ABSTRACT
In the present study, the Gordonia terrae was subjected to chemical mutagenesis using ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 5-bromouracil (5-BU) and hydroxylamine with the aim of improving the catalytic efficiency of its nitrilase for conversion of 3-cyanopyridine to nicotinic acid. A mutant MN12 generated with MNNG exhibited increase in nitrilase activity from 0.5 U/mg dcw (dry cell weight) (in the wild G. terrae) to 1.33 U/mg dcw. Further optimizations of culture conditions using response surface methodology enhanced the enzyme production to 1.2-fold. Whole-cell catalysis was adopted for bench-scale synthesis of nicotinic acid, and 100% conversion of 100 mM 3-cyanopyridine was achieved in potassium phosphate buffer (0.1 M, pH 8.0) at 40 °C in 15 min. The whole-cell nitrilase of the mutant MN12 exhibited higher rate of product formation and volumetric productivity, i.e., 24.56 g/h/g dcw and 221 g/L as compared to 8.95 g/h/g dcw and 196.8 g/L of the wild G. terrae. The recovered product was confirmed by HPLC, FTIR and NMR analysis with high purity (> 99.9%). These results indicated that the mutant MN12 of G. terrae as whole-cell nitrilase is a very promising biocatalyst for the large-scale synthesis of nicotinic acid.
