title: Cloning, expression, and characterization of a wide-pH-range stable phosphite dehydrogenase from Pseudomonas sp. K in Escherichia coli.
abstract: A phosphite dehydrogenase gene (ptdhK) consisting of 1,011-bp nucleotides which encoding a peptide of 336 amino acid residues was cloned from Pseudomonas sp. K. gene ptdhK 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.
authors.affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, China.
authors.forename: Dan-Feng
authors.initials: DF
authors.lastname: Liu
authors.forename: Hai-Tao
authors.initials: HT
authors.lastname: Ding
authors.forename: Yi-Qing
authors.initials: YQ
authors.lastname: Du
authors.forename: Yu-Hua
authors.initials: YH
authors.lastname: Zhao
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Jia
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009930:Organic Chemicals / Q000494:pharmacology; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012997:Solvents / Q000494:pharmacology; D055672:Static Electricity; D013696:Temperature
chemical_list: D008670:Metals; D009930:Organic Chemicals; D011994:Recombinant Proteins; D012997:Solvents; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase