硫酸铵沉淀和层析法分离纯化纳豆激酶的研究.docx

文章编号：1003-9015(2006)01-0063-05硫酸铵沉淀和层析法分离纯化纳豆激酶的研究高大海1,梅乐和1,盛清2,许静3,林东强1,姚善泾1(1.浙江大学化学工程与生物工程学系,浙江杭州310027;2.浙江理工大学生命科学学院,浙江杭州310018;3.杭州中美华东制药有限公司,浙江杭州310011)摘要：利用生产纳豆激酶的Bacillussubtilis进行深层发酵。采用发酵液离心除菌，20％~60％饱和度的硫酸铵沉淀，Superdex75凝胶过滤层析和SPSepharoseFastFlow离子交换层析对活性组分进行分离提纯。用纤维平版法测定了活力，并用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法(SDS)对分离纯化的效果进行了检验。结果表明在SDS中观察到单一条带，分子量27.7kD，最终纯化倍数和酶活回收率分别为8.4和49%。关键词：纳豆激酶；分离纯化；凝胶层析；离子交换中图分类号：Q814.1文献标识码：ASeparationandPurificationofNattokinaseProducedbyBacillusSubtiliswithAmmoniumSulfatePrecipitationandChromatographyGAODa-hai1,MEILe-he1,SHENGQing2,XUJing3,LINDong-qiang1,YAOShan-jing1(1.DepartmentofChemicalandBiochemicalEngineering,ZhejiangUniversity,Hangzhou310027,China;2.CollegeofLifeScience,ZhejiangUniversityofScienceandTechnology,Hangzhou310018,China;3.HangzhouEast-ChinaPharmaceuticalCompany,Hangzhou310011,China)Abstract:InordertoobtainhighpuritynattokinasefromthefermentationbrothofBacillussubtilis,someseparationandpurificationtechniqueswereexplored.Theoptimizedseparationandpurificationprocessincludesthefollowingsteps:removingcellsbythecentrifugation,20%~60%saturationammoniumsulfateprecipitation,gelfiltrationchromatographywithSuperdex75andion-exchangechromatographywithSPSepharoseFastFlow.Sodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS)wasusedtoexaminethepurificationeffect,andtheresultsindicatethatthefinalproductobtainedishomogeneousandhasamolecularweightaround27.7kD.Thefibrinolyticactivityofthenattokinasewasmeasuredbymeansoffibrinplatemethod.Thepurificationfactorandactivityrecoveryofthenattokinaseare8.4and49%,respectively.Thespecificactivitiesofpurifiednattokinasecouldreach28530IU?mg?1.TheresultsdemonstratethatthecombinationofbioseparationprocessofammoniumsulfateprecipitationwithgelfiltrationandionexchangechromatographyissuitablefortheseparationandpurificationofnattokinasefromculturebrothofBacillussubtilis.Especiallyforlabscale,theprocessmentionedabovecanbeeasilyperformedasanefficientwaytopreparepurenattokinasewithhighyield.Keywords:nattokinase