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Co-production of S-adenosylmethionine and glutathione under salt-stress by Candida utilis

S-腺苷蛋氨酸和谷胱甘肽联产发酵中盐胁迫的作用



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2012
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ChineseJournalofBioprocessEngineering
Vol.10No.3
May2012
doi:10.3969/j.issn.1672-3678.2012.03.007
OPQI
:2011-09-25
KCR!

no1o1oM¯‰oÅÑÆ)ÒÕÖ
(KY2011094A)
ST.U

ABÜ
(1987—),
ß

%nÝÂã

påÆÇM

ÆÇæç

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‚êã
),
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,Email:weigy@suda.edu.cn

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ŠŽ

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–p9: —˜™:=;
–p
215123)
7
 
l

q¾KLvœk²¬¿
(NaCl,Na2SO4,KCl,K2SO4)ÀÁ"¨Â2æĥ¦—¨ S ŨƇ«
(SAM)
ÓÇÈÜÉ
(GSH)
¬EF

ë쥽pÊòó¬
Na+
Ó
K+
"
SAM
Ó
GSH
)‹Ë#•ÖÌæàå

Í
Cl
¬àåÎ%,

Û
Na2SO4á«À,œÖZ¥¦KLv¿ÀÁ¬àå,ëìÀ#:ù¦Ä~4§ŽSÏ(15h)¹r
10g/LNa2SO4,SAM、GSH۟ªÐ¬I—¨¸á2525、2859Ó5219mg/L,C"ÑÖ×ÏО88%、226%
Ó
139%。
ISÖ×`®¸«¬Ó¥¦‰ŠŒÒó"ÖZ¥¦¬ëìæçžÖÓ

mno
:S
ŨƇ«

—¨¥¦

ÇÈÜÉ

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:A    
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:1672-3678(2012)03-0033-06
CoproductionofSadenosylmethionineandglutathioneunder
saltstressbyCandidautilis
WANGYulei,YEZiyou,HEQiuping,WANGWenlong,WEIGongyuan
(SchoolofBiologyandBasicMedicalSciences,SoochowUniversity,Suzhou215123,China)
Abstract:EfectsofsaltstressofNaCl,Na2SO4,KClandK2SO4oncoproductionofSadenosylmethi
onine(SAM)andglutathione(GSH)wasinvestigatedinflaskebyCandidautilisCCTCCM209298It
wasshownthatNa+andK+enhancedthecoproductionofSAMandGSHonappropriateconcentrations,
whileCl-inhibitedcelgrowthandproductbiosynthesis.Basedontheresults,saltstressresultedfrom
Na2SO4wasfurtherstudiedunderbatchcultivation.TheconcentrationsofSAM,GSH,thecoproduction
ofSAM,andGSHreached2525mg/L,2859mg/Land5219mg/Lafteradding10g/LofNa2SO4at
15hwereincreasedby88%,226% and139%,respectively.ComparedwiththosewithoutNa2SO4
addition.Furthermore,theresultswereanalyzedbyconcentratingontheenergymetabolismandthefer
mentativekineticsinbatchfermentationofSAMandGSH.
Keywords:Sadenosylmethionine;coproduction;glutathione;saltstress
  S
|w}h=
(SAM)
íM¶–²Aî×·>
Mª®¯òi-¶—

~ÞS‹&LßG*©Q

Nùà~âá8>HI
[1]。
€H
(GSH)
íA
îàQγ â)>®¯K,L߉~*ÏÐks,
0*¾›–²eY>DX`a$%ÚÌ×·>†
G
[2],
~ÞSjo

Õmˆf

0ùʓøVãÏQ
ÌLß>‡G
[3]。
Ö

9y89‰íM:
SAM
~
GSH
>_·æ‰
[4-5],
FGAB4Brù
SAM
~
GSH
>‚YM:Œ~?@9y
[6]
~:äÔV9
y
[7]
sØÿrù

çýÈA–‘û‚::˜
íðØÆÇ>xD

  
~üM¶89ËÌs

å{‰k$%„…æ
õ‡:M®¯D
(ROS),
ó1çè
ROS
>Š1

ü
M¶Ÿ YòÿË.UÏß>Z[\]ÿÈ
‡Ñ
[8]。GSH
~
SAM
0*PþŸ é«‰
k$%æõàQ×·>†G


GSH
òÿËP
ÊÏß?@)éêËDXõ„…>?@
[9];SAM
ò
ÿ~=„…‘’"‘ûŸ Kzˆ
ATP
V>®¯

xyK‹úT>×ÀYZ

S͑û
SAM
>:
˜
[10]。
ƒ„…íAîYÄ>‰k„…æõæ‰

(
$)ËÌÆÇ>9G1’

