29355.粘質(zhì)沙雷氏菌脂肪酶包涵體變復(fù)性及活性包涵體研究

1、華東理工大學(xué)碩士學(xué)位論文粘質(zhì)沙雷氏菌脂肪酶包涵體變復(fù)性及活性包涵體研究姓名：龐懷宇申請學(xué)位級別：碩士專業(yè)：生物化學(xué)與分子生物學(xué)指導(dǎo)教師：李素霞20091228第Ⅱ頁華東理工大學(xué)碩士學(xué)位論文TheStudyofRefoldingofSerratiamarcescensLipasefromInclusionBodyExpressedbyEscherichiacoliandActiveLipaseInclusionBodiesAbstract

2、ThelipasefromSerrat／amarcescenspossessanNterminala／phydrolasedomainTheextracellularlipasefromSelTatiamarcescensECUl010isallimportantbiocatalystforthestereospecificsynthesisofdiltiazernprecursorInthispapertherefoldingcond

3、itionoftheproductionofpET24alipAexpressedasinclusionbodiesinEscherichiacoilBL21(DE3)wasoptimzedInadditionthepreparationandsomepropertiesofthelipaseinclusionbodieswimactivitywerestudied8mol／Lureawasoptimizedasdenaturerege

4、ntAnorthogonalexperimentwasdesignedinordertoscreenthekeyfactorsforlipaserefoldingThepHandionstrengthofrefoldingbufferwerethekeyfactorsonlipaserefoldingTheeffectofconcentrationofproteinandca’inrefoldingbufferrefoldingtemp

5、eratureandrefoldingtimewerefurtheroptimized刀Ieoptimizeddenaturationandrefoldingconditionswereasfollowing：8mol／Lureaasdenatureregent，10mmol／LTris—HCI(pH7O)，lmmo／LCa，proteinconcentration0204mg／mL，30℃，refoldingfor12hOnestep

6、ofanionexchangechromatography(DEAEFF)WasperformedforcondensationandpurificationoftherefoldedlipaseWiththelinearconcentrationofNaCIfrom0—1000mmol／Linelutionbufferabout60％ofproteinand80％lipaseactivityrecoveryWasgotafterDEA

7、EpurificationTwoseparatedactiveUpasepeakswereobtained，thefirstelutedlipaseWasactivelipasepolymerandthesecondwaslipasemonomers，basedonresultofactivestainingfollowingnativePAGEanalysisThepolymerlipaseactivitywasincreasedin

8、50％(v／v)isopropyletherorpetroleumetherwhiletheactivityofmonomerlipasekeptunchangedThemechanismwasinvestigatedwithFTIRandactivestainingfollowingnativePAGEWiththepresenceofisopropyletherorpetroleumetherpolymerlipasecouldco

9、nver∞intomonomerlipase硼1elipaseWasactivatedbyCaandinhabitedbycuandZn2nerefoldingoflipaseWasimprovedbyisopropyletherandpetroleumethermorethan2foldSThecontentofpolymerlipaseWasreducedinthepresentofisopropytetherorpetroleum

10、etherinrefoldingbufferTherefoldedlipasecouldenantioseleetivelyhydrolyze(士)一MPGMTheproductionofpET24alipAexpressedasinclusionbodiesinEscherichiacoliBL21(DE3)andthelipaseactivityWasfoundinthelipaseinclusionbodiessuspension