Ensifer meliloti 1021烟酰胺酶的酶学特性及3-氰基吡啶调控机理的研究

Research on the NAMase of Ensifer meliloti 1021 and Regulation Mechanism of 3-Cyanopyridine

克隆和异源表达固氮菌Ensifer meliloti 1021烟酰胺酶(NAMase),研究其酶学特性,并探究3-氰基吡啶(3-CP)对该酶的调控机制。PCR扩增获得E.meliloti 1021烟酰胺酶基因(nam)序列,构建含nam的重组质粒并导入Escherichia coli Rosetta(DE3)中异源表达,用Ni-NTA亲和层析纯化蛋白,探究温度、pH、有机溶剂及金属离子对NAMase活性的影响,并对烟酰胺酶进行了固定化研究。结果显示,克隆得到长636bp的nam,编码的蛋白分子量为22.6kD,等电点(PI)为5.5。NAMase的最适pH为7.0,在30-50℃温度范围内孵育2h酶活维持在97.1%以上,最适温度在30-70℃。Ag2+和异戊醇对NAMase的活性抑制率最大。在E.meliloti 1021野生菌株及NAMase过表达菌株转化烟酰胺的过程中3-CP会抑制烟酸的产量,这种效应并非底物抑制作用。

This work aims to clone and express the NAMase of Ensifer meliloti 1021,and to investigate its enzymatic properties and explore the enzymatic mechanisms regulated by 3-CP. PCR was used to amplify the gene nam of E. meliloti 1021 and the constructed recombinant nam-containing plasmid was imported to Escherichia coli Rosetta(DE3)cells for heterologous expression. Ni-NTA affinity chromatography was employed to purify the protein. The effects of temperature,pH,organic solvents and metal ions on NAMase activity were explored,and the immobilization of NAMase was further investigated. The results showed that the full-length of nam was 636 bp,encoded a 22.6 kD protein with PI of 5.5. The optimal pH for NAMase was 7,it retained over 97.1% of its initial activity after incubation at 30-50℃ for 2 h, and the optimal temperature ranged in 30-70℃. Ag2+ and isoamyl alcohol demonstrated the highest inhibitory effect on NAMase activity. 3-CP inhibited nicotinic acid production during the transformation of nicotinamide by E. meliloti 1021 and over-expressed NAMase in E. coli Rosetta (DE3),and this was not a substrate inhibition.