浑球红细菌Rhodobacter sphaeroides LHS-305腈水解酶基因的克隆及表达

Cloning and Expression of Nitrilase Gene from Rhodobacter Sphaeroides LHS-305

以筛选的产腈水解酶浑球红细菌Rhodobacter sphaeroides LHS-305为出发菌株,根据已知腈水解酶基因保守区域设计简并引物,从总DNA中成功扩增得到腈水解酶基因的部分片段(402 bp)。通过染色体步移扩增片段的上下游序列,经拼接,得到的腈水解酶基因全长为969 bp(在Genebank数据库中的登录号为JN635494)。该基因与已知腈水解酶序列的最高相似性为82%。构建pET28a-nit表达载体,转化大肠杆菌Rosetta(DE3),得到重组菌。SDS-PAGE电泳结果表明该基因在大肠杆菌中成功表达。重组酶对底物3-氰基吡啶催化结果显示了较高的酶活力,具有跟原始菌相同的特性。

Degenerate primers were designed by comparing homologous sequences of reported ni- trilase. A 402 bp fragment of nitrilase gene was successfully amplified using these primers from Rhodobacter sphaeroides LHS-305 which was isolated by our lab. Then, upstream and downstream se- quences were cloned using genome walking technique. Finally, a complete nitrilase gene consisting of 969 bp nucleotides was obtained （its GenBank accession number is JN635494）. The nucleotide se- quence of Rhodobacter sp. LHS-305 had the highest similarity of 82% to nitrilase known. The heter- ologous expression of nitrilase was achieved in Escherichia coli Rosetta （ DE3 ） with pET28a-nit re- combinant vector. SDS-PAGE result showed that the gene successfully expressed in Escherichia coli, and the result of catalytic reaction with 3-cyanopyridine showed the recombinant nitrilase had activity towards nitrile substrate.