四个肠杆菌植酸酶基因的克隆、表达及性质研究

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1、中国农业科学院博士学位论文四个肠杆菌植酸酶基因的克隆、表达及性质研究姓名：谷维娜申请学位级别：博士专业：生物化学与分子生物学指导教师：姚斌20070601AbstractPhytases(myo-inositolhexakisphosphatephosphohydrotases)hydrolysephytatetolowermyo-inositolphospharesandinorganicphosphate．Asatypeofanimalfeedadditives，additionofphytaseinanimalf

2、eedallowsmonogastricanimaltoutilizethephytinphOSphomsinfeedandreducestheenvironmentalphosphomspoHufiomPhyta∞has丽dccoralapplicationsinindustryproccs$essuchas．foodstuffandpharmaceuticalindustry．Microorganismisanimportantsourceofphytase．Screeningofnewandgoodnaturep

3、hytasesfromthemicroorganismisanimportantresearchdirection．Apairofdegeneratepfinl∞(FIandRI)wasdesignedthroughidentityanalysisofpublishedanthlOacidsequencesofphytase．UsingpriI础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