Benzoate Degradation at low temperature by psychrotrophic Pseudomonas orientalis KOPRI 25931

Abstract

The psychrotrophic Pseudomonas orientalis KOPRI 25931 was originally isolated from soil on the Alps, Austria, for the ability to grow on benzoate as the sole carbon and energy source. KOPRI 25931 was able to grow on a wide range of temperatures (5-30 °C), having an optimal temperature of 30 °C and showing a capacity to degrade benzoate even at 5 °C. It indicates that KOPRI 25931 is a cold-adapted hydrocarbon degrader possessing cold-active catalytic enzymes. It was identified using phylogenetic analysis on the basis of 16S rRNA gene sequence. The growth substrate range experiments demonstrated that it has also the ability to grow on various hydrocarbon substrates including phenol, toluene, phthalate, and naphthalene. To study the benzoate degradation pathway in molecular level, PCR amplification was carried out with KOPRI 25931 genomic DNA and a degenerate primer set, which has been designed for the genes of aromatic dioxygenase large subunits. Successfully, a PCR product containing a gene fragment of multi-component aromatic dioxygenase was amplified, and then analyzed to be similar (93% identity) to an anthranilate dioxygenase large subunit from a Pseudomonas fluorescens strain. Now, RACE- and Inverse-PCR experiments are in progress to obtain a full ORF encoding a multi-component aromatic dioxygenase system, which might be a useful cold-active enzyme used in future industrial bioconversion processes.