突变微小毛霉天冬氨酸蛋白酶的纯化及性质研究.pdfVIP

农产食品科技 2011，5(1)：35～43 uhuralFoodProductsScienceandTechnol PurificationandCharacterizatiOnofaMutatedFungalAspartic ProtejnasefrOm M ucorPusillus LIYu—qiu，WANGJing—hui，LITie—zhu，ZHENG Li，ZHANGLi，LIZhou一¨n YANGZhen-nai (1．CenterofAgro-foodTechnology，JilinAcademyofAgriculturalSciences，Changchun130033，China；2． InstituteofBiologicalandAgriculturalEngineering,JinlinUnivercity，Changchun130024，China) Abstract：Site——directed mutagenesiswascarried outto alterpropertiesofMucorPusillusrennet (M )forasubstitutionofcommercialchymosin．Onemutant，E13D，withdecreasedproteolyticactivity wasexpressedinPichiapastoris，purified andcharacterized．Themolecularweightofthepuriedenzyme wasestimated tobe46 kDa bySDS—PAGE．Themaximum enzymeactivity wasatawide range ofpHs (5．0—7．0)and60 ．Themilk—clottingactivity(MCA)increasedwithadecreaseinthepHfrom8．0to5．0． TheenzymewasinhibitedbyheavymetalionsfFe ，Fe ，Cu andZn。)，1．10-Phenantrolinandpepstatin A．ReactivationoftheenzymeactivitywithCa ．Mn andMg indicatedtheimportanceofthesemetalsin the catalytic function oftheenzyme．Furthertextureanalysisofcheddarcheesesmadebynon—mutant rennet，mutantE13D rennetandcommercialrennetfoundthattherewasnosignificantdifferencebetween mutantrennetcheese and commercialcoagulantcheese，exceptforlowersoluble nitrogen contentand highervalueofhardnessofmutantcheese than thoseofthenon—mutant．The resultimplicated that，in someextent，themutantrennetcoulddecreasehydrolysisofproteinduringripeningofcheese，probablyto becandidateforausefulmilkcoatulant． Keywords：Asparticproteinase；MucorPusillusrennin；Mutation；Proteolyticactivity 突变微小毛霉天冬氨酸蛋 白酶的纯化及性质研 究 李玉秋 ，王景会 ，李铁柱 1，郑 丽 ，张 莉 ，李倬林 ，杨贞耐 1※ (1．吉林省农业科学院农产品加工中心，长春 130033；2．吉林大学生物与农业丁程学院，长春 130024) 摘 要 ：本文利用定点突变技术改造微小毛霉凝乳酶 ，将突变体 (E13D)在毕赤酵母 中表达，并对其进行 纯化及性质研究 ，此突变酶水解活性有明显降低。通过 SDS—PAGE电泳鉴定纯化后酶的分子质量为46kDa。