产漆酶菌株的筛选与酶活性的测定论文.doc

产漆酶菌株的筛选与酶活性的测定目录摘要IAbstractII前言11材料与方法21.1实验材料与方法21.2培养基31.3实验方法31.3.1产漆酶菌株的筛选31.3.2漆酶液态发酵及其制备32漆酶酶活性的测定42.1ABTS-分光光度计法42.2分光光度法具体步骤42.3酶活性的计算43菌株的活化43.1培养基及主要试剂的配置43.2菌种活化步骤54改良CTAB法提取待测菌株全基因组54.1基因组提取55结果与分析66讨论和小结91.讨论92.小结9参考文献10致谢11I摘要本实验从南昌农药厂土壤中分离筛选出一株能够产漆酶活性的细菌菌株，并初步分析和鉴定了该菌株。此菌株具有生长快速、遗传稳定、菌落规则的特性。为了从土壤中筛选产漆酶酶活相对较高的菌株，采用以愈创木酚为底物，利用平板筛选法和定性测定酶活力法筛选得到高产漆酶菌株。以ABTS〔2,2’-连氮基-双（3-乙基苯丙噻唑啉-6-磺酸）〕为底物测定漆酶活得到产漆酶活性的菌株，菌株的产酶高峰期出现在发酵培养基培养后的第6天，最高的产漆酶活为5.33U。通过测序和序列比对得到此菌株为溶血葡萄球菌JCSC1435（hemolyticStaphylococcusJCSC1435）。适宜的发酵培养基为：葡萄糖2%，酒石酸铵1%，磷酸二氢钾0.2%，七水硫酸镁0.05%，无水氯化钙0.0075%，五水硫酸铜0.001%。适宜的培养条件：50ml/250ml发酵培养基，培养温度为28℃，转速为200r/min，培养时间为7d。关键词：漆酶、愈创木酚、溶血葡萄球菌JCSC1435IIAbstractThecollectedsoilfromthepesticidefactoryinNanchangasmaterial,andin2014JulytoSeptember,toisolateabacterialstrain,rapidgrowth,geneticstability,withtheactivityoflaccase,andthephysiologicalandbiochemicalpropertiesofthestrainwerestudied.Inordertoselectthelaccaseenzymeactivityisrelativelyhighstrain,byusingguaiacolassubstrate,usingplatescreeningmethodandqualitativedeterminationofenzymeactivityoflaccaseproducingstrainwasselected.Screeningofastrainoflaccaseproducingstrainswithhighactivityfromthesoil,thesixthdaypeakofenzymeproductionstrainsinculturemediumafterinoculationwith2,2,ABTS'-(eventheamino-(3-ethylphenylthiazole-6-sulfonicacid))assubstratebydeterminationoflaccaseactivityoflaccaseproducingstrains,thehighestthelaccaseactivitywas5.33U.Throughsequencingandsequencealignmentbythisstrain,thebacteriaisStaphylococcushaemolyticusJCSC1435.Thesuitableculturemediumforfermentationofglucose2%,ammoniumtartrate1%,potassiumdihydrogenphosphate0.2%,magnesiumsulfateseven,water0.05%,anhydrouscalciumchloride0.0075%,fivecoppersulfate0.001%.Thesuitableculturecondition:50ml/250mlculturemedium,culturetemperaturewas28C,thespeedof200r/min,theculturetimewas7d.Keywords：Laccase,guaiacol,StaphylococcushaemolyticusJCSC143512前言漆酶是一种结合多个铜离子的多酚氧化酶，其肽链一般由500个左右氨基酸组成，糖配基占整个分子的10%-45%，是一种结合多个铜离子的糖蛋白，是铜蓝氧化酶蛋白家族的一员漆酶是一种绿色的生物催化剂[12-15]。漆酶广泛存在于自然