Screening and characterization of a nitrilase with significant nitrile hydratase activity

Abstract Purpose We screened nitrilases with significant nitrile hydratase activity to exploit their potential in aromatic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on aromatic amide production by nitrilase.Methods A semi-rational screening method based on previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as “amide-formation hotspots”) was built and used for nitrilases identification. Results Based on the previously reported “amide-formation hotspots,” we identified a nitrilase NitPG from Paraburkholderia graminis DSM 17151 that could produce a significant amount of mandelamide toward mandelonitrile and exhibited general hydration activity toward various aromatic nitriles. The time-course experiment with NitPG demonstrated that amide was also a true reaction product of nitrilase, suggesting that the nitrile catalysis by amide-forming nitrilase could be a post-transition state bifurcation–mediated enzymatic reaction. Further research demonstrated that low temperature, metal ion addition, and specific substrate structure could profoundly improve the amide formation capability of nitrilase. Conclusions NitPG with broad hydration activity is a potential candidate for the enzymatic synthesis of aromatic amides for biotechnological applications. Studying the effect of nitrilase hydration activity could promote our understanding of the factors that influence amide and acid distribution.