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Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

Indian Journal of Microbiology volume 53, pages 297–302 (2013)Cite this article

Abstract

The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40 kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40 kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K m and V max values of recombinant P. putida ProDH were estimated to be 31 mM and 132 μmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30 °C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.

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Acknowledgments

We thank to the Biochemistry Dept., Pasteur Institute of Iran. The authors are grateful to Dr. Esfahani for his helpful discussions during this project.

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Affiliations

  1. Department of Biochemistry, Pasteur Institute of Iran, Pasteur Street, Tehran, Iran

    Eskandar Omidinia & Hamid Shahbaz Mohammadi

  2. Department of Marine Biology, Bandar Abbas Branch, Islamic Azad University, Bandar Abbas, Islamic Republic of Iran

    Rahman Mahdizadehdehosta

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  1. Eskandar Omidinia
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  2. Rahman Mahdizadehdehosta
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  3. Hamid Shahbaz Mohammadi
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Correspondence to Rahman Mahdizadehdehosta.

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Omidinia, E., Mahdizadehdehosta, R. & Mohammadi, H.S. Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84. Indian J Microbiol 53, 297–302 (2013). https://doi.org/10.1007/s12088-013-0375-2

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  • DOI: https://doi.org/10.1007/s12088-013-0375-2

Keywords

  • Characterization
  • Expression
  • Proline dehydrogenase (ProDH)
  • Pseudomonas putida