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Purification and characterization of complete and truncated forms of Ca2+ independent α-amylases

2种不同形态不依赖Ca2+α-淀粉酶的纯化与表征



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2012
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ChineseJournalofBioprocessEngineering
Vol.10No.5
Sep.2012
doi:10.3969/j.issn.1672-3678.2012.05.009
NOPH
:2011-12-01
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ãäÓ

šÉÊJAÓÉKL
Ca2+
Óα MN€,Ï%€™
ã2â(HPLC)
ìh

Ö×ØÙ

NOÇOPQ

rR
、(NH4)2SO4SMyìÌFTh,8,®à
kmH>Uíïš
36
UÓËVH‡WXJY α MN€ Amy7Byïš75UÓZùÎJY α MN€ Amy7S;
Amy7B
y
Amy7S
Óþ[
pH
!
65,
þ[Æú!
65℃,
€†®\ÉKL°œ]^þœ—
(EDTA)
y
Ca2+;
€™ã2
bJ/y_¼/]‡
。Amy7B
Ó¯ÏìÌcÜ!
67×104,`
a÷Æú!
596℃,
6†‡!
(90599±
9652)U/mg。
qƒ¯6
,Amy7S
Ó¯ÏìÌcÜb”
20×104(
!
47×104),`
a÷Æúš
27℃(
!
623
℃),
6†‡š
105
U

ÿð
(185387±7561)U/mg)。
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:1672-3678(2012)05-0044-06
Purificationandcharacterizationofcompleteandtruncated
formsofCa2+independentαamylases
WANGChenghua1,2,SHENNaikun2,QINYan2,ZHUJin2,WANGQingyan2,
HEBingfang1,HUANGRibo1,2
(1ColegeofBiotechnologyandPharmaceuticalEngineering,NanjingUniversityofTechnology,Nanjing210009,China;
2StateKeyLaboratoryofNonFoodBiomassandEnzymeTechnology,NationalEngineeringResearchCenterfor
NonFoodBiorefinery,GuangxiKeyLaboratoryofBiorefinery,GuangxiAcademyofSciences,Nanning530007,China)
Abstract:TwoformsofCa2+independentαamylases(Amy7BandAmy7S),fromBacilussubtilisCN7,
werepurifiedandcharacterized.TheenzymaticstarchhydrolysisproductswereanalyzedbyHPLC.The
resultsshowedthattheAmy7Bwaspurifiedby36foldandAmy7Swaspurifiedby75foldsimultaneously
afterthermaldenaturation,ultrafiltration,ammoniumsulfateprecipitation,andmolecularsievechroma
tography.BothformshadthethesameoptimumpH65,thesameoptimumtemperatureof65℃ and
verysimilaramylolyticpatern.CalciumionsandEDTAhadnosignificantefectsontheiractivities.
However,theAmy7S(47×104)wasapproximately20×104,shorterthantheAmy7B(67×104),
thehalfinactivationtemperatureandthespecificactivityofAmy7Swere623℃ and(185387±
7561)U/mg,theywerehigherthanthatat596℃ and(90599±9652)U/mgofAmy7B.
Keywords:aciduric;αamylase;Bacilussubtilis;calcium;starchhydrolysis
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Sh201GH"ÛKL
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–

î·´?
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ëX
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[\t
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pH
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123
c‚FF_º»
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pH
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QñR5,ÚÆÀ
50~95℃
R5

æmR5_åT
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tm。
  
…1uօ£
(Km)PG9£(kcat)º»:£¤
&ϣ7

º»ŠLp(´µè
04~9g/L
2æŒ
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Km,kcatPê9x%A(kcat/Km)。
  
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80℃
[\tªöœ
30min,
À
4℃

2h
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¹ÖF_

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50%
IT-52öœÚµ

t3050。
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½˜Ï*P¥Áˆj¥X
(EDTA)
234
  
ƒ9F©²Ý„½˜Ï*
(1mmol/L
P
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8
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À
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2h
Ç

ÖÛ
123
cÔ7Öº¹ÖF_

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FK>L2mx©€ÓÔ
(HPLC)
²³
  2mL
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mol/L
ëX ©ªXBæ©
(pH65),2U
ƒ9F©

N
1μg/mL)。R5,ÚÀ50℃b­cR524h。
À®b­c
10min
DíR5Ç

À
4℃、12000r/min
ÏÐ
5min,¯
’©@µ
022μm·Òµ·,µ·©@
µ
AminexHPX 87H
~XØ

Ë·
BioRad


d

HPLC
Öº

Öº[:RI
Úµ
50℃,

05
mL/min,
Vº€è
5mmol/LH2SO4。
2 
xy%z)
21 B.subtilisCN7α ~Î^€¡% SDS
PAGE
õØ
  B.subtilisCN7
ˆª~²¤…2cÚ α ‚
F
[6],
whµ´ªK¸–”“

î·´?

