Cloning, Expression And Characterization Of A Phosphite Dehydrogenase From Pseudomonas Sp.M In Escherichia Coli

Phosphite dehydrogenase involved in the metabolism of phosphite in organisms, it catalyzed oxidation of phosphite coupled with reduction of NAD(P)+ to generate NADPH. In the common used coenzyme regeneration system, the phosphite dehydrogenase has many advantages such as low K_m, almost no reverse reaction, high concentration of the product phosphate does not inhibit the reaction, wide pH stability etc., these properties bring about higher using value and broad application prospect in coenzyme regeneration process of industrial biocatalysis.In this dissertation, we started from homologous sequences of phosphite dehydrogenases, using some bacteria preserved in our laboratory as the target, cloned phosphite dehydrogenase gene from Pseudomonas sp. M strain by molecular biology method, constructed recombinant expression plasmid of phosphite dehydrogenase gene, finally got the recombinant phosphite dehydrogenase by inducing expression and affinity chromatography purification, and then we studied the detailed properties of this phosphite dehydrogenase.The phosphite dehydrogenase gene consists of 1011bp nucleotides encoding a peptide of 336 amino acid residues. The upstream gene was ptxC and downstream gene was ptxE indicated that the phosphite dehydrogenase gene was situated in ptxABCDE operon, which is homologous in Pseudomonas stutzeri WM88.Recombinant phosphite dehydrogenase was purfied by Ni-NTA affinity chromatography. The purified phosphite dehydrogenase displayed a single band in SDS-PAGE. Compared with the standard protein marker, molecular weight of the recombinant enzymes was calculated as 35kDa.The properties of this recombinant phosphite dehydrogenase was intensively studied. The results showed that: The optimum temperature was 40℃, after treating at different temperatures at pH 7.0 in MOPS buffer for 30min, residual enzyme activity was measured and we found that the recombinant enzyme is most stable below 40℃; it's optimal pH value is 7.0, after treated at different pH at 25℃for 1h, we measured the residual enzyme activity and found that the enzyme is most stable at pH 7.0; the Km and Kcat for phosphite of the recombinant enzyme were 0.633±0.036 mM and 2.057±0.018 s-1, the Km and Kcat for NAD were 0.171±0.013 mM and 2.789±0.029 s-1 respectively.These studies have provided a reliable data support for the industrial production, and has laid the foundation for further study of the catalytic mechanism and three-dimensional structure of this enzyme.