辅酶Q10高产菌Rhizobium radiobacter的选育 及发酵条件优

45Vol.4No.5200410TheChineseJournalofProcessEngineeringOct.2004Q10Rhizobiumradiobacter1,221,2(1.2.214036)(Rhizobiumradiobacter)WSH2601DWSH−F06.pHWSH−F06Q10.WSH−F06Q1034mg/L2.4mg/g16%.Q10DTQ929A1009−606X(2004)05−0451−061Q10(CoQ10)2,3−−5−−1,4−10[1]..Q10..[2−4].Q10[5].Q10.Q10[1].Q10.Q10Q10Q10[6]Q102.50mg/g.Q10Q10[7].(Rhizobiumradiobacter)WSH2601Q10DWSH−F06.22.1(Rhizobiumradiobacter)WSH2601.2003−09−252003−12−18(1976−)E-mail:[e-mail];HPLC.45242.2(g/L)2010105pH7.2,121oC15min.20g/L.(g/L)5(NH4)2SO421KH2PO46,K2HPO4420pH7.2121oC15min.(g/L)30101010,KH2PO40.4,Na2HPO4⋅12H2O1.5,MgSO4⋅7H2O0.3;pH7.2,121oC15min.2.32.3.130oC24h30oC200r/min24h.250mL50mL.2.3.25%(ϕ)30oC200r/min50h().3.2.4Q1015mL3000r/min20min.15mL0.5h3000r/min10minHPLC[8].2.54mL10000r/min10min260oC48h.2.624h5mL10000r/min5min0.1mol/L(pH6.5)2107/mL.10mL(UV)5s(NTG)NTG300mg/L.NTG20min0.1mol/LpH6.53.12h3D30oC4∼5dD.33.1D(ActinomycinDActD)12DNA[9].ActDActD.Q10ActDQ10.R.radiobacterWSH260170D.75Q10Rhizobiumradiobacter453Q1010%.731DR.radiobacter(ActDr)WSH−F06Q1011±0.2mg/L(9.5±0.2mg/L)16%.5Q1010.8mg/L.3.2R.radiobacterWSH-F06Q103.2.1Q1030g/LR.radiobacterWSH−F06Q101.Q10Q1011.1mg/L.20304050891011CoQ10(mg/L)Glucoseconcentration(g/L)8.08.59.09.510.010.5DCW(g/L)024681012CarbonsourceCornstarchMolassesSolublestarchMaltoseSucroseGlucoseDextrinCoQ10(mg/L)1Q102Q10Fig.1EffectofcarbonsourcesonCoQ10productionFig.2Effectofglucoseconc.onCoQ10production3.2.2Q10Q102.30g/LQ1010.2g/L10.8mg/LQ10.3.2.3Q10Q10Q10.3Q10.30g/L600246810121416Peptone(NH4)2SO4YeastextractBeancakeKNO3NH4NO3UreaCornsteepliquorCoQ10(mg/L)Nitrogensources809010011020:2020:4020:6030:2030:4030:6030:8030:0CoQ10relative(%)Glucose/molassessconcentrationratio3Q104Q10Fig.3EffectofglucoseandmolassesonCoQ10productionFig.4EffectofnitrogensourcesonCoQ10production4544g/LQ1030g/L13%Q10.3.3R.radiobacterWSH-F06Q103.3.1Q1030g/L60g/L160mmol/LCoQ104.R.radiobacter(ActDr)WSH−F06()[(NH4)2SO4NH4NO3]Q10Q1013.8mg/L.3.3.2R.radiobacter(ActDr)WSH−F06(NH4)2SO4Q10.Q10(NH4)2SO4Q10.130g/L60g/L.1Table1Arrangementoforthogonaldesign(g/L)Level(NH4)2SO4PeptoneYeastextractCornsteepliquor12357105101551015304050Q10(NH4)2SO4(NH4)2SO47g/L5g/L5g/L30g/L.30g/L60g/L50hQ1024.1mg/L(4)75%.3.4R.radiobacterWSH-F06Q103.4.1pHQ10pHQ105.pHQ10.pH6.8~7.5Q101.9mg/gQ10pH7.2.6.06.57.07.50481216202428CoQ10(mg/L),DCW(g/L)InitialpH0.00.51.01.52.02.5CoQ10DCWCoQ10(mg/g)3040506070809010010152025CoQ10(mg/L),DCW(g/L)Mediumvolume(mL)1.41.51.61.71.81.92.0DCWCoQ10CoQ10(mg/g)5pHQ106Q10Fig.5EffectofinitialpHonCoQ10fermentationFig.6EffectofmediumvolumeonCoQ10fermentation5Q10Rhizobiumradiobacter4553.4.2Q10.500mLQ10.650mLQ10R.radiobacter(ActDr)WSH−F06Q10[10].3.4.3Q10pH7.23%,5%,7%,9%11%(ϕ)Q10().7%∼9%Q107%.3.5R.radiobacterWSH-F06Q10Q1030g/L60g/L7g/L(NH4)2SO45g/L5g/L30g/LpH7.250mL7%,30oC200r/minR.radiobacterWSH−F06Q107.R.radiobacterWSH−F06Q10.Q1083hQ10Q1034.0mg/L2.4mg/g.50h11.0mg/L().83hQ10210%0.22mg/(L⋅h)0.41mg/(L⋅h).02040608010005101520253035DCWCoQ10CoQ10(mg/L),DCW(g/L)Time(h)0.00.51.01.52.02.5CoQ10(mg/g)7Q10Fig.7TimecoursesofCoQ10fermentationR.radiobacterWSH2601DR.radiobacter(ActDr)WSH−F0683hQ1029.334.0mg/LQ102.12.4mg/gQ1016%UVNTGD.Q10.Yoshida[6]AgrobacteriumtumefaciensAU−55Q10Q10110mg/L2.5mg/g0.76mg/(L⋅h).DR.radiobacter(ActDr)WSH−F06Q10.A.tumefaciensAU−55Q10R.radiobacter(ActDr)WSH−F06.4(1)RhizobiumradiobacterWSH2601DQ10WSH−F06.Q10Q10456434mg/L3.6.WSH−F06Q102.4mg/gYoshida[6]A.tumefaciensAU−55.(2)DQ10Q10.[1].[M].,2002.130−136.[2].Q10[J].,2001,14(2):85−88.[3]ErnsterL,DallnerG.Biochemical,PhysiologicalandMedicalAspectsofUbiquinoneFunction[J].Biochim.Biophys.Acta,1995,1271:195−204.[4]KawamukaiM.Biosynthesis,BioproductionandNovelRolesofUbiquinone[J].J.Biosci.Bioeng.,2002,94:511−517.[5]GingoldEB,KopsidasG,LinnaneAW.CoenzymeQ10andItsPutativeRoleintheAgeingProcess[J].Protoplasma,2002,214:24−32.[6]YoshidaH,KotaniY,OchiaiK.ProductionofUbiquinone-10UsingBacteria[J].J.Gen.Appl.Microbiol.,1998,44:19−26.[7].Q10[J].,2001,27(7):49−53.[8].Q10[J].,2002,21(4):420−423.[9].DDNA[J].,2002,38(3):87−89.[10].WSH260lQ10[J].,2003,22(1):65−69.ScreeningofHighProductivityCoQ10StrainandOptimizationofFermentationConditionsforCoQ10ProductionwithRhizobiumradiobacterPANChun-mei1,2,DUGuo-cheng2,CHENJian1,2(1.KeyLab.IndustrialBiotechnol.,MinistryofEducation,SouthernYangtzeUniv.,Wuxi,Jiangsu214036,China;2.SchoolofBiotechnology,SouthernYangtzeUniversity,Wuxi,Jiangsu214036,China)Abstract:RhizobiumradiobacteWSH2601wasmutagenizedbyusingUV-radiationandNTGmutationconsecutively.Anactinomycin-Dresistantmutant,R.radiobacteWSH-F06,wasisolatedaftertworunsofscreening.TheCoQ10yieldofmutantWSH-F06increasedby16%comparedtothatoftheparentstrain.TheeffectsofnutritionalandenvironmentalconditionsontheproductionofCoQ10byR.radiobacteWSH-F06wereinvestigatedinshakingflasks.Acomplexcarbonsourceconsistingofglucose(30g/L)andmolasses(60g/L)wasshowntobesuitable.Inaddition,R.radiobacterWSH-F06couldassimilateavarietyofnitrogensources,includingorganicandinorganicnitrogencompounds.Acompoundnitrogensourceconsistingof(NH4)2SO4(7g/L),peptone(5g/L),yeastextract(5