Enzymatic degradation of poly[( R)-3-hydroxybutyrate]: secretion and properties of PHB depolymerase from Pseudomonas stutzeri

Abstract

The secretion mechanism and properties of an extracellular polyhydroxybutyrate (PHB) depolymerase of Pseudomonas stutzeri YM1006, which had been isolated from sea water, were studied in the culture containing poly [( R)-3-hydroxybutyrate] [P(3HB)], ( R)-3-hydroxybutyrate [( R)-3HB] or (S)-3-hydroxybutyrate [( S)-3HB] as sole carbon source. P. stutzeri YM1006 was able to grow on P(3HB), ( R)-3HB and ( S)-3HB. The bacterium secreted a PHB depolymerase into the culture supernatant during growth on P (3HB) and ( R)-3HB, while it did not secrete the enzyme on ( S)-3HB. The PHB depolymerase was purified to electrophoretic homogeneity from the culture medium of P. stutzeri by hydrophobic interaction chromatography and its molecular weight was determined as 60 kDa by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures up to 50 °C in aqueous solution at pH values of 6 to 12. The optimum activity of degrading P (3HB) by the enzyme was observed at pH 7.0. A kinetic study of enzymatic hydrolysis of P(3HB) film was carried out at temperatures from 30 to 45 °C at pH 7.4 in 0.1 M potassium phosphate buffer. The enzymatic degradation product contained monomer and dimer of 3-hydroxybutyric acid, and the monomer was a major product over the whole range of enzyme concentration.