CAJ | 학술논문

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。
이사선적산정수해매혼구홍세균Rhodobacter sphaeroides LHS-305위출발균주,근거이지정수해매기인보수구역설계간병인물,종총DNA중성공확증득도정수해매기인적부분편단（402 bp）。통과염색체보이확증편단적상하유서렬,경병접,득도적정수해매기인전장위969 bp（재Genebank수거고중적등록호위JN635494）。해기인여이지정수해매서렬적최고상사성위82%。구건pET28a-nit표체재체,전화대장간균Rosetta（DE3）,득도중조균。SDS-PAGE전영결과표명해기인재대장간균중성공표체。중조매대저물3-청기필정최화결과현시료교고적매활력,구유근원시균상동적특성。
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.