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1、中國農(nóng)業(yè)科學(xué)院博士學(xué)位論文四個(gè)腸桿菌植酸酶基因的克隆、表達(dá)及性質(zhì)研究姓名:谷維娜申請學(xué)位級別:博士專業(yè):生物化學(xué)與分子生物學(xué)指導(dǎo)教師:姚斌20070601AbstractPhytases(myo-inositolhexakisphosphatephosphohydrotases)hydrolysephytatetolowermyo-inositolphospharesandinorganicphosphate.Asatypeofanimalfeedadditives,additionofphytaseinanimalf
2、eedallowsmonogastricanimaltoutilizethephytinphOSphomsinfeedandreducestheenvironmentalphosphomspoHufiomPhyta∞has麗dccoralapplicationsinindustryproccs$essuchas.foodstuffandpharmaceuticalindustry.Microorganismisanimportantsourceofphytase.Screeningofnewandgoodnaturep
3、hytasesfromthemicroorganismisanimportantresearchdirection.Apairofdegeneratepfinl∞(FIandRI)wasdesignedthroughidentityanalysisofpublishedanthlOacidsequencesofphytase.UsingpriI礎(chǔ)fs,fragmentsofthreephytasegeneswmobtainedbyPCRmethodfromgenomeofstrainsHafniaalveiB125,D
4、ickeyadadamiiB187andDickeyaparadisiacaB188.耵”full—lengthofthreegeneswereobtainedbymethodofTAILrPCRandnameriasB125appA,B187appAandB188appA.AnovelphytasegenerevealedbygenomeanalysisofYersiniapestisWaSobtainedbyPCR.namod硒YlappA.ThehighestidentityofnucleicacidWas75%
5、amongthesefourgenes∞187appAandB188appA),thelowestidentityofnucleicacidWas48%(B188appAandB125appA,B188appAandYlappA).IncomparisonwiththesequencesofpreviouslyisolatedphytasesbyBLASTsearch,thededucedaminoacidsequenceofBl25appAshowed95%identitytophytasefromObesumbac
6、teriumproteus.thededucedaminoacidsequenceofBl87appAandBl88appAshowed53%and52%identitiestoaphytasefromKlebsiellapneumoniae,reSpectively.TheresultprovedthatBl87appAandBl88appAaretwonewphytasegenes.11lededucedaminoacidsequenceofYlappAshowed81%identitytophytasefromY
7、ersiniainrermedia.ThegeneB125appA,8187appAandB188appAwereexpressedinEscherichiacoliBL21(DE3).ThegeneylappAwasexpressedinPichiapastoris.Recombinantproteinsw啪purifiedandtheirenzymaticpropertieswefedetermined.TheoptimumpHfortheB125r-APPAWas4.5andtheoptimumtemperatu
8、reWas60℃.TherelativephytaseactivityWasabove80%aftertreatedinhlffersofpH2.0-pH10.0.硼1especificactivity0fBl25r-APPAWas356.7l-O/rag.11峙‰andK峨valuesforBl25r-APPAw粥0.49lnl