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Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from Pseudomonas rhodesiae CIP 107491 and Production of the Recombinant Protein in Escherichia coli

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Abstract

The alpha-pinene oxide lyase (Prα-POL) from Pseudomonas rhodesiae CIP107491 belongs to catabolic alpha-pinene degradation pathway. In this study, the gene encoding Prα-POL has been identified using mapping approach combined to inverse PCR (iPCR) strategy. The Prα-POL gene included a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7 kDa protein, with a theoretical isoelectric point (pI) of 5.23. The amino acids sequence analysis showed homologies with those of proteins with unknown function from GammaProteobacteria group. Identification of a conserved domain in amino acid in positions 18 to 190 permitted to classify Prα-POL among the nuclear transport factor 2 (NTF2) protein superfamily. Heterologous expression of Prα-POL, both under its native form and with a histidin tag, was successfully performed in Escherichia coli, and enzymatic kinetics were analyzed. Bioconversion assay using recombinant E. coli strain allowed to reach a rate of isonovalal production per gramme of biomass about 40-fold higher than the rate obtained with P. rhodesiae.

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Funding

This work was supported by grants of Ministère de la Recherche et de l’Enseignement Supérieur. This study was developed in partnership with students of the Engineering School of Polytech’Clermont-Ferrand at University of Clermont-Auvergne (UCA).

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Authors and Affiliations

  1. Institut Pascal UMR CNRS 6602, Polytech Clermont-Ferrand, Université Clermont Auvergne (UCA), F-63178, Aubière, France

    Pascal Dubessay, Christian Larroche & Pierre Fontanille

Authors
  1. Pascal Dubessay
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  2. Christian Larroche
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  3. Pierre Fontanille
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Correspondence to Pascal Dubessay.

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Dubessay, P., Larroche, C. & Fontanille, P. Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from Pseudomonas rhodesiae CIP 107491 and Production of the Recombinant Protein in Escherichia coli. Appl Biochem Biotechnol 185, 676–690 (2018). https://doi.org/10.1007/s12010-017-2685-z

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