üM¶0ƒ„…>@‡
RbM+Þë
[11],
̓„…æõíìFGŒÈüM
¶Ÿ YZÕ©>
SAM
~
GSH
íQ¬ÆÇ

QRVW9;î¤>ƒ„…æõ0:äÔV
9y89‚:
SAM
~
GSH
>?@

!§YSF˜
Z[~89mnoTU0SNÂÃÈæiž

1 
~ƀ
11 

  
:äÔV9y
(Candidautilis)CCTCCM
209298,
no1oüM¶“Ìrst>}!ˆ•

12 
ƒ„K
  
î-wx)
(g/L):
tuK
20,
}{
10,
9y
š
10;pH60。
  
89wx)
(g/L):
IK
35,(NH4)2SO410,
KH2PO4123,MgSO4005,CaCl2005;pH50。E
7Ziû™rs·žü£

13 
ƒ„€
131 
î-wx
  
‡
-70℃
³îˆ•>î-®Xޑ’î-w
x)s
,30℃、200r/min
­Swx
24h。
132 
­®89wx
  
î-wx‘î•
500mL
­®s

“˜
50mL,
‘î˜
10%,30℃、200r/min
­Swx
30h。
133 
ij89wx
  
î-wx‘î•
5L
ïð(89”
(BIO
TECH 5BGZ,
‹ºˆñM¶c“ÌQ¸´µ

s

“˜
3L,
‘î˜
10%,
ïð³™
350r/min,

¸˜
30L/min,pH50(
ËÏmÂÊ
3mol/L
H2SO4~3mol/LNaOH¡Èæ.ù)。
14 
r…€
141 
9yM¶˜>££
  
ÿŸ ¡—˜
(DCW)
zè9yM¶˜

’
25mL
89
,3500r/min
°
10min,
žŸÂHI

b

°

ýÁ4–
,70℃
v¡•¨—˜Þ+
—˜

142 
 ²
GSH、SAM
>‘’~££
  
à–‘’~££æ‰¤`¥¦
[12]。
143 
IK…U££
  
FGå#>
DNS
‰
[13]
££

144 
 ²
ATP、NADH
>££
  
’
10mL
89

*
4℃
"
,8000r/min
°
10min,
hª‹®

4–)’òs©ª
1min,
H
EZ[óÊôÒ
,-70℃
ˆ‰*¼ÚG*
ATP
~
NADH
>££
。ATP
~
NADH
>££æ‰i†¤
`¥¦
[14]
~
[15]。
145 
89mno¤ìÚô
  
Ÿ þM—™š
(μ)–:¶þYZ™š(qp)i
†Û(
(1)
~
(2)
Úô

μ=1X
dX
dt=

XlimΔt→0
ΔX
Δt
(1)
qp =


dP
dt=

XlimΔt→0
ΔP
Δt
(2)
(s
:X
~

i†z衟 —˜…U
(g/L)
~:
¶>:˜
(mg/L)。
146 
ì™iž
  
­®89ì™)Ï

Eõ/§s§¨>ðÇ
ð

ij89wx왒Ï

]ð槨>ðÇð

§sì™>òöU·G

¢sÈæiž

È
P<005
Î
001

qÚižD
’ó
StatisticalAnalysisSystem(SAS90)。
2 
†‡%ˆ)
21 NaCl
SAM
Æ
GSH
„edijk
  
VW­®wx‘’"9;—˜…U>
NaCl
0
C.utilisCCTCCM209298
‚:
SAM
~
GSH
>?
@

ÂÃçY

µè

/Y

òÅ

AL~
0h`
í
15h(
Ÿ M—Þ#
),NaCl
>ÆÊÏâb14–>


ûË̅U>‘û

âbí‡Ü8Þê

È
NaCl
—˜…Uó
100g/L
<

Ÿ ¡—˜Í0`i
†ú‹1
136%(0h)、121%(15h)。
0*:¶
SAM
~
GSH
ÍÈ

‹…U
NaCl
?@Íò

.û…U
NaCl`
íú‹1
SAM
~
GSH
>‚:˜

È
NaCl
—
˜…Uó
100g/L
<
,SAM
~
GSH
‚:˜i†ó
2458mg/L(0h)、3605mg/L(15h),
Í0`i†
ú‹1
461%(P<002)
~
217%(P<005)。
43
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10
"
 
q1 NaCl
\eˆ‰Šd‹„e
SAM/GSH
ijk
Fig.1 EfectsofNaClonthecoproductionofSAM
andGSHinflasks
22 Na2SO4\SAMÆGSH„edijk
  
VW9;—˜…U
Na2SO40C.utilisCCTCCM
209298
89‚:
SAM
~
GSH
>?@

ÂÃçY

µè

/Y

òÅ
:Na2SO4>—˜…U≤10g/L
<

0
SAM
~
GSH
>MZQü*>ŒÈ†G

È
Na2SO4>—˜…Uû* 20g/L<,4–˜÷øú
‹

:¶>YZî{âb

~
15h
ÆÊ
10g/L
Na2SO4<,SAM~GSH‚::˜ó4908mg/L,þ
0`‘û1
73%(P<01)。
  NaCl
~
Na2SO4µù)>ƒ„…0* SAM~
GSH
>89‚:zùå9ÆS;>í‡

íì,ó
    
SO2-4 ~Cl

>‰BÍy8>

ÿ"‡Ëúƒ„…
)ÈA–VW

q
2 Na2SO4\eˆ‰Šd‹„eSAM/GSHijk
Fig.2 EfectsofNa2SO4onthecoproductionofSAM
andGSHinflasks
23 KCl
Æ
K2SO4\SAMÆGSH„edijk
  
89½¾È惄…Ç9F*:¶>YZ~
?@

ó^~
15h
ÆÊ9;…U>
KCl
~
K2SO4,
ÿVWE0
SAM
~
GSH
‚:89>?@

ÂÃç
Y

µè

/Y

òÅ
:KCl
>ÆÊ;¨âb1:
¶>YZ

zùå~
NaCl
¤‹>í‡

Í
K2SO4´
~A£ÌU‹òÿøiŒÈ:¶>YZ

È
K2SO4
—˜…Uó
10g/L
<
,SAM
~
GSH
‚:˜þ0`
       
q
3 KCl
Æ
K2SO4\eˆ‰Šd‹„eSAM/GSHijk
Fig.3 EfectsofKClandK2SO4onthecoproductionofSAM andGSHinflasks
53 
!

# ABÜø
:S
|w}h=~€H‚:89sƒ„…>†G
‘û1
59%。
/^òÿ8ù

ÈÈ>
Na+
~
K+
„
…QF*
SAM
~
GSH
>YZ

Í
Cl-
>‰~Aû¿
FڏSÐ>†G

,^

ÿ"‡ÿ
Na2SO4óI,
~89”)ًÈA–VWƒ„…>†G

24 Na2SO4†‡0iràƒ„„eSAMÆGSH
241 
ij89ËÌiž
  
FGM¶—alM:QGXoù<

Y·GÆ
H2SO40alÈæ=éª,ÈA–ÿ NaOHs~
=Ð

,^FG^¤K‡=:M
Na2SO4„…。
ó^

ÂY¥ÆÇÂÃ

~
5L
89”sÈA–V
W
10g/LNa2SO4~ijwx‘’"0SAM~GSH
‚:>?@

ÂÃçY

µè

/Y

òÅ

~
0h
ÆÊ
Na2SO4<,Ÿ M—Óé,;<.ËÌ:¶Y
Z˜>ú‹

Í
15h
ÆÊ
Na2SO4üQF* SAM~
GSH
>?@
,SAM、GSH
ÿ–«:˜i†{
2525、2859
~
5219mg/L,
þ0`i†‘û1
88%、226%
~
139%(P<005,
z
1)。
242 Na2SO4ÆÊ0C.utilisF˜Z[>?@
  SAM
~
GSH
>M¶YZÇóóí
ATP

Ì
[16-17]。
~=„…‘’"
,SAM
òÿ‘û
ATP
V
>®¯

SÍòÿŒÈ
ATP
>YZ
[10]。
‚ý
Na2SO4„…=0Ÿ F˜Z[:Mþ¨>?@?
’0ìM—s#
(12h)
~:¶YZ#Ÿ 
(21h),
££ ²
ATP、NADH
¼˜

ÂÃçz

µè

/z

òÅ
:0h
ÆÊ
Na2SO4< ² ATP~ NADH¼˜
ÏÞê‹*0`

Í~
15h
ÆÊ
Na2SO4<,:¶Y
Z#Ÿ >
ATP
ý
NADH
¼˜Í0`i†‘û1
388%
~
27%,
òÿÕÖ×ËÌ89Þ#:¶Y
Z0F˜>¶ž

