巧克力微杆菌中酯酶的克隆表达及性质研究
毕业论文关键词: 酯酶；克隆；表达；性质；分析
Cloning, Expression and Characterization of a Esterase from Microbacterium chocolatum
Abstract: Esterase，capable of catalyzing the hydrolysis of ester bonds, is considered as a very important kind of biocatalyst. It has been widely used in agriculture, medicine, food industry and so on. Based on the result of homologous sequence alignment, we predict four proteins may exist esterase activity from Microbacterium chocolatum SIT 101, then cloning and expression in E. coli, After esterase activity experiment, three active enzyme were found. Enzymatic aynthesis shows that EstMC1, EstMC3 and EstMC4 optimum activity is at pH 9 and 40 °C. Under this condition, three esterase activity were significantly improved to 27.750 U / mg,20.520 U / mg and 79.282U / mg.
Key words: Esterase；Cloning; Expression; Nature; analysis
目录
1 前言 7
1.1 酯酶的介绍 7
1.1.1 酯酶的分类 7
1.1.2 酯酶的立体结构和催化机制 7
1.1.3 酯酶在合成中的其他应用 9
1.2 基因工程简介 9
1.3 实验方案 10
2 实验材料 10
2.1 菌种和质粒 10
2.1.1 菌种 10
2.1.2 质粒 10
2.2 试剂 10
2.3 培养基： 11
2.3.1 巧克力微杆菌斜面培养基(PH7.0) 11
2.3.2 巧克力微杆菌发酵培养基(pH7.0) 11
2.3.3 LB液体培养基 11
2.3.4 LB固体培养基 11
2.4 溶液配置 12
2.5 主要仪器 12
2.6 软件及网站 13
3 实验方法 13
3.1 序列的分析 13
3.2 重组菌的构建 14
3.2.1 巧克力微杆菌基因组的提取 14
3.2.2 引物的设计 15
3.2.3 PCR扩增目的基因与酶切 15
3.2.4 PCR产物与T载体的连接 18
3.2.5 表达质粒pET-21a的提取与酶切 18
3.2.6 氯化钙法制备感受态细胞 19
3.2.7 目的基因与pET21a的连接 20
3.2.8 连接产物的转化 20
3.2.9 质粒的提取并验证 20
3.3 工程菌的发酵及酶活测试 20
3.3.1 种子培养 20
3.3.2 发酵培养 21
3.3.3 细胞的破碎 21
3.3.4 Bradford考马斯亮兰法测定蛋白质浓度 21
3.3.5 对硝基苯酚酯测定方法 22
3.4 重组酯酶的酶学性质表征 22
3.4.1 聚丙烯酰胺凝胶电泳 22
3.4.2 温度对酶活力的影响 23
3.4.3 pH对酶活力的影响 23
4 结果及讨论 23
4.1 EstMC1，EstMC2， EstMC3， EstMC4性质的分析 23
4.2 基因组提取的结果 26