Molecular Engineering of Nicotinamide Riboside Kinase and Process Optimization for Efficient Nicotinamide Mononucleotide Production.

Nicotinamide mononucleotide (NMN) plays vital physiological roles as a vitamin B derivative, with nicotinamide riboside kinase (NRK) serving as a key enzyme for its efficient and environmentally friendly synthesis. In this study, semi-rational design was employed to modify the <i>Hi</i>-NRK enzyme at the molecular level, leading to the identification of a critical mutant, <i>Hi</i>-NRK^G8S. This variant exhibited a twofold increase in enzymatic activity and significantly enhanced thermal stability, extending its half-life at 40 °C from 4 to 8 h. By optimizing reaction conditions, NMN yield reached 94.17% at a nicotinamide riboside (NR) substrate concentration of 50 g/L. Further addition of adenylate kinase (ADK) to facilitate ATP recycling increased the yield to 97.24% at 75 g/L NR. This study establishes a foundation for industrial-scale, efficient, and green NMN production.