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EffectofproteinaseApropeptidedeletiononitsenzymeactivityinSaccharomycescerevisiaeDeguangWu,YefuChen,JunLu,YananQi,CuiyingZhangandDongguangXiao*KeyLaboratoryofIndustrialFermentationMicrobiology,MinistryofEducation,TianjinIndustrialMicrobiologyKeyLaboratory,CollegeofBiotechnology,TianjinUniversityofScienceandTechnology,Tianjin300457,[e-mail]lasmidYCP-54A,whichcontainstheincompletePEP4lackingtheprosequence(pep4-23△76).Fig.1ConstructionofexpressionplasmidYCP-54AFig.3PCRverificationofyeasttransformantsThePrAactivityofhoststrainW303-K22andthreeyeasttransformants(WYCP,WYCP-EPandWYCP-54A)cultivatedinwortmediumweremeasured.TheintracellularandextracellularPrAactivityoftwomutantstrains(W54AandW54A-K)andtheparentalstrain(W303-1A)weremeasuredinthesamewortmedium.(a)DeletionofthepropeptideofPrAcouldsignificantlydecreasethePrAactivityandthedeclinetrendwasnotinfluencedbythepresenceoftheKanMXgene.(b)Therewerenosignificantdifferencesinthefermentationcharacteristicsbetweenmutantstrainsandtheparentalstrain.SaccharomycescerevisiaeproteinaseA(PrA,EC3.4.23.25)candirectlydegradefoamproteinsinbeer,sotheextracellularmaturePrAisthemainreasonthatbeerfoamstabilityisdecrease.However,theproPrAwouldbecompletelydegradedintheERincaseoflackingthepropeptide.Therefore,thefollowingstudyworksareperformed:(a)TodeletethepropeptideofPrA;(b)TodeterminethePrAactivityofmutantwithoutpropeptide.IntroductionExperimentalmethodFig.2SchematicdiagramofrecombinantplasmidpUC-ABKResultsanddiscussion3PrAactivityofmutantstrainsConclusionsReferences[1]Teichert,U.,Mechler,B.andMuller,H.:Lysosomal(vacuolar)proteasesofyeastareessentialcatalystsforporteindegradation，differentitation，andcellsurvival.JournalofBiologicalChemistry264,16037-16045(1989).[2]Parr,C.L.,Keates,R.A.B.,Bryksa,B.C.,Ogawa,M.,andYada,R.Y.:ThestructureandfunctionofSaccharomycescerevisiaeproteinaseA.Yeast24,467-480(2007).[3]Kondo,H.,Shibano,Y.,Fukui,N.,andNakatani,K.:DevelopmentofanovelandsensitivemethodformeasurementofproteinaseAinbeer.OxfordUniversityPress,669-676(1995).ConstructarecombinantplasmidpUC-ABK,whichwasusedtodeletetheprosequenceofPEP4geneviahomologousrecombination.Fig.4PCRverificationoftheyeastrecombinantstrains2ConstructionofPrApropeptidedeletionmutants(Fig.4)StrainsPrAconstructinvectorIntracellularPrAactivity1（×10-5U/mg）W303-K22—0.104±0.002WYCPnone0.324±0.005WYCP-EPPrA5.128±0.022WYCPPrA△pro0.639±0.003Tab.2PrAactivityofhoststrainanddifferentyeasttransformants1ThePrAactivityin1mgintracellulartotalproteinswasdefinedastheintracellularPrAactivity(UPrAactivity/mg).StrainsGenotypeExtracellularPrA1activity（×10-5U/mL）IntracellularPrAactivity（×10-5U/mg）W303-1APEP412.208±0.43412.308±0.272W54Apep4-23△76::KanMX0.716±0.0060.347±0.005W54A-Kpep4-23△76::loxP0.102±0.0050.431±0.004Tab.3TheintracellularandextracellularPrAactivitiesofmutantstrainsandtheparentalstrain1ThePrAactivityin1mLfermentationliquorwasdefinedastheextracellularPrAactivity(UPrAactivity/mL).1ConstructionofyeasttransformantsWYCP-EPandWYCP-54A(Fig.3)4FermentationcharacteristicsStrainsCO2reduction(g)Ethanol(%vol)Residualsugar(g/L)W303-1A1.18±0.020.8±0.0642±0.6W54A1.15±0.010.7±0.0643±1.0W54A-K1.14±0.020.7±0.0642±0.6Tab.4CO2reduction,ethanolandresidualsugarofmutantstrainsandtheparentalstrainInordertoinvestigatetheimpactoftheprosequencedeletiononthefermentationperformance,CO2reduction,ethanolandresidualsugarofmutantstrainsweremeasuredaftertheprimarybeerfermentation.