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(+)-tartaricacidproductionbyCorynebateriumsp.infed-batchcultureWenpengLi，JinlunXieKeyLaboratoryofIndustrialMicrobiology&FermentationTechnologyofYunnan,YunnanUniversity,Kunming650091,P.R.ChinaE-mail:[e-mail]roductionofcis-epoxysuccinatehydrolasefromCorynebateriumsp.YNUCC0211andbioconversionconditionsforL(+)-tartrateproductionwereoptimizedbymeansofstatisticalmethodsrespectively.ResultsrevealedthattheyieldsofL(+)-tartratewereimprovedbyfeedingfreshsynthesizedcis-expoxysuccinatesolutionwith12.0ofpHorwith3%ofhydrogenperoxidetotheoptimummediumafterbeinginoculatedfor32-48hours.Theoptimummediumcontains(g/litre):glucose15,cornsteepliquor3.0,yeastextraction0.3,K2HPO4.3H2O3.5,dipotassiumcis-epoxysuccinate7.0,NH4NO34.0,(NH4)2SO41.0,MgSO4.7H2O1.0,FeSO4.7H2O0.02,pH7.0-7.2.Keywords:OADs,Corynebaterium,L(+)-tartaricacid,cis-Expoxysuccinate,Bioconversion1IntroductionL(+)-tartratewasusedwidelyinfoodindustryasemulsifye,naturalacidifye,preserverandingredient.Itwasalsousedinpharmaceuticalindustryandotherindustrialfields.Bioconversionofcis-expoxysuccinateisoneofthemostimportantsourcesofL(+)-tartrate.Variousmicroorganismscanbeusedtoconvertcis-expoxysuccinatetoL(+)-tartrate.Theessentialprerequisitetotheprocesswasthatcis-epoxysuccinatehydrolasemustbeproducedbymicroorganismstrainsofgeneraAgrobacterium,Acinetobacter,RhizobiumPseudomonas(Kamatanietal.,1977),Nocardia(Miuraetal.,1977a),Achromobacter,Alcaligenes(Sato&Yanai,1976),andCorynebaterium(Zhang&Qian,2000).Cis-epoxysuccinateasthesubstrateofcis-epoxysuccinatehydrolasecanbesynthesizedbyepoxidingmaleicanhydridewithhydrogenperoxideinthepresenceoftungstateion.Beforebioconversion,thetungstateioncatalyst,DL–tartrate,maleateandhydrogenperoxideresiduemustberemovedfromthereactionsmixturesincetheyseverelyinhibitthegrowthofbacteriaandsuppresstheactivityofcis-epoxysuccinatehydrolase(Kamatanietal.,1977).Crystallizationofthestartingmaterialoranionexchangewasutilizedtogetoutofthetrouble.However,thisalsoincreasedtheoperatingcost.Severaldifferentstrategiestoenhancecis-epoxysuccinatehydrolaseactivitiesduringbioconversionhavebeensuccessfullyappliedbytheadditionofdetergents(Miuraetal,1977b)andvariousions(Mikováetal,1998).-1-(+)-tartratebyCorynebateriumsp.YNUCC0211,usingfreshsynthesizedsolutionofdipotassiumcis-expoxysuccinateassubstateinthepresentoftungstrateioncatalyst,DL-tartrate,maleateandhydrogenperoxideinhibitors.2MaterialsandMethods2.1StrainandcultivationCorynebateriumsp.YNUCC0211whichpreservedatKLIMFTY(KeyLaboratoryofIndustrialMicrobiology&FermentationTechnologyofYunnan,YunnanUniversity,P.R.China)wasgrownoninoculamedium(50ml)in250-mlErlenmeyerflaskscontaining(g/L)glucose,5;CornsteepLiquor,5;dipotassiumcis-expoxysuccinate,5,distilledwater1liter,thepHofthemediumwasadjustedto7.3with1MKOHpriortoautoclaving(121°C,15min),200rev/minshakingflaskfor18hat30°C.2.2Bioconversionofcis-expoxysuccinatetoL(+)-tartrateAliquotsof35mlofenzymeproductionmediumwereplacedin500mlconicalflasksandseededwithinoculumtoaninitialconcentrationofabout106bacteria/ml.Theexperimentswerecarriedoutinarotaryshaker(150rev/min)at30°Cfor48h,adding15mloffreshsynthesizedsolutionofdipotassiumcis-expoxysuccinate,continuetoincubatefor96h.For5000mlconicalflasks,350mlofenzymeproductionmediumwasusedand150mlofsubstratewasfed.2.3Preparationofcis-expoxysuccinateThisisamendedmethodforpreparationofdipotassiumcis-expoxysuccinatebasedonPayne&Williams’method(1959).Itwascarriedoutina5-liter,3-neck,roundbottomflaskwithstirrer,thermometer,anddroppingfunnel.Theflaskwasfilledwith636mlof2Mmaleicanhydridetowhich300mlof10MKOHwasadded.Theheatofneutralizationcausedariseintemperaturetoabove70°C.Add13.2gofNa2WO4and308mlofhydrogenperoxide(30%)tothereactionsolutionwhichwascooledtoabout70°C.Maintainthetemperatureat60°CandmaintainpHat4.5-5.0for2-3h.Attheendofreaction,themixturewascooledto40°C,andpHwasadjustedto7.5.Thefinalvolumeofthereactionsolutionwasabout1600mlatroomtemperature.2.4AnalyticalMethodsGrowthwasmonitoredbymeasuringtheopticaldensityofthecultureat660nminaglasschamber10mmthickusingaS23ASpectrophotometer.ThevaluesofpHweremeasuredbyPHS-3CPrecisepHMeter(ShanghaiPrecision&ScientificInstrumentCo.,Ltd.).A‘Petit-Salumbéni’haemocytometerwasusedfordirectcellcounting.Thenumberofcellspersquareofgridwascountedusingamicroscope(X400magnification).L(+)-tartratewasdeterminedaccordingtoAmmoniummetavanadatemethod(Huang&Qian,1989).2.5Orthogonalarraydesign-2-(OADs)wereusedforoptimizationofmediumcomponents.ThematrixofL64(421)orthogonalarraywasemployedtodeterminethekeyfactors,whichinfluencetheyieldsofL(+)-tartrateandbacteriacellconcentration.20componentswereselected(listedinTable1).Inthedesignedmatrix,eachrowrepresentsatrial(64media),andeachcolumnrepr