产漆酶菌株的筛选与酶活性的测定论文.docVIP

目 录 摘要 I Abstract II 前言 1 1 材料与方法 2 1.1 实验材料与方法 2 1.2 培养基 3 1.3 实验方法 3 1.3.1 产漆酶菌株的筛选 3 1.3.2 漆酶液态发酵及其制备 3 2 漆酶酶活性的测定 4 2.1 ABTS-分光光度计法 4 2.2 分光光度法具体步骤 4 2.3 酶活性的计算 4 3 菌株的活化 4 3.1 培养基及主要试剂的配置 4 3.2 菌种活化步骤 5 4 改良CTAB法提取待测菌株全基因组 5 4.1 基因组提取 5 5 结果与分析 6 6 讨论和小结 9 1. 讨论 9 2. 小结 9 参考文献 10 致谢 11  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（hemolytic Staphylococcus JCSC1435）。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 JCSC1435  Abstract The collected soil from the pesticide factory in Nanchang as material, and in 2014 July to September, to isolate a bacterial strain, rapid growth, genetic stability, with the activity of laccase, and the physiological and biochemical properties of the strain were studied. In order to select the laccase enzyme activity is relatively high strain, by using guaiacol as substrate, using plate screening method and qualitative determination of enzyme activity of laccase producing strain was selected. Screening of a strain of laccase producing strains with high activity from the soil, the sixth day peak of enzyme production strains in culture medium after inoculation with 2,2, ABTS - (even the amino - (3- ethyl phenyl thiazole -6- sulfonic acid)) as substrate by determination of laccase activity of laccase producing strains, the highest the laccase activity was 5.33U. Through sequencing and sequence alignment by this strain ,the bacteria is Staphylococcus haemolyticus JCSC1435.The suitable culture medium for fermentation of glucose 2%, ammonium tartrate 1%, potassium dihydrogen phosphate 0.2%, magnesium sulfate seven, water 0.05%, anhydrous calcium chloride 0.0075%, five copper sulfate 0.001%