Protein Engineering of NADH Pyrophosphatase for Efficient Biocatalytic Production of Reduced Nicotinamide Mononucleotide

NADH pyrophosphatase, a hydrolase catalyzing the phosphate bond of NADH to reduced nicotinamide mononucleotide, has potential applications in the food, cosmetic and pharmaceutical industry. Here, we investigated the effects of vector screening, promoter and RBS strategies on NADH pyrophosphatase expression and protein engineering on its enzymatic activity and thermal stability. In this study, we describe a NADH pyrophosphatase derived from (). Strategies focusing on expression regulation including screening vectors, optimizing promoters and ribosome binding sites were utilized to enhance the productivity of (1.8 U/mL). Moreover, protein engineering was adopted to further improve the catalytic properties of , achieving 3.3-fold higher activity and 3.6-fold greater thermostability at 50°C. Furthermore, fermentation for the combined mutant R148A-H149E () production in a 7 L fermenter was implemented and the enzyme activity of reached 33.0 U/mL. Finally, the was applied in the catalysis of NADH with the highest NMNH yield of 16.65 g/L. In conclusion, we constructed a commercially available genetically engineered strain with high activity and thermal stability of NADH pyrophosphatase, laying a broad foundation for the biocatalytic industrial production of NMNH and expand its application range.