Acidovorax facilis 72W细菌腈水解酶基因的克隆
关键词： 腈水解酶；重组大肠杆菌；发酵优化；HPLC；酶活
Cloning and Expression of Nitrilase in Escherichia coli from Acidovorax facilis 72W
Abstract: The nitrilase gene from Acidovorax facilis 72W was over-expressed in Escherichia coli, generating a microorganism that efficiently and regioselectively catalyzes the conversion of aliphatic dinitriles to cyanocarboxylic acids. The high yields obtained, mild reaction conditions used, and robustness observed make this biocatalyst suitable for industrial applications. This experiment mainly studied the cloning and expression of nitrilase genes those in E.coli and the industrial bioconversion processes of recombinant E.coli BL21 (DE3) contained nitrilases turn the dinitrile into carboxylic acid. We extracted plasmid from E.coli DH5α that contains the nitrilases and plasmid pET-21a, put it into host bacterial E.coli BL21 (DE3), in order to gain the recombinant E.coli BL21 (DE3). SDS-PAGE showed that there w as the special protein band. Then we optimized it with fermentation conditions. In the process this fermentation conditioned optimization, employing HPLC to single-factor-test the inducer IPTG concentration, IPTG temporal induction and fermentation culture component, respectively. The results turned out that under the conditions of shaker incubator temperature was 30℃, 180rpm, YP culture(replacing the glucose with glycerol), initial PH7.0, adding IPTG 0.2mM after fermented 4h, the optimum condition of enzyme activity in this experiment was available.
Keywords: Nitrilases; recombinant E. coli; fermentation optimization; HPLC; enzyme activity
目录
1 绪论 1
1.1 腈水解酶简介 1
1.2产腈水解酶的菌种以及植物 1
1.3 腈水解酶的作用机理 2
1.4腈水解酶的应用 3
1.5 重组大肠杆菌的利用 4
1.6重组腈酶发酵的优化 5
1.7 本课题的研究内容和意义 5
2 材料与方法 7
2.1 实验材料 7
2.1.1 菌种 7
2.1.2培养基与试剂 7
2.1.3 仪器和设备 7
2.2 实验方法 8
2.2.1 基本操作 8
2.2.2 质粒的抽提、检测、转化 9
2.2.3 腈酸标准曲线的绘制 11
2.2.4 发酵工艺的研究 11
2.2.5发酵工艺参数的优化 13
3 实验过程及结果分析 14
3.1 重组菌E.coli BL21 (DE3) 14
3.1.1 E.coli DH5α质粒的提取 14
3.1.2转化新菌株 14
3.2 发酵实验 15
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