A Triple-Focus Workflow of a Novel Nitrilase for Simultaneously Optimizing Activity, Thermostability, and Specificity.

Nitrilases provide a sustainable route for directly converting nitriles to carboxylic acids. However, efficient biocatalytic synthesis of the agrochemical intermediate 3,6-dichloropicolinic acid(3,6-DCPA) from 3,6-dichloropicolinonitrile (3,6-DCPN) remains unreported. We identified a novel nitrilase from Rhodococcus pyridinivorans (RpNIT) and developed the integrated Feature Cluster-Multi-Force field-Evolutionary Co-Localization (FMEC) strategy to simultaneously enhance activity, thermostability, and specificity. FMEC employs hierarchical clustering, molecular dynamics simulations, and multiforce field algorithms, identifying 19 candidate residues. Using Parallel Iterative Saturation Mutagenesis, we generated the quintuple mutant M5a-DGPAT. This variant exhibited a 17.2 °C increase in melting temperature and a 3.3 × 104-fold improvement in hydrolytic efficiency (kcat/Km) over wild-type RpNIT, while abolishing hydratase activity. Structural analysis attributed the superior performance to an optimized rigidity-flexibility balance, refined substrate binding pocket geometry, and strengthened interaction networks. M5a-DGPAT achieved complete hydrolysis of 347.9 mM 3,6-DCPN to 3,6-DCPA under mild conditions, demonstrating significant industrial potential. This study establishes a robust computational framework for designing highly active, stable, and specific engineered enzymes.