Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli.

Nitrilases constitute an important class of biocatalysts for chiral synthesis. This work was undertaken with the aim to optimize nitrilase production in a host that is well-studied for protein production. Process parameters were optimized for high cell density fermentation, in batch and fed-batch modes, of Escherichia coli BL21 (DE3) expressing Pseudomonas fluorescens nitrilase with a T7 promoter based expression system. Effects of different substrates, temperature and isopropyl β-D-1-thiogalactopyranoside (IPTG) induction on nitrilase production were studied. Super optimal broth containing glycerol but without an inducer gave best results in batch mode with 32 °C as the optimal temperature. Use of IPTG led to insoluble protein and lower enzyme activity. Optimized fed-batch strategy resulted in significant improvement in specific activity as well as volumetric productivity of the enzyme. On a volumetric basis, the activity improved 40-fold compared to the unoptimized batch process.