Improvement of Activity, Stability, and Soluble Expression of Nitrilase through Stepwise Sequence Optimization for...

Nitrilases are important industrial enzymes that catalyze the conversion of nitrile compounds into carboxylic acids. This study employed a stepwise sequence optimization strategy to enhance nitrilase performance, combining PROSS for combinatorial mutation design with SPIRED-Fitness for fitness-guided optimization. Beneficial distal mutations were identified and iteratively combined. Ultimately, optimal mutant PD1F4 showed over 2-fold increase in specific activity compared to the starting variant C57E10, and its half-life at 50 °C improved from 10.53 ± 1.13 to 123.40 ± 5.94 min. Owing to improved soluble expression and product tolerance, PD1F4-expressing cells catalyzed the production of 272 g/L nicotinic acid. Molecular dynamics simulations revealed that the distal mutations stabilized the oligomeric assembly of nitrilase, confined substrate binding to the near attack conformations, and reduced detrimental interactions between residues with nicotinic acid. This work demonstrates an effective strategy for enhancing enzyme robustness and provides an efficient biocatalyst for nicotinic acid synthesis.