編碼草菇中性內(nèi)切葡聚糖酶Ⅰ基因（egⅠ）的克隆與表達研究.pdf

1、江南大學碩士學位論文編碼草菇中性內(nèi)切葡聚糖酶Ⅰ基因（egⅠ）的克隆與表達研究姓名：王超凱申請學位級別：碩士專業(yè)：食品科學指導教師：李劍芳鄔敏辰20080601AbstractAbstractAnendoglucanaseI(EGI)cDNAincludingsignalpeptidewasisolatedfromⅣvolvaceaV23byRTPCRThefulllengthcDNAcontainedanORFof1167bpwhich

2、encodedasignalpeptideof23aminoacidsandamaturepeptideof366aminoacidsSimilarityanalysisshowedtheEGIbelongedtofamily5ofglycosylhydrolasesThecDNAencodingthematurepeptidewasclonedintoexpressionplasmidpET28aandexpressedinEsche

3、richiacoliBL21(DE3)byIPTGinduction，SDS—PAGEshowedthatEG1wasexpressedsuccessfullythemolecularweightofEGIWasabout395kDbutwithoutenzymaticactivityThematurepeptidecDNAWasclonedintothesecretiveexpressionvectorpPIC9KTherecombi

4、nantplasmidwasintroducedintoPichiapastor&GS115byelectroporationafterlinearizedbySalIAfterscreening，ahighyieldingrecombinantstrainPpastor西EGI1WasobtainedSDSPAGEshowedthemolecularweightofEGIWasabout42kD，itWashigherthanthet

5、heoreticmolecularweightwhichmaybeduetoglycosylationaftertranslationTheconditionsofexpressingEGIinPichiapastor括wereoptimized，TheoptimumconditionsforEGIaccumulationwereasfoll6ws：inducedtime96hby2O％methanol；theinitialinduce

6、dpH75；theconcentrationofglycerininBMGY40％；glycerinadditionquantityper24hinBMMY075％；theconcentrationofthesurfaceactiveagenttween20inBMMYO15％，theenzymeactivityWasupto4612U／mLattheseconditionsPartoftheenzymaticpropertiesofE

7、G1weredetermined，TheresultsrevealedthattheoptimaltemperatureandoptimalpHforenzymereactionwere55。Cand75；theenzymehadgoodstabilitywhenthetemperaturebellow55。Cwhiledeclinedquicklyabove55。C，butstillholded60％oftheenzymaticact