AUTHOR=Liu Ye , Gong Jin-Song , Marshall George , Su Chang , Hall Michael , Li Heng , Xu Guo-Qiang , Shi Jin-Song , Xu Zheng-Hong 

TITLE=Protein engineering of NADH pyrophosphatase for efficient biocatalytic production of reduced nicotinamide mononucleotide

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=Volume 11 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1159965

DOI=10.3389/fbioe.2023.1159965

ISSN=2296-4185

ABSTRACT=NADH pyrophosphatase, a hydrolase catalyzing the phosphate bond of NADH to reduced nicotinamide mononucleotide (NMNH), has potential applications in the food, cosmetic and pharmaceutical industry. In this study, we describe a NADH pyrophosphatase derived from Escherichia coli (EcNudc). Strategies focusing on expression regulation including screening vectors, optimizing promoters and ribosome binding sites were utilized to enhance the productivity of EcNudc (1.8 U/mL). Moreover, protein engineering was adopted to further improve the catalytic properties of EcNudc, achieving 3.3-fold higher activity and 3.6-fold greater thermostability at 50℃. Furthermore, fermentation for the combined mutant R148A-H149E (EcNudc-M) production in a 7 L fermenter was implemented and the enzyme activity of EcNudc-M reached 33.0 U/mL. Finally, the EcNudc-M was applied in the catalysis of NADH with the highest NMNH yield of 16.65 g/L. Overall, the mutant NADH pyrophosphatase obtained in this paper will lay a foundation for the biocatalytic industrial production of NMNH and expand its application range.