+
3
•žÎ¬å¢šQ¦hð9

u0íÎK+h©
On
(Km@ß),2P_u(íÎhŒˆÍ*(Vmax
u0
),
ÖT#¬‡\u0#¬+\Žu0
[16]。
D
16 Mn2+
cŠ;þª).
Fig.16 ActivatedefectsofMn2+onChA
Zn2+
§ÎhSTh¸nŽÏ3@46
17。
75 
!

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>Îk.h
KmOu0,VmaxO


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。Zn2+
§ÎhåcË5
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áƒb
Zn2+
O+KÎñX3•ð9÷2

 
9rsíΧ+h©On

ÖTcˏ‡\hu
0cË+\ŽT"u0

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D
17 Zn2+
cŠ;{|).
Fig.17 InhibitedefectsofZn2+onChA
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[1] ShahidiF,ArachchiAKV,JeonYJ.Foodapplicationsofchitin
andchitosans[J].TrendsFoodSciTechnol,1999,10:3751.
[2] LeeHW,ParkYS,JungJS.Chitosanoligosaccharides,dp28,
haveprebioticefecton the Bifidobacterium bifidium and
Lactobacilussp[J].Anaerobe,2002,8(6):319324.
[3] MengíbarM,GananM,MiralesB.Antibacterialactivityof
productsofdepolymerizationofchitosanswithlysozymeand
chitosanaseagainstCampylobacterjejuni[J].CarbohydrPolym,
2011,84(2):844848.
[4] MoloyC,CheahLH,KoolaardJP.Inducedresistanceagainst
Sclerotiniasclerotiorum incarotstreatedwithenzymaticaly
hydrolysedchitosan[J].PostharvestBiolTechnol,2004,33(1):
6165.
[5] AbdElmohdyFA,ElSayedZ,Essam S,etal.Controling
chitosanmolecularweightviabiochitosanolysis[J].Carbohydr
Polym,2010,82(3):539542.
[6] KimSK,RajapakseN.Enzymaticproductionandbiological
activitiesofchitosanoligosaccharides(COS):areview[J].
CarbohydrPolym,2005,62(4):357368.
[7] 
NS‘



ö“

{

àø÷ÎÏh78
[J].
ˆ)lä

2002,21(6):381386.
[8] SunY,HanB,LiuW,etal.Substrateinductionandstatistical
optimizationfortheproductionofchitosanasefromMicrobacterium
sp.OU01[J].BioresourTechnol,2007,98:15481553.
[9] NgoKX,UmakoshiH,ShimanouchiT,etal.Heatenhanced
productionofchitosanasefrom Streptomycesgriseusin the
presenceofliposome[J].JBiosciBioeng,2005,100(5):
495501.
[10] StruszczykK,SzczesnaAntczakM,WalczakM,etal.Isolation
andpurificationofMucorcircineloidesintracelularchitosanolytic
enzymes[J].CarbohydrPolym,2009,78:1624.
[11] Somashekar D, Joseph R. Chitosanasesproperties and
applications:areview[J].BioresourTechnol,1996,55:3545.
[12] 
+¼,
YW§

à;½

&ˆŽàLOF
[M].
ö


ö0Žæ„˜
,1994.
[13] GaoX A,Ju W T,JungW J,etal.Purification and
characterizationofchitosanasefrom BaciluscereusD11[J].
CarbohydrPolym,2008,72:513520.
[14] ChiangCL,ChanCT,SungHY.Purificationandpropertiesof
chitosanasefrom amutantofBacilussubtilisIMRNK1[J].
EnzymeMicrobTechnol,2003,32:260267.
[15] 
”•

Ŗ—

H&àø÷Îh78]Ž
[J].
v,0ŽŽ
)

â&K&A΄
,2002,21(6):381386.
[16] ChenXE,XiaWS,YuXB.Purificationandcharacterizationof
twotypesofchitosanasefromAspergilussp.CJ22326[J].Food
ResInt,2005,38(3):315322.
[17] WangSL,ChenSJ,WangCL.Purificationandcharacterization
ofchitinasesand chitosanasesfrom anew speciesstrain
Pseudomonassp.TKU015usingshrimpshelsasasubstrate[J].
CarbohydrRes,2008,343(7):11711179.
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