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《擴(kuò)展青霉脂肪酶“蓋子”結(jié)構(gòu)域突變對(duì)其活性影響的分析》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在行業(yè)資料-天天文庫(kù)。
1、福建師范大學(xué)林瑞鳳碩士論文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(fā)
7、id”mutantlipase，theconcentratedfermentationbrothofthisrecombinant^鈺jlipasedonotshowanylipaseactivity．■Inaword，Penicilliumexpansumlipase”lid-’domainplayveryimportantroleinitsactivity,especiallythemutationson”lid”，therighthingeregion,T81P,K94E，whichwillresuLtin
8、thelossorwhole-foldimprovementoflipaseactivityKeywords：Penicilliumexpansumlipase，circularplasmidstepPCR，overlapextensionPCR，liddomainIIIr福建師范大學(xué)林瑞鳳碩士論文，"'a隊(duì)J-1IV中文文摘中文文摘擴(kuò)展青霉脂肪酶是一類(lèi)中溫堿性脂肪酶，能
