Cloning, Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from Pseudomonas sp. K in Escherichia coli

• Published in: Applied biochemistry and biotechnology 2012-03, Vol.166 (5), p.1301-1313
• Main Authors: Liu, Dan-Feng, Ding, Hai-Tao, Du, Yi-Qing, Zhao, Yu-Hua, Jia, Xiao-Ming
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Base Sequence; Biochemistry; Biotechnology; Chemistry; Chemistry and Materials Science; Cloning; Cloning, Molecular; Dehydrogenase; E coli; Enzyme Stability; Escherichia coli; Escherichia coli - genetics; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Metals - pharmacology; Models, Molecular; Molecular Sequence Data; NADH, NADPH Oxidoreductases - chemistry; NADH, NADPH Oxidoreductases - genetics; NADH, NADPH Oxidoreductases - isolation & purification; NADH, NADPH Oxidoreductases - metabolism; Organic Chemicals - pharmacology; Organic solvents; Protein Conformation; Pseudomonas; Pseudomonas - enzymology; Pseudomonas - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Solvents - pharmacology; Static Electricity; Temperature; Temperature effects
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-011-9518-2

A phosphite dehydrogenase gene ( ptdh K) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdh K was expressed in Escherichia coli BL21 (DE3) and the corresponding recombinant enzyme was purified by metal affinity chromatography. The recombinant protein is a homodimer with a monomeric molecular mass of 37.2 kDa. The specific activity of PTDH-K was 3.49 U mg −1 at 25 °C. The recombinant PTDH-K exhibited maximum activity at pH 3.0 and at 40 °C and displayed high stability within a wide range of pHs (5.0 to 10.5). PTDH-K had a high affinity to its natural substrates, with K m values for sodium phosphite and NAD of 0.475 ± 0.073 and 0.022 ± 0.007 mM, respectively. The activity of PTDH-K was enhanced by Na + , NH 4 + , Mg 2+ , Fe 2+ , Fe 3+ , Co 2+ , and EDTA, and PTDH-K exhibited different tolerance to various organic solvents.