*Article* **Highly Efficient Biosynthesis of Nicotinic Acid by Immobilized Whole Cells of** *E. coli* **Expressing Nitrilase in Semi-Continuous Packed-Bed Bioreactor**

**Xue-Jiao Liu, Bao-Di Ma, Xiao-Mei Wu and Yi Xu \***

School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China **\*** Correspondence: xuyi@sit.edu.cn; Tel.: +86-21-60873024

**Abstract:** 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 CaCl<sup>2</sup> . Finally, 95 g of nicotinic acid were obtained at 90% yield after separation and purification. The developed technology has potential application value.

**Keywords:** nitrilase; cell immobilization; nicotinic acid; reaction optimization; semi-continuous packed-bed bioreactor
