A Basic Application Of Nicotinic Acid Production By Nitrilase

Nitrilase which was screened from nitrilase library of New World Institution of Biotechnology was studied in this paper. The optimum reaction condition, substrate specificity and catalytic reaction kinetics was investigated, and the final choice is thel5th nitrilase, i.e. the nitrilase from Alcaligenes faecalis ATCC 8750. The optimum pH is pH 7 and the optimum temperature is 60℃. So it is a heat-resistant nitrilase. Michaelis constant of the enzyme is 2.86mmol·L-1, the maximum velocity rmax is 0.17μmol·mg-1·min-1.Nicotinic acid was prepared by free cell of recombinant E. Coli. By analyzing the factors influencing the catalytic process, the optimum conditions for hydrolyzing 3-cyanopyridine were confirmed at pH 7, PB buffer and 45℃. The maximum tolerated concentration was 500mmol·L-1 and no product inhibition. In the batch reaction, the conversion of second batch was up to 95% and it began to decrease from third batch when the substrate concentration is 500mmol·L-1. By batch reactions with substrate continuously added, the conversion rate can reach 96.16% when the substrate concentration was increased to 1050mmol·L-1 after 6h.The application of immobilized recombinant E.coli. with glutaraldehyde was studied. The optimized conditions for immobilization was 0.5g·L-1 addition of glutaraldehyde and the cross-linking time was 45min. After immobilization, the thermal stability and batch-reaction stability was improved significantly, e.g.90% of the enzyme activity can be maintained within 48h at 45℃and the activity of resting cells was 4% left in the same time; the batches upgraded to six, more than 3 batches compared with free cells when the substrate concentration was 500mmol·L-1 and the reaction conversion was up to 90%. The immobilized cells was applied to the continuous catalytic reaction with an additional substrate. After 10h, the substrate concentration was increased to 2mol·L-1 and the conversion rate was 98.5%.