Expression of Nicotinamide Phosphoribosyltransferase in Escherichia coli and Catalytic Synthesis of Nicotinamide Mononucleotide
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Abstract:
Nicotinamide phosphate ribose transferase (Nampt) is an important enzyme in the bioenzymatic synthesis of nicotinamide ribotide (NMN), and is used to catalyze the synthesis of NMN from nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP). In this study, Nampt from Meiothermus ruber was expressed intracellularly in the Escherichia coli expression system, and the expressed product was purified for the examinations of enzymatic properties and catalysis for the synthesis of NMN. Recombinant strains were induced in shake flasks at a low temperature (16 ℃) for 21 h. The fermented cells were collected and disrupted ultrasonically. After the disruption, the supernatant was purified by Ni-NTA chelation affinity chromatography. SDS-PAGE results showed that the size of the expressed and purified product was about 55 ku (which was consistent with the expected protein molecular weight). The optimal reaction temperature of the recombinant Nampt was 45 ℃, with the optimal pH as 6. Enzyme kinetic analysis showed that the Km, Vmax, and kcat of the enzyme on the substrate NAM were 0.39 μmol/L, 3.20 μmol / (mg·min), and 1.39 1/s, respectively. The enzyme was used to catalyze the production of NMN, and the product NMN yield could reach 30 mg/L with an addition of the substrate PRPP within the 10 min reaction. In this study, a Nampt with excellent enzymatic properties and thermal stability was successfully expressed, and applied to a single enzyme-catalyzed production of NMN with a higher yield and production efficiency. This research has laid the foundation for the application of bioenzymatic synthesis of NMN.
