Enhancing the Robustness of Nitrilase from Betaproteobacteria bacterium by Multistrategy Synergistic Evolution.
TL;DR
Molecular dynamics revealed that enhanced thermostability resulted from reduced loop flexibility, strengthened hydrophobic interactions, and new hydrogen bonds, which provided mechanistic insights into nitrilase thermostability.
Abstract
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.