邻苯二酚2,3-双加氧酶基因克隆、定位和高效表达

采用特异性引物，以菲、芘降解菌株ZL5的代谢性质粒为模板，扩增出邻苯二酚2，3—双加氧酶(C23O)基因，将该基因和表达载体pET—30a( )连接，转化E.coli JM109(DE3)，获得了高效表达的转化子，SDS—PAGE结果表明，转化子的C23O蛋白不仅在细胞内存在，而且能被分泌到胞外，薄层扫描显示，转化子细胞内和细胞外表达蛋白总量占细胞总蛋白的42％，酶活分析表明，分布在转化子细胞内、外的表达蛋白都具有较高的C23O比活力，Southern杂交将菌株ZL5的C23O基因定位在内生质粒的不同酶切片段上。图5表1参12。

CLONING,LOCATION AND OVEREXPRESSION OF CATECHOL 2,3-DIOXYGENASE GENE

Catechol 2,3-dioxygenase(C23O) gene was amplified with the specific primer and the template from plasmid in the strain ZL5 degrading phenanthrene and pyrene. The cloned gene was connected with pET-30a(+) vector carring T7 promotor and transformed to E.coli JM109(DE3), and the overexpressing recombinants were obtained. SDS-PAGE showed that the expressed protein was also secreted to the supernant. The total C23O protein outside and inside the recombinant cells was about 42% of the total cell proteins according to the thin layer scanning. The C23O specific activity was high both in and out of the recombinant cells. Southern blotting showed that the C23O gene was located on different fragments of the resident plasmid digested by different restriction enzymes. Fig 5, Tab 1, Ref 12