扩展青霉脂肪酶“盖子”结构域突变对其活性影响的分析

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1、福建师范大学林瑞凤硕士论文IIAbstractAbstractInordertobringfacilityformoleculardesigningandalteratingPenicilliumexpansumlipasegene，weamplifythematurepeptideofPenicilliumexpansumlipase，andinsertintopPIC9Kvectortoconstructrecombinantexpressionplasmid．ByaligningPenicilliumexp

2、ansumlipasewithferuloylesterase，wemutagenesisthesixty-sixth，^eihty-first，ninty-fourthaminoacids，the”lidt．，therightsideofhingeregionof-．IPenicilliumexpansumlipase，SOaStostudytheireffectonlipaseactivity．Firstly,bymeansofthepreviousconstructedrecombinantexpressi

3、onplasmidtemplate，wesite—directedmutagenesisthreeaminoacidsinbothsidesofthehingeregionof”lidf’(T66G,T81P,K94E)bycircuLarplasmidsstepPCR，andabtainthreerecombinantexpressionplasmidscontainingmutantaminoacids．AfterexpressedinHchiapastorisGSll5strain，wepurifiythe

4、expressionproductbyNi—column,thenmeasuretheirhydrolysisactivityon4·nitrophenyldecanoatelaurate．Theresultsshowthat：PELT66Grecombinantlipaserelativeactivity(5．09U／mg)issimilartothewild-typePELlipase，whilePELT81PandPELK94Erecombinantlipaserelativeactivityhavebee

5、nimproved2．95timesand2．57times，respectivelyupto19．08966U／mgand17．2391U／mg·Secondly,weexchangethe”lid”domainbythewayofintroducingrestrictionsitesthenbackmutationbycircuLarplasmidstepPCRandconstructrecombinantexpressionplasmidwithpPIC9Kvector．However,afterexpre

6、ssedinPichiapastorisGSll5strain，itdoesnotshowanylipaseactivityonoliveoil-rhodamineBagarplate．WhilethepurifiedlipasebyNi．columncanbeseenonestrapabout28KDabySDS-PAGEelectrophoresis．Atlastwealsoreplacetherighthingeregionoflipase”lid”byoverlapextensionPCR．Asthe“l

7、id”mutantlipase，theconcentratedfermentationbrothofthisrecombinant^钰jlipasedonotshowanylipaseactivity．■Inaword，Penicilliumexpansumlipase”lid-’domainplayveryimportantroleinitsactivity,especiallythemutationson”lid”，therighthingeregion,T81P,K94E，whichwillresuLtin

8、thelossorwhole-foldimprovementoflipaseactivityKeywords：Penicilliumexpansumlipase，circularplasmidstepPCR，overlapextensionPCR，liddomainIIIr福建师范大学林瑞凤硕士论文，"'a队J-1IV中文文摘中文文摘扩展青霉脂肪酶是一类中温碱性脂肪酶，能