Degradation of polyhydroxyalkanoate (PHA) by three Pseudomonas spp., isolated from fuel-oil contaminated soil and identified as Ps. fluorescens, Ps. aeruginosa and Ps. putida on the basis of 16S rRNA sequence analysis, was investigated. Degradation of PHAs was determined as molecular weight loss measured by gel permeation chromatography and morphological changes in poly-3-hydroxybutyrate films observed by scanning electron microscopy. Both, decrease in molecular weight (7-17%) and weight loss (up to 29%) of solution-cast films were evidence of the isolates secreting an active depolymerase responsible for degradation of PHA. The kinetic parameters, V-max and Km, for depolymerase activity of Ps. aeruginosa in the presence of p-nitrophenylbutyrate as substrate were determined to be 2.8 mumol min(-1) and 2.8 mM, respectively, in 50 mM phosphate buffer, pH 7.9, at 30degreesC. Stimulation of activity by Na+, K+, Ca2+ and Mg2+ at 1 mM concentration indicated a requirement for metal ions as a cofactor for activity. Inhibition of the extracellular depolymerases of the three strains in the presence of p-methylphenyl sulfonylfluoride, cyanide, azide, deoxycholate or EDTA confirms the presence of an enzyme in the isolates similar to poly(3-hydroxybutyrate) degrading hydrolases reported earlier. (C) 2003 Elsevier Ltd. All rights reserved.