耐熱堿性磷酸酶的亞克隆和耐熱機制的研究.pdf

1、復旦大學碩士學位論文耐熱堿性磷酸酶的亞克隆和耐熱機制的研究姓名：白曉陽申請學位級別：碩士專業(yè)：遺傳學指導教師：毛裕民2001.5.18FudanUniversityGraduateThesissubcIoningandThermostabilityMechanismStudyonThermostableAlkalinePhosphataseAbstractOurlabhasscreenedandclonedthegenecodingfo

2、rthermostablealkalinephosphatase(TAP)fromthermophileThermussp3041ThispaperconcentratesOOthefollowingworkwiththermostablealkalinephosphatase：24Withmolecularcloningmethod，aseriesofmutantswereobtainedwith24，25，26and27aminoa

3、cidsdeletedinFD—TAPN—terminalsignalpeptidesequenceamongwhichmutantsTAPND25，1APND26and1APND27getenhancedexpressionlevelinEcoliThemutantproteinTAPND27wasinducedandpurifiedthroughaseriesofproceduresThepurificationefficiency

4、wasabove90％withaspecificactivity9736U／mgKmvalueiS1281O～mol／m1anditiShighlythermostablewithaTmvalue91℃WefonndthationandsolventpHinfluenceTAPND27activityandthermostabilityAftertreatmentwithEDTAtheiondeprivedTAPND27complete

5、ly10NeSitsthermostabilitywithgreatchangeinitssecondarystructureThatsuggeststhatTAPNl3127iSanionenzymeTheremaybeion～bindingsitesinthestructureTAPND27iSthermostableunderawiderangeofpHfrom65to80Whileitsthermostabilitydecrea

6、sesdramaticallywhenpHiSbelow65WefoundthatinorganicphosphateCangreatlyimproveTAPND27thermostabilityInthepresenceof10mmol／LPiitsTmwasenhancedfrom91℃to97℃EnzymatickineticexperimentshowsthatPiiSareversiblecompetitiveinhibito

7、rofTAPND27withKivalue099104mol／LnleresultsindicateaPibindingsiteintheenzymeCDspectrumscanninghasmeasuredTAPND27secondarystructurechangesinphosphatebuffer(pH8O)duringheatdenaturation，whichiSquitedifferentfromthatofTAPND27

8、inTris—HClbufrer(pH8O)，indicatingthatproteinthermostabilitycloselyrelatestoitssecondarystructureTheTAPND27crystallizationconditionswerescreenedusinghangingdropvapordiflusionmethodCrystalsusedforX—raydiffractiondatacollec

9、tionweregrownagainstreservoirscontaining40mmol／LMgCl2，100mmol／LHepesBufferfpH75，298K)，285％(W，V)polyethyleneglyc01Andtheygrewtomaximumdimensionsof080501mmXraydifiractiondataarecollectedto254AItwasfoundthatthecrystalsbelon