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

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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 , Mg2+, Fe2+, Fe3+, Co2+, and EDTA, and PTDH-K exhibited different tolerance to various organic solvents.

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Acknowledgments

This study was supported by the National Hi-Tech Research and Development Program (863) of China (no. 2007AA06Z329), National Natural Science Foundation of China (31070079), the Science and Technology Project of Zhejiang Province (2008 C13014-3, 2011 C13016), and the International Cooperation Project in Science and Technology of Zhejiang Province (no. 2008 C14038).

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Authors and Affiliations

  1. Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China

    Dan-Feng Liu, Hai-Tao Ding, Yi-Qing Du, Yu-Hua Zhao & Xiao-Ming Jia

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  1. Dan-Feng Liu
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  2. Hai-Tao Ding
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  3. Yi-Qing Du
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  4. Yu-Hua Zhao
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  5. Xiao-Ming Jia
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Correspondence to Yu-Hua Zhao or Xiao-Ming Jia.

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Liu, DF., Ding, HT., Du, YQ. et al. Cloning, Expression, and Characterization of a Wide-pH-Range Stable Phosphite Dehydrogenase from Pseudomonas sp. K in Escherichia coli . Appl Biochem Biotechnol 166, 1301–1313 (2012). https://doi.org/10.1007/s12010-011-9518-2

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  • DOI: https://doi.org/10.1007/s12010-011-9518-2

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