过氧化氢酶基因在大肠杆菌中的克隆表达及发酵优化

Cloning,expression and fermentation optimization of a catalase gene in E.coli

利用PCR扩增技术得到枯草芽孢杆菌(Bacillus subtilis)过氧化氢酶基因katA,将该基因与表达载体pET-20b(+)连接构建重组质粒,经测序验证后,在大肠杆菌JM109中进行表达得到重组大肠杆菌基因工程菌E.coli BL21(DE3)(pET-20b(+)-katA)。SDS-PAGE电泳结果显示出明显的特异性表达条带,大小与过氧化氢酶吻合。摇瓶实验获得重组菌的最佳碳、氮源成分:5 g/L甘油,40 g/L安琪酵母粉,产酶水平最高达到20 000 U/mL。

The catalase gene katA from Bacillus subtilis WSHDZ-01 was amplified by PCR. It was then inserted into the expression vector pET-20b（ ＋ ） to create the recombinant plasmid pET-20b（ ＋ ）-katA and the modified vector was transformed into E. coli JM109 for expression. The size of the expressed specific band by SDS-PAGE electrophoresis corresponded wetl to that of the catalase gene katA. For batch fermentation in flasks, the medium composition regarding the carbon and nitrogen was optimized and the results indicated that the maximal enzyme activity of 20 000 U/mL was obtained with 5 g/L glycerol and 40 g/L yeast extract, respectively.