title: Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506.
abstract: 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. (PtxD(R4506)) appeared in the soluble fraction in Escherichia coli. 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°C, and its half-life of thermal inactivation at 45°C was 80.5h, 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.
authors.affiliation: Department of Molecular Biotechnology, Graduate School of Advanced Sciences ofMatter, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8530, Japan.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Hirota
authors.forename: Sho-taro
authors.initials: ST
authors.lastname: Yamane
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Fujibuchi
authors.forename: Kei
authors.initials: K
authors.lastname: Motomura
authors.forename: Takenori
authors.initials: T
authors.lastname: Ishida
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ikeda
authors.forename: Akio
authors.initials: A
authors.lastname: Kuroda
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006207:Half-Life; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D043367:Ralstonia / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase