敏捷食酸菌腈水解酶在大肠杆菌中的组成型表达及其催化高浓度烟腈水解的研究

Constitutive Expression of Acidovorax facilis Nitrilase in E. coli and Its Application in the Hydrolysis of Nicotinonitrile in High Concentration

中文关键词:  腈水解酶  组成型表达  烟腈  烟酸  生物催化

英文关键词:nitrilase  constitutive expression  nicotinonitrile  nicotinic acid  biocatalysis

中文摘要:

      对来源于敏捷食酸菌的腈水解酶在大肠杆菌中的组成型表达进行了研究, 构建了组成型表达腈水解酶重组质粒pET3b-NIT, 确定了其表达的最佳宿主菌株为E. coli BL21Gold(DE3), 所得组成型菌株发酵产酶活性和比酶活分别为4392.39 U·L^-1和953.25 U·g^-1, 是原始诱导表达菌株的2.19和1.94倍. 在此基础上系统研究了该重组菌静息细胞催化高浓度烟腈水解合成烟酸的工艺条件, 发现12 g·L^-1的细胞在35 ℃条件下, 2 h可将2 mol·L^-1的烟腈完全转化为烟酸, 时空产率达246.22 g·g^-1·d^-1. 并进行了烟酸克级规模的制备实验, 最终得到2.23 g烟酸, 收率为90.65%, 纯度达99%以上, 实现了高底物浓度下烟酸的高效制备.

英文摘要:

      The constitutive expression of nitrilase derived from Acidovorax facilis in E. coli was studied, and the constitutively expressed nitrilase recombinant plasmid pET3b-NIT was constructed, and the optimal host strain for its expression was determined to be E. coli BL21Gold(DE3). The enzyme activity and specific activity of recombinant E. coli were 4392.39 U·L^-1 and 953.25 U·g^-1, which were 2.19 and 1.94 times higher than those of the original inducible expression strain, respectively. On this basis, the process conditions for the hydrolysis of high concentration of nicotinonitrile for the synthesis of nicotinic acid by resting cells were systematically investigated. It was found that 2 mol·L^-1 nicotinonitrile could be completely converted to nicotinic acid by 12 g·L^-1 cells for 2 h at 35 ℃, and the space-time yield was up to 246.22 g·g^-1·d^-1. Preparation experiments on the gram scale of nicotinic acid were also performed, and 2.23 g of nicotinic acid was obtained in 90.65% yield and over 99% HPLC purity, achieving efficient preparation of nicotinic acid at high substrate concentration.