大肠杆菌表达腺苷蛋氨酸合酶及产酶条件优化

Heterologous expression of adenosylmethionine synthase in Escherichia coli and optimization of enzyme-producing conditions

腺苷蛋氨酸(adenosymethionine,SAM)是人体中重要的代谢中间体,在医疗上被广泛应用于治疗肝炎,抑郁症以及关节炎等疾病。酶法进行腺苷蛋氨酸的生产具有良好的工业前景。将来源于酿酒酵母BY4741中的腺苷蛋氨酸合酶基因(sam2)进行密码子优化后,与p ET-28a(+)和p ET-3b(+)进行连接获得重组质粒p ET-28a(+)-sam2和p ET-3b(+)-sam2,分别实现其在E. coli BL21(DE3)和E. coli Rosetta(DE3)中的异源表达,结果表明,重组菌E. coli BL21/p ET-28a(+)-sam2的SAM酶活较高,达到0. 184 U/m L。对该重组菌株进行发酵条件优化,在最优条件下,SAM合酶最高酶活为0. 245 U/m L,比优化前提高34%。

Adenosylmethionine(SAM) plays a vital role in the organisms’ metabolic activity as an important intermediate metabolite and has been widely used in pharmaceutical industry involving the therapies for liver,nervous systems,osteoarthritis and so on. Thus,production of SAM by enzyme transformation process exhibits potential industrial prospects. Codon optimization of the adenosylmethionine synthase gene sam2 derived from S. cerevisiae BY4741 was performed,and the gene was ligated to pET-28 a( +) and pET-3 b( +),resulting in recombinant plasmids p ET-28 a( +)-sam2 and pET-3 b( +)-sam2,respectively,and was heterologous expressed in E. coli BL21( DE3) and E.coli Rosseta( DE3) respectively. Results showed that the recombinant strain E. coli BL21/pET28 a( +)-sam2 exhibited higher enzymatic activity up to 0. 184 U/m L. Thus,the recombinant strain E.coli BL21/p ET-28 a( +)-sam2 was chosen for the optimization of fermentation condition to further improve the enzymatic activity. Under optimal conditions,the enzyme activity of the recombinant adenosylmethionine synthase reached 0. 245 U/m L,which was 34% higher than that before optimization.