Enhancing the Robustness of Nitrilase from Betaproteobacteria bacterium by Multistrategy Synergistic Evolution

Nicotinic acid is vital in pharmaceuticals, feed, food, and cosmetics. Nitrilase can catalyze the conversion of 3-cyanopyridine to nicotinic acid, but its industrial application is limited by poor thermostability and substrate tolerance. In this study, nitrilase from Betaproteobacteria bacterium was engineered via multistrategy synergistic evolution, including C-terminal loop truncation, consensus mutation, loop engineering and protein surface engineering. Through iterative saturation mutagenesis, we obtained mutant 4 M (L194F/A201Q/S208L/M180Q-Δ47). Its activity reached 12.78 U/mL. After 1 h at 50 °C, 4 M retained 10.46 U/mL residual activity─3.25-fold higher than WT. Its melting and aggregation temperatures increased by 8.3 and 6.6 °C, respectively. Molecular dynamics revealed that enhanced thermostability resulted from reduced loop flexibility, strengthened hydrophobic interactions, and new hydrogen bonds. An engineered Vibrio natriegens strain produced 564.3 g/L nicotinic acid, a 5.8-fold increase over WT. This work provides mechanistic insights into nitrilase thermostability.