,Íòÿ~A£ÌU‹ŒÈ
SAM
~
GSH
:˜>‘û

q
4 
ràƒ„ÇÈ0
Na2SO4\SAMÆ
GSH
„edijk
Fig.4 EfectsofNa2SO4additiononthecoproductionof
SAM andGSHunderbatchcultivation
 
1 Na2SO4š›LM\SAMÆGSH„edŸ3MN
Table1 EfectsofNa2SO4additiontimeonparametersofbatchfermentationforcoproductionofSAM andGSH
wx
æ(
ρ/(mg·L-1) ؚ/(mg·g-1)  ²—˜iì/% M:þU/(g·L-1·h-1)
SAM GSH SAM+GSH SAM GSH SAM GSH SAM GSH
0`
2321 2332 4583 663 667 170 172 859 1110
Ⅰ 1946 2068 3803 556 591 153 181 649 862
Ⅱ 2525 2859 5219 721 817 185 203 765 1059
 
ñ
:Ⅰzè0hÆÊ10g/LNa2SO4,Ⅱzè~8915hÆÊ10g/LNa2SO4,0`zè9ÊNa2SO4Èæ89。
63
M
 

 
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10
"
 
 2 
ràƒ„ÇÈ0±-
ATP
Æ
NADH
Œ
Table2 IntracelularATPandNADHcontentsunderbatchculture
¤ì
0`
0h
ÆÊ
Na2SO4 15hÆÊNa2SO4
Ⅰ Ⅱ Ⅰ Ⅱ Ⅰ Ⅱ
ATP
>¼˜
/(mg·g-1) 211 085 173 072 219 118
NADH
>¼˜
/(mg·g-1) 432 256 362 241 429 263
         
ñ
:Ⅰzè0ìM—s#Ÿ (12h),Ⅱzè:¶YZ#Ÿ (21h)。
243 
89mnoiž
  
mno¤ì

çþM—™š
(μ)、þ:¶YZ™
š
(qp)øòÿÐ)Ÿ M—~:¶YZFn>1
ò

ó1g*þÍ

ÿ"‡ij89ËÌió
15h
~
15h
Þ

]67

9;ij89ËÌmno¤
ìÄz
3。
/z

òÅ

~
0h
ÆÊ
Na2SO4<,89
>#:¶YZFnÍþ

.
15h
Þ:¶>þYZ
™šüÞê‹*0`

ÐÍ
15h
ÆÊ
Na2SO4üQF
*‘ûŸ M—Þ#
SAM
~
GSH
YZ>Fn

,
Í΄òÿ5ØÕû>
SAM
~
GSH
‚::˜

 
3 
ràdŸ3°±y`Æ
SAM/GSH
÷ì1Y¬MN—Ø
Table3 KineticsparametersofcelgrowthandbiosynthesisofSAM andGSH
wxæ(
þM—™š

h-1
SAM
þYZ™š

(mg·g-1·h-1)
GSH
þYZ™š

(mg·g-1·h-1)
15h

15h
Þ
15h

15h
Þ
0
 
`
0161 143 062 141 058
0h
ÆÊ
Na2SO4 0087 154 049 158 021
15h
ÆÊ
Na2SO4 0159 138 068 139 093
 
3 
†
 
)
  
9;>ƒ„…0:äÔV9y‚:
GSH/SAM
>?@9ÆS;
,Na+
~
K+
òÿøiŒÈ
SAM
~
GSH
>‚:

Í
Cl-
üâbŸ M—~:¶YZ

~ij89>
15h
Ab¯ÆÊ
10g/LNa2SO4,
SAM、GSH
ÿ–‚:˜{
2525、2859
~
5219mg/L,
þ0`i†‘û1
88%、226%
~
139%。
M©6u

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.S
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m!

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Y¤É

ø

p‰  γÒ YxZ>}SAM
~
GSH
‚:894B
[J].
"ÒÆÇý"ғ”o*
,2010,
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73 
!

# ABÜø
:S
|w}h=~€H‚:89sƒ„…>†G
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siaemutantunabletoconvertglucosetoglucose6phosphateaccu
mulatesexcessiveglucoseinendoplasmicreticulumduetocoreoli
gosaccharidetrimming[J].EukaryotCel,2003,2(3):534541.
[16] MurataA,TaniK,KatoJ,etal.GlycolyticpathwayasanATP
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[17] ShiozakiS,ShimizuS,YamadaH.Sadenosyllmethioninepro
ductionbySaccharomycessake:optimizationoftheculturecondi
tionsfortheproductionofcelswithahighSadenosyllmethio
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