Enhancing the catalytic efficiency and thermostability of NADH oxidase by a multi-strategy computational approach.

NADH oxidase (NOX) plays a crucial role in diverse fields, including medicine, food processing, and environmental protection, by catalyzing the oxidation of NADH to NAD⁺. This reaction is essential for biosensor performance, metabolic regulation, anti-aging processes, and the degradation of industrial pollutants. In this study, a NOX (EfNOX) was obtained through gene cloning from Enterococcus faecium. However, its limited thermal stability and catalytic efficiency constrained its industrial applications. To address these limitations, rational design strategies were employed to generate a double mutant, N211M/Q293L. The mutant exhibited a half-life of 27 min at 50℃ and a specific activity of 24.9 U/mg, representing 2.08-fold and 1.87-fold improvements, respectively, over the wild-type EfNOX. These enhancements are expected to promote the applicability of EfNOX in high-temperature environments and its industrial application potential. Collectively, this work offers a practical and efficient strategy for enhancing NOX and other industrial enzymes.