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1、天然产物研究与开发NatProdResDev2010,22:268273文章编号:10016880(2010)02026806冬瓜蛋白酶的分离纯化及其部分性质研究*孔令营,杜桂彩,郭道森,李荣贵青岛大学生物系,青岛266071摘要:冬瓜的果肉中发现了丰富的蛋白水解酶。用硫酸铵将冬瓜果肉汁分级盐析,得到粗酶液。再经DEAESepharoseFF离子交换层析和Superdex75柱层析等步骤得到一种电泳纯的冬瓜蛋白酶。SDSPAGE测得其分子量为64kDa。以酪蛋白做底物时,该酶的最适反应温度为70,最适作用pH为6.5,在pH4.5~10.
2、5,402+2+~70范围内较稳定。PMSF强烈抑制该蛋白酶的活性。另外,Hg对该酶有强烈的抑制作用,Mn离子对2+2+2+其有保护作用,Zn、Ca和Cu等离子对其活性没有影响。关键词:冬瓜;蛋白酶;纯化;性质中图分类号:R284.2;Q946.5;S642.3文献标识码:APurificationandCharacterizationofaProteasefromtheSarcocarpofBenincasahispida[L.]Lam*KONGLingying,DUGuica,iGUODaosen,LIRongguiDepartmentofBio
3、logy,QingdaoUniversity,Qingdao266071,ChinaAbstract:InordertoinvestigatetheproteasesinBenincasahispida[L.]Lamthatmightbeusedformedicalandfoodindustry,aproteasewaspurifiedanditscharacteristicswerestudied.AnovelproteasewaspurifiedfromthesarcocarpofB.hispidatoelectrophoretichomogeneityb
4、ythreepurificationsteps:ammoniumsulfateprecipitation,chromatographyonDEAESepharoseFFcolumnfollowedbychromatographyonaSuperdex75column.ThemolecularweightwasdeterminedbySDSPAGE,andcaseinolyticactivitywasusedforanalyzingtheprotease.Aproteasewithamolecularweightof64kDawaspurified.Th
5、eproteaseshowedrelativelygoodthermalandpHstabilitywithoptimumpHof6.5andoptimumtemperatureof70.TheproteasehadgoodthermalstabilityandpHstability.ItcouldbestronglyinhibitedbyPMSF.2+Noothertestedinhibitorsorcompoundshadanysignificanteffectontheproteolyticactivity.Hgwasobservedtohave2+2
6、+2+2+obviousinhabitationonitsactivity.Mncouldactivatetheprotease,butotherionssuchasZn,CaandCuhadnoeffectonitsactivity.AquickmethodforpurificationofaproteasefromB.hispidawasestablished,andtheproteasecouldbeusedinmedicalandfoodindustrybasedonitsgoodthermalandpHstability.Keywords:Benin
7、casahispida[L.]Lam;protease;purification;characteristicstiesareaffectedbyairoxidationormetalions,andbyIntroductionreducingorchelatingagents.Incontras,tvariousserineEnzymeshaveextensiveapplicationsinvariousindusproteasesincludingplantproteaseshavenorequirementtrialprocesses.Protease
8、saccountforapproxim
