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 Improvement of porcine interferonα production by Pichia pastoris and NADH reutilization efficiency using methanol/sorbitol cofeeding strategy

甲醇/山梨醇共混诱导改善重组毕赤酵母表达猪α干扰素发酵过程和NADH再生效率



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ChineseJournalofBioprocessEngineering
Vol.10No.2
Mar.2012
doi:10.3969/j.issn.1672-3678.2012.02.012
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:2011-08-22
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:1672-3678(2012)02-0055-08
ImprovementofporcineinterferonαproductionbyPichiapastorisand
NADHreutilizationeficiencyusingmethanol/sorbitolcofeedingstrategy
DAIKeke1,DONGShijuan2,YURuisong2,SHIZhongping1
(1.KeyLaboratoryofIndustrialBiotechnologyoftheMinistryofEducation,SchoolofBiotechnology,
JiangnanUniversity,Wuxi214122,China;2.InstituteofAnimalVeterinarymedicine,
AcademyofAgriculturalScienceofShanghai,Shanghai201106,China)
Abstract:Theefectsofmethanol/sorbitolcofeedingstrategyonporcineinterferonα(pIFNα)produc
tionbyrecombinantPichiapastorisandtherelevantenergy(NADH)regenerationeficiencyduringinduc
tionphasewereinvestigatedina10LfermentorTheresultsshowedthatmethanol/sorbitolcofeeding
weakenedthemethanolmetabolismincelsandrelievedaccumulationofthetoxicintermediatemetabo
lites(formaldehyde,H2O2,etc.).Comparedwith30℃ puremethanolinductionstrategy(maximum
pIFNαantiviralactivity10×104IU/mL)and20℃ lowtemperaturepuremethanolinductionstrategy
(maximumpIFNαantiviralactivity14×106IU/mL),methanol/sorbitolcofeedingstrategyandsorbi
tolfeedingrateat0785g/(L·h)couldimprovepIFNαantiviralactivity,andthehighestpIFNαanti
viralactivityreached18×107IU/mLInaddition,thetargetedproteindegradationwasalsorelieved
whenusingthecofeedingstrategy.Withthecofeedingstrategy,theresistantabilityagainsthighmetha
nolconcentrationofthefermentationprocesswasenhancedforensuringthefermentationstability.Using
thecofeedingstrategy,energyutilizationratewasimprovedandNADHreutilizationeficiencywasraised
from29% to84%
Keywords:sorbitol;Pichiapastoris;porcineinterferonα
  
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CH3OH(MeOH)+

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r(2)O2 +r
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O2 =OUR(T,t)-r
(1)
O2 (5)
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(2)
O2 (6)
r(C2)NADH(T,t)=2r
(3)
O2 (7)
r(F1)NADH(T,t)=2r
(1)
CO2 =
2(CER(T,t)-rCO2)
(2) (8)
r(F2)NADH(T,t)=
11
6r
(2)
CO2 =11rsor(T,t) (9)
η=
r(C)NADH(T,t)
r(F)NADH(T,t)
r(C1)NADH +r
(C2)
NADH
r(F1)NADH +r
(F2)
NADH

2OUR(T,t)-rMeOH(T,t)
2CER(T,t)-rsor(T,t)
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Table1 Cofactorregenerationeficiencyandotherrelatedparametersondiferentinductionstrategies
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(g·L-1·h-1)
§¨
ç)
t/h
rMeOH(t)/
(mmol·L-1·h-1)
rsor(t)/
(mmol·L-1·h-1)
r(C)NADH(t)/
(mmol·L-1·h-1)
r(F1)NADH(t)/
(mmol·L-1·h-1)
r(F2)NADH(t)/
(mmol·L-1·h-1)
η
20 894 112 565 020
0 40 928 86 575 015
60 860 141 584 024
20 780 27 340 280 297 059
0390 40 586 31 530 300 335 083
60 540 34 575 255 374 090
20 1926 48 657 706 528 053
0785 40 976 55 1230 660 605 097
60 900 55 1098 505 605 098
20 1520 90 1080 328 982 082
1437 40 785 103 1520 423 1133 097
60 780 100 1323 300 1101 094
  




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NADH
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r(F1)NADH(t)féy5,>öJ0E¢4.^&b7pu
ŽewÛx0X¢’P/9@A
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kpuµ¶‚æ7Qç
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A
NADH
Y*Ò¶
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18×107IU/mL
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29%~84%,
+
æ
pIFNαŒïەv¯3 18×107IU/mL,$
20℃
mª¢4‘€§¨=+æ•v7
10
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26
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