Characterization of a poly(3-hydroxybutyrate) depolymerase from Aureobacterium saperdae: Active site and kinetics of hydrolysis studies

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase was purified from Aureobacterium saperdae cultural medium by using hydrophobic interaction chromatography. The isolated enzyme was composed of a single polypeptide chain with a molecular mass of 42.7 kDa as determined by SDS-PAGE and by native gel filtration on TSK-HW-55S. The enzyme was not a glycoprotein. Its optimum activity occurred at pH 8.0 and it showed a broad pH stability, ranging from pH 3 to pH 11. N-Bromosuccinamide and 2-hydroxy-5-nitrobenzyl bromide completely inactivated the enzyme, suggesting the involvement of tryptophan residues at the active site of the protein. The enzyme was very sensitive to diisopropyl fluorophosphate and diazo-DL-norleucine methyl ester, showing the importance of serine and carboxyl groups. The modification of cysteine residues by p-hydroxy mercuricbenzoate did not cause a loss of activity, whereas dithiothreitol rapidly inactivated the enzyme, revealing the presence of disulfide bonds. A saperdae depolymerase acted on the surface layer of PHB films and the degradation proceeded by surface erosion releasing monomers and dimers of 3-hydroxybutric acid. The degradation of PHB films by A. saperdae depolymerase was partially inhibited in the presence of excess amounts