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摘要：在大肠杆菌中过量表达Acidovorax facilis 72W细菌腈水解酶基因，会产生一种微生物，它可以高效而有选择性的催化脂肪族腈到氰基羧酸。产量高，反应少和稳定性使这种生物催化剂非常适用于工业生产。本实验研究主要就是腈水解酶基因在大肠杆菌中的克隆表达以及含腈水解酶的重组菌将底物腈变成产物腈酸的工业生物转化过程。我们从含有腈水解酶和pET-21a质粒的E.coli. DH5α菌株中提取质粒转入宿主菌E.coli. BL21 (DE3)中，获得了重组菌E.coli. BL21(DE3)，通过琼脂糖凝胶电泳鉴定其重组克隆，然后对其进行发酵条件的优化。在对含有腈水解酶基因的重组E.coli. BL21(DE3)菌株进行发酵优化的过程中，分别对诱导剂IPTG浓度，IPTG加入时间以及发酵培养基成分利用HPLC进行单因素实验。实验结果表明，在摇床温度30℃，180rpm，采用YP培养基（甘油替代葡萄糖），初始PH7.0培养条件下，发酵4h后加入0.2mM的IPTG，可得到本实验中酶活最佳条件。7449
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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