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高温蛋白酶产生菌的筛选及其产酶条件和酶学性质分析 摘要 高温蛋白酶是一种新型的热稳定酶,可广泛应用于食品、医药、工业和环境等领域。本论文通过筛选高温蛋白酶产生菌,研究了其产酶条件和酶学性质。结果显示,菌株TH-18产生的高温蛋白酶经过优化产酶条件后,其最适反应温度为80℃,最适反应pH为9.0,具有良好的热稳定性和耐碱性。该研究为高温蛋白酶产业的发展提供了实验基础和理论指导。 关键词:高温蛋白酶,产生菌,产酶条件,酶学性质 Introduction Proteasesareatypeofenzymethatcatalyzesthehydrolysisofpeptidebondsinproteins.Thermophilicproteasesareagroupofproteasesthatareactiveathightemperatures,usuallyabove50°C,andhavebeenwidelyusedinvariousindustriesduetotheirthermalstabilityandhighactivity.Amongthem,high-temperatureproteaseshavethecharacteristicsofbeingthermostableandalkaline-resistant,whichhavegreatpotentialinmanyfieldssuchasfood,pharmaceuticals,industry,andenvironmentalprotection. Inthisstudy,weaimtoscreenforhigh-temperatureprotease-producingstrainsandinvestigatetheirenzymeproductionconditionsandproperties.Thestudyprovidesexperimentalandtheoreticalguidanceforthedevelopmentofthehigh-temperatureproteaseindustry. MaterialsandMethods IsolationandScreeningofHigh-TemperatureProtease-ProducingStrains Soilsampleswerecollectedfromhigh-temperatureenvironments,suchashotsprings,andwereusedastheinoculumforscreening.Thesamplesweredilutedseriallyandinoculatedonnutrientagarplatescontaining1%caseintoselecttheprotease-producingstrains.Theplateswereincubatedat60°Cfor24hours,andtheappearanceofatranslucentcirclearoundthecolonywasconsideredapositiveresult. IdentificationoftheProtease-ProducingStrains 16SrRNAgenesequencingwasperformedtoidentifytheprotease-producingstrains.TheDNAofbacterialstrainswasextractedusingabacterialgenomicDNAextractionkit,andthe16SrRNAgenewasamplifiedbyPCRusinguniversalprimers.TheamplifiedPCRproductwassequencedandidentifiedusingtheBLASTalgorithm. OptimizationofEnzymeProductionConditions Theprotease-producingstrainTH-18wasselectedforoptimizationofenzymeproductionconditions.Theeffectsofdifferentcultureconditions,suchastemperature,pH,andincubationtime,onenzymeproductionwereinvestigated. Enzymepurificationwascarriedoutbyammoniumsulfateprecipitationanddialysis.Theenzymeactivitywasdetermi