(NH4)2SO4²、²*qD³Pî·´?,ƒ9žý
/

z}~2α ‚F Amy7BP Amy7S,%‹²
݃9/
36
¿P
75
¿
,SDS PAGE
ÖºyÚÞ
1,
ƒ9yڅ
1。
›Þ



ƒ92
Amy7B
P
Amy7S
ý/)õ

[pŸÏ²Ý‚À
67×
104
P
47×104
·Œ

*„a{À:;š¡S˜S
h2α ‚F2ªz¸P´@,}K2ŸÏ€
‘
[10]。
TJPJeº
,Amy7B
P
Amy7S
²Ý-5
À
B.subtilisCN7α ‚F2ªz¸}KP´@,
}K

22 
€¡hα ~Î^΂޼
221 pH
-
Amy7B
P
Amy7S
234
  
ƒ9Ç
Amy7B
P
Amy7S
2‚F_`ì
pH
„9UÞ





PJäN
:Amy7B
P
Amy7S
F_ì
pH
„9óG¯~2

 }

#$

s
æ¾
pH

65,

pH45~70
âã}

%‹:
P9:
80%
2æŸ_å

…GN/”³2X`
pH
,ü`

(a)M—
”“pQñR
;1—BacilussubtilisCN7
01©
;2—20μgƒ92Amy7S。
(b)M—
”“pQñR
;1—10μgƒ92Amy7B;2—1μgƒ92Amy7B;3—5μgƒ92Amy7S
f
1 B.subtilisCN7α ~ÎAmy7SqAmy7B€ÎSDS PAGEV;
Fig.1 SDSPAGEanalysisofpurifiedAmy7SandAmy7BfromB.subtilisCN7
œ
1 B.subtilisCN7α ~΀¡xy
Table1 PurificationresultsofαamylasesfromB.subtilisCN7
\獕 Î_å
/U
°_å
/(U·mg-1) ƒ9¿£ m(”“)/mg -.%/%
ŒÆ01©
2527114 2459 0  102770 10000
´ª¸–
2407489 3165 129 76066 9527
î·´?
1925981 3776 154 51012 7621
(NH4)2SO4² 969401 16328 664 5937 3836
g³¨s
866144 16524 672 5243 3427
D³²Ï
Amy7B 108073 70177 2854 154 428
Amy7S 488491 133467 5428 366 1933
î·´?
Amy7B 52047 89736 3649 058 206
Amy7S 370482 183407 7459 202 1466
64
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!
10
"
 
f2 pH
Z
Amy7B
q
Amy7Sα ~ÎÖÞ^_`
Fig.2 EfectsofpHonpurified
Amy7BandAmy7S
222 
Úµ-
Amy7B
P
Amy7S
2_`PY»`
234
  
Úµ-
Amy7B
P
Amy7S
2_`PY»`23
4UÞ


。Amy7B
P
Amy7S
æ¾Úµè
65℃,

60~70℃
âã}

%‹:P9:
90%
J¯2æŸ


Þ
3(a))。
÷‚#ڵ݅†F©ªY»`2
ÞŇßPQ

ÖÛ
124
c÷‚#Úµº»Ô7

ªöœ
30min
2[\t
,Amy7B
P
Amy7S
2÷‚
#Úµ²Ýè
596
P
623℃(
Þ
3(b))。Amy7S
2
÷‚#Úµ°
Amy7B
m
27℃,
±¹í
Amy7S
ã;
êm2Úµ,ü`

f
3 
!‹Z
Amy7B
q
Amy7S^ α ~ÎÖÞqƒYÙÞ^_`
Fig.3 EfectsoftemperatureonactivityandthermalstabilityofpurifiedAmy7BandAmy7S
223 
æ¾
pH、
Úµ[\t2F¼"£
  
æ¾
pH65
Pæ¾Úµ
65℃
[\t

JP&
`‚èŠLºž2
Amy7B
P
Amy7S
2F¼"£
څ
2。
›…


:Amy7B
P
Amy7S
ã;Ÿ,€å
2
KmA,²Ýè(344±042)P(301±038)g/L,
éÝ
Amy7S
2°_å°°
Amy7B
m
105
¿

²Ýè
(185387±7561)
P
(90599±9652)U/mg。Amy7B
P
Amy7S
2
kcatA°è1∶143,ê9x%(kcat/Km)
°Nè
1∶164。
  
Š‹ÅӁy„ghij2a{À
B.subtilis
X 23
2ªz¸P´@, α ‚FÅӁy€
°

€|öݪz¸P´@,:ã;€Œ2
Km
A
,´
@,}K°ªz¸ã;êm2°_å

{|
öÀa¹
B.subtilisCN7
2ªz¸„´@,2
G9£Pê9x%Ÿ,€å
[10],^
ÄÅÓc2
Amy7S
;;mÀ
Amy7B。
+z

ÄÅÓc
Amy7B
P
Amy7S
2æ¾Úµ

Úµ,ürê9x%{|
ÀŠ‹"#ˤ¨F©
[6]
PŸ ¡ScÀ{…
2‡ˆ α ‚F[7]º»2F¼"£。
œ
2 Amy7B
q
Amy7S^
΂޼
Table2 EnzymaticparametersofpurifiedAmy7BandAmy7SfromB.subtilisCN7
α ‚F Km/(g·L
-1) kcat/s
-1
€-²*p(
°_å

(U·mg-1)
kcat
Km

(L·g-1·s-1)
Amy7B 344±042 102105±4851 67620 90599±9652 29682
Amy7S 301±038 146224±5624 47325 185387±7561 48579
 
74 
!

# 5ª-y
:2
z{|}~{€
Ca2+α ‚F2ƒ9„…†
23 
Q„:W®
EDTA
Z~ÎÖÞ^_`
  Ca2+
y½˜Ï*r
EDTA
-
Amy7B
P
Amy7S
234yUÞ



›Þ



½˜Ï*P
EDTA
-
Amy7B
P
Amy7S
234Þ,

彘Ï
*´µ±)
(1mmol/L)
I
,Co2+
PJ,m
20%

‚F_å
,^ Mn2+

10%
2‚F_å

sÖ2
3
Ca2+
}2mŸW£½˜Ï*-F_234{
ñ/
。10mmol/L
2½˜Ï*´µ-

z‚F2
34IĄ
1mmol/L´
µI€|
。1~100mmol/L
2
EDTA
-

zFF_234¸À{ñ/
(p>
005,t
֖
)。Amy7B
P
Amy7S
T…GN2{
€À
Ca2+
P
EDTA
2|`

„ghij2a{À
BacilusspKR8104[3]
P¶ë
BacilusKSM K38[11]
2α ‚F€Þ,。
f
4 
Q„:W®
EDTA
Z
Amy7B
q
Amy7S^ α ~ÎÖÞ_`
Fig.4 EfectsofmetalionsandEDTAonαamylase
activityofAmy7BandAmy7S
24 
Îïë^
HPLC

  α ‚FPJ(}”ì†JK‚c α 1,4

Tª α ~NO<、·š<、·š¡UNO<
。Amy7B
P
Amy7S
3ÆbKP&`‚
>L²³ÔÚÞ
5。
›Þ

PJäN

%‹3ÆJ
KP&`‚2D>L:›NO<

·š<

·š
¡
æŸ2ˆª”²²ÝÝNO

{|öÀ

zªº2ˆª°*á
Ñ;¸À

*z¸ÀíPé› α ‚F(P&`
‚}”2솀»Zª

1—
·šj<
;2—
·š¡<
;3—
·š<
;4—PO3-4 ;5—NO<
f
5 Amy7B
q
Amy7S
Îï…}Þ~^
3†ë
HPLC

Fig.5 HPLCanalysisoffinalproductsliberated
fromsolublestarchbypurifiedAmy7B
andAmy7S
3 
x
 
)
  
ƒ9…†/
BacilussubtilisCN7
cÚº$2

z}~2{€
Ca2+
2 α ‚F Amy7BP
Amy7S,
²³/sFKP&`‚2>LÔÞ


y…ð

@µ´ª¸–

î·
、(NH4)2SO4²P²*
qD³€©

Þwµž/ƒ9¿£²Ý,m
36
¿
P
75
¿2)Ã
Amy7B
P
Amy7S,
ƒ92
Amy7S
2
€-²*p(°
Amy7B
ÏN
20×104,
%‹ã;€
|2æ¾
pH65、
æ¾Úµ
65℃、
IĀ|2P&`
‚FK>L
,10mmol/LCa2+
P
1~100mmol/L
2
EDTA
-

z‚F_`234{ñ/

æ¾
pH
65
Pæ¾Úµ
65℃
[\t
,Amy7S
2°_å°
Amy7B
m
105
¿

÷‚#Úµm
27℃。
L6j

[1] vanderMaarelMJ,vanderVeenB,UitdehaagJC,etal.Proper
84
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