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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• 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