嗜冷杆菌低温脂肪酶分离纯化及表征

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1、万方数据嗜冷杆菌低温脂肪酶分离纯化及表征PurificationandCharacterizationofCold·adaptedLipasefromPSychrobactersp．AbstractLipase，whichcallcatalyzethehydrolysisoflongchaintriglycerides，iswidelyappliedinmedical／pharmaceuticalindustry。foodindustry．finechemicalsynthesisandenvironmentalgover

2、nance．Lipasecatalyzessynthesisofchiralorganiccompounds，aswellasesterification，hydrolysis，alcoholysisreactions，SOmoreandmoreattentionsarepaidtolipase．25strains，derivedfromtheChinesefourthArcticscientificexpeditionsamples，wereobtainedfromtheNationalMarineEnvironment

3、alMonitotingCentertoselectthecold．adaptedlipase．Characterizationofthecold·adaptedlipasewouldhelptoimproveitsfurtherapplicationandexploration．Overall25strainswereplatedontheVitotiablueandbromocresolpurplelipaseselectiveplates．ThentheselectedsinglecolonyWasdetermine

4、dbytheactivityoflipasedetectiontoobtainahigherlipaseproducingstrain．ThespeciesWasidentifiedby16SrDNAsequence．Afterthesequencealignmentandphylogenetictreeanalysis，thesimilaritybetweentheselectedstrainandPsychrobactersp．is99％．indicatingthestrainbelongstoPsychrobacte

5、rsp．、whichWasnameditPsvchrobactersp．ZY214．Withoptimizedcultureconditions．Psychrobactersp．ZY214fermentationbrothwasprecipitatedby40％．60％ammoniumsulfatefractionation．TheprecipitateWasdissolvedin50mMTris．HCl，whichpHiS8．0andthenloadedonaPhenylSepharoseFFcolumn．Afterpu

6、rification．thelipaseenzymeactivitypositivesinglecomponentonSDS—PAGEWasanalyzedbyLC—MS—MSandtheestimatedmolecularweightoftheenzymeiS38．1KDaandnameditLipZCl2．TheoptimumreactiontemperatureofLipZCl2Was40"C，theoptimumpHofLipZCl2Was8．0．TheactivityofLipZC12Wasincreasedby

7、additionofionCa2+andM92+．LipZC12hadastrongtolerancetoorganicsolventsandexhibitedgoodstability．After3h’Sincubationwith50％DMSO．theenzymeactivityremained95％．SubstratescreeningofLipZC12demonstratedthatoptimumsubstratecarbonchainlengthofLipZC12WasC12．KineticstudiedofLi

8、pZCl2showedthatitsKmvalueiS13．1mM，andkatvalueiS1．20x103s．1．T11econservedsequencesoflipaseactivitycenter(ACR)(Ser—Asp—His)andoxyanionhole(OXF)weredesigne