Robust nitrile hydratase design via molecular dynamics simulations: synergistic enhancement of stability, activity, and aromatic substrate tolerance

Nitrile hydratase (NHase) is a promising biocatalyst for the industrial synthesis of high value amides, yet its practical application is often limited by conformational instability and activity loss under high product concentrations. In this study, we employed a rational engineering strategy guided by molecular dynamics simulations in high amide concentrations to elucidate the structural dynamics of NHase acrylamide/water mixed solvents and identify key residues governing its stability. Through systematic mutagenesis, we developed a double mutant, βV4I/βI177K (designated M1), which exhibited a 2.2-fold increase in specific activity toward acrylonitrile, a 1.7-fold improvement in catalytic efficiency, and a 9.6-minute extension (corresponding to a 37% increase in enzyme durability) in thermal inactivation half-life compared to the wild-type enzyme. Under simulated industrial conditions, the superior catalytic stability and conversion efficiency of M1 resulted in a 1.3-fold increase in acrylamide yield. Furthermore, the mutant displayed broadened substrate adaptability, with activities toward 1-naphthonitrile and cinnamonitrile enhanced by 2.98- and 3.34-fold, respectively. These findings not only reveal the molecular mechanism underlying NHase inactivation acrylamide/water mixed solvents but also establish a robust engineering framework for developing highly efficient and stable biocatalysts for industrial amide synthesis.