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 degrees C on a minimal medium containing phosphite as the sole source of phosphorus. A recombinant PtxD of Ralstonia sp. (PtxD(R4506)) appeared in the soluble fraction in Escherichia coil. The purified PtxD(R4506) showed 6.7-fold greater catalytic efficiency (V-max/K-m) than the first characterized PtxD of Pseudomonas stutzeri (PtxD(PS)). Moreover, the purified PtxD(R4506) showed maximum activity at 50 degrees C, and its half-life of thermal inactivation at 45 degrees C was 80.5 h, which is approximately 3,450-fold greater than that of PtxD(PS). Therefore, we concluded that PtxD(R4506), which shows high catalytic efficiency, solubility, and thermostability, would be useful for NADH regeneration applications. (C) 2011, The Society for Biotechnology, japan. All rights reserved.