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17420017ChineseJournalofBiotechnologyVol.17No.4July2001:2000212225,:2001204204:(39870204)3Tel:[phone];Fax:[phone];E2mail:[e-mail]11312AnthonyEGCass31(430071)2(110015)3(BiochemistryDepartment,ImperialCollegeofScience,Technology&Medicine,London,SW72AY,UK)(GOD)2pPIC9,Pichiapasto2risGS115,GOD2FactorAOX1,GOD,34d,GOD3040uPmLSDS2PAGEGOD,60%70%,QSepharoseTMFastFlowGOD426163uPmg,GOD116,GODKmkcat38125mmolPL3492166s-1,GOD,GOD,Pichiapastoris,,,,Q789A100023061(2001)0420400206(Glucoseoxidase,EC1.1.3.4,GOD),H2O2,,,GOD,GOD,[1],GOD,GOD,FrederickKR[2]WhittingtonH[3]GOD,GODWittS[1]GOD,Pichiapastoris8090[4],PichiapastorisGS115,GOD,GOD,GODGOD11.11.1.1:PichiapastorisGS115pPIC9T9029Promega(ATCC),E.coliDH5pPIC9T1.1.2:LBYPDMDMMRD:LB(L-1):10g,5g,10g;YPD(L-1):20g,10g,20g;MD(L-1):(YNB)117g,5g,20g,400g;MM(L-1):YNB117g,5g,12mL,400g,10g,pH516;©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.RD(L-1):YNB117g,5g,20g,400g,30g,18414g1.1.3:DNAT4DNATaKaRaSangon;GOD(BSA)Sigma;Bio2Rad1.21.2.1:[5]262d,,10mL10mL(100mmolPLTris2HCl,40mmolPLEDTA,pH815)110mL,2mL10%SDS6mL,,501h,10min1,6mL3molPL,15min46000rPmin15min,,DNA1.2.2:[6]GS115YPDOD600=012,10mL110mLSED(1molPL,25mmolPLEDTA,50mmolPLDTT)110mL1molPL2,10mLSPE(1molPL,10mmolPLEDTA,011molPL,pH715),512mgPmL20L,3080%90%10mL1molPL210mLCaS(10mmolPLCaCl2,1molPL)1,016mLCaS100L4gDNA(Stu)20gDNA,20min1mL20%3350(10mmolPLCaCl2,10mmolPLTris2HCl,pH714),15min,150LSOS(1molPL,10mmolPLCaCl2,0133YPD),30min,1molPL1mL,200L5510mLRD,MD,,8mL(011%SDS,10mmolPLTris2HCl,5mmolPLEDTA,50mmolPLNaCl,pH714)KRNA100gPmL,372h7010min,,018mL5molPL,30min,,DNA1.2.3GOD:[23],SigmaGODpH516011molPL2mgPmL2mgPmL100uPmL5mL1mL2mL100L,3010min,20LGOD,,303min,2mL5molPL,OD5251.2.4SDS2PAGE:10%,R2250,Bio2RadGelDoc20001.2.5GOD:QSepharoseTWFastFlow,pH610NaCl(0011molPL)1.2.6:Bio2Rad,BSA1.2.7:[7]3L20%BSA,10L2mgPmLGOD2L215%2cmTeflon,1hTeflon,,22.1GOD[2],GOD,SnaBHindNot,52TACGTAAGCAATG2GCATTGAAGCCAGC2352AAGCTTCATGGAAG2CATAATCTTCCAAG25SnaBHind117kbGODPCR,T,T4DNADH5,,PCRGODTT2GODTNot,T2GODGODSnaB2Not,pPIC9,DH5,GODpPICGOD1(1)1044:©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.2.2GOD[6]pPIC2GOD1GS115,RD3040P4,PCR,:52TACGTAAGCAATGGCATTGAAGCCAGC2352AAGCTTCATGGAAGCATAATCTTCCAAG23GOD,2,3,pPICGOD1GS1151pPICGOD1Fig.1RestrictionpatternofrecombinantpPICGOD11,6.DL15000DNAmarker;2.pPIC9digestedwithBgl;3.pPICGOD1digestedwithBgl;4.pPICGOD1digestedwithSal;5.pPIC9digestedwithSal2DNAPCRFig.2PCRassayofrecombinantyeastgenomicDNA1,6.DL2000DNAmarker;24.Positivetransformants;5.Negativetransformant,pHGOD,3,4pH(pH48),pHGODpH56,GOD,(pH=8),GOD,GOD,GODpH510615,[8]4,GOD,,GOD,112%MMGOD,112%,GOD,MM24h,GOD,GOD,96hGOD,,,112%pH516MM4dGOD3pHGODFig.3EffectofinductionpHonGODyield424hGODFig.4EffectofmethanolcontentonGODyield,MDpH=516MM4d,,,,GS1152Factor,,9517%GOD,120417©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.1GS115GODTable1ActivityofGODproducedbyGS115PichiapastorisGS115ActivityP(uPmL)ExtracellularPelletTotal341481155361032.3GOD5,,GODGS115,60%70%(),01002molPLpH610,QSepharoseTMFastFlowpH6100101molPL,,pH6100011molPLNaClGOD,SDS2PAGEGOD6GODQSepharoseTMFastFlow,SDS2PAGEGODGODGOD[2][9]5SDS2PAGEFig.5SDS2PAGEofcrudeproteinextract1.GS115carryingpPICGOD1;2.CommercialGOD;3.GS115carryingpPIC9;4.HMWProteinmarker2.4GODGOD,30pH516,,GODGOD10121416182025303540506080100mmolPL,,2GOD,GOD,426163uPmg,(266182uPmg)116GODGOD,Km38125mmolPL33144mmolPL,kcatkcatPKm,GODkcatkcatPKm3492166s-191131(s-1mmol-1L),2303156s-168189(s-1mmol-1L),GOD6GODSDS2PAGEFig.6SDS2PAGEofpurifiedGODfromyeast1.HMWproteinmarker;2.PurifiedrecombinantGOD;3.CommercialGOD2GODGODTable2KineticparametersofGlucoseoxidaseSourceofGODSpecificactivityP(uPmgprotein)KmP(mmolPL)kcatP(s-1)(kcatPKm)P(s-1mmol-1L)A.niger26618233144230315668189P.pastoris426163381253492166911312.5GOD15uPmL(pH516)GODGOD1h,,77,GODGOD,440,40,7GODGODFig.7ComparisonofGODtemperaturestability3044:©1995-2005TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.,70,0GODGOD,pH(pH8,pH8pH8Tris2HCl)GODGOD15uPmL,3012h,GOD(8)GODGODpH47,pH47,8GODGODFig.8ComparisonofGODpHstability2.6GODGOD,GOD,(EFIA)[7]GODGOD(9)210mLPmin,25L,GOD050mmolPL,GOD035mmolPL,GODGOD9GODGODFig.9ComparisonofGODelectroderesponses3,GODGOD,[1012]GODPichiapasto2risGS115,GODGOD:a.MD130gPL;b.AOX1,,GOD,60%70%;c.2Factor,,95%GOD,34d,GOD3040uPmL;d.,,,QSepharoseTMFastFlowGOD,pH,GODGODKm,GODkcatkcatPKm,GOD,GOD,,GODGOD,,REFERENCES()[1]WittS,SinghM,KaliszHM.StructuralandkineticpropertiesofnonglycosylatedrecombinantPenicilliumamagasakienseglucoseoxi2daseexpressedinEscherichiacoli.AppliedandEnvironmentalMicro2biology,1998,64(4):14051411[2]FrederickKR,TungJetal.GlucoseoxidasefromAspergillusniger.JBiolChem,1990,265:37933802[3]WhittingtonH,Kerry2WilliamsSetal.ExpressionoftheAspergillusnigerglucoseoxidasegeneinA.nidulansandSaccharomycescerevi2siae.CurrGenet,1991,18:531536[4]CreggJM,VedickTS,RaschkeWC.Recentadvancesintheexpres2sionofforeigngenesinPichiapastoris,BIOPTECHNOLOGY,1993,11:905910[5]ZHUH(),QUF(),ZHULH().ExactingfungiDNAformolecularbiologyanalysiswithbenzylchloride.Ac