Structure-oriented engineering of a nitrile hydratase to improve the biotransformation performance toward amide

Acrylamide possesses extensive application value in modern industry. Nitrile hydratase is a powerful biocatalyst for industrial amide synthesis, but its application is hindered by enzyme inactivation at high acrylamide concentrations. To address this, an enzyme tailoring strategy based on structure-oriented engineering was proposed to precisely modify key tunnel of nitrile hydratase from Aurantimonas manganoxydans. In order to rapidly and efficiently screen for mutants, a novel high-throughput screening method based on color reaction and a colorimetric HTS coupled to an amidase was developed to screen solvent-tolerant NHase variants. The best mutant D3 (R55C/S51W/G41D/R98L) demonstrated remarkable catalytic efficiency (increased by 3.2-fold) and exceptional acrylamide tolerance (retaining >35 % activity at 50 % (v/v) acrylamide), whereas the wild-type was completely inactivated. In a process mimicking industrial acrylamide production, D3 catalyzes the formation of 482.5 g/L of product within 250 min. Ultimately, a productivity of 756 g/L/h/g DCW can be achieved through process optimization. Kinetic parameter analyses and molecular dynamic simulations revealed that strategic tunnel-widening mutations facilitated substrate ingress and enhanced tolerance to organic solvent. This structure-guided multi-optimization strategy not only resolves long-standing challenges in NHase mediated acrylamide synthesis but also lays a solid foundation for its industrial application.