Highly Efficient Biosynthesis of Nicotinic Acid by Immobilized Whole Cells of <i>E. coli</i> Expressing Nitrilase in Semi-Continuous Packed-Bed Bioreactor

A recombinant E. coli, expressing nitrilase from Acidovorax facilis 72W with dual-site expression plasmid pRSFduet (E. coli pRSF-AfNit2), was constructed. It showed higher soluble expression of nitrilase than that in the pET21a plasmid. The recombinant nitrilase can efficiently catalyze the hydrolysis of 3-cyanopyridine to nicotinic acid. The whole cells of E. coli pRSF-AfNit2 were immobilized by using sodium alginate/glutaraldehyde/polyethylene imine as the best immobilized reagents. The immobilized cells showed 95% activity recovery and excellent mechanical strength, with improved thermal stability and pH stability. They also retained 82% of initial activity after nearly two months of storage at 4 °C. A semi-continuous packed-bed bioreactor (sPBR) filled with the immobilized cells was studied for efficient production of nicotinic acid. After optimization, the highest space–time yield of 1576 g/(L·d) was obtained on 0.8 M substrate concentration at 2 mL/min of flow rate. The sPBR was repeatedly operated for 41 batches, keeping 100% conversion in the presence of 30 mM CaCl2. Finally, 95 g of nicotinic acid were obtained at 90% yield after separation and purification. The developed technology has potential application value