Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506

Phosphite dehydrogenase (PtxD), which catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD+ to NADH, has great potential for NADH regeneration in industrial biocatalysts. Here, we isolated a soil bacterium, Ralstonia sp. strain 4506, that grew at 45°C on a minimal medium containing phosphite as the sole source of phosphorus. A recombinant PtxD of Ralstonia sp. (PtxDR4506) appeared in the soluble fraction in Escherichia coli. The purified PtxDR4506 showed 6.7-fold greater catalytic efficiency (Vmax/Km) than the first characterized PtxD of Pseudomonas stutzeri (PtxDPS). Moreover, the purified PtxDR4506 showed maximum activity at 50°C, and its half-life of thermal inactivation at 45°C was 80.5 h, which is approximately 3,450-fold greater than that of PtxDPS. Therefore, we concluded that PtxDR4506, which shows high catalytic efficiency, solubility, and thermostability, would be useful for NADH regeneration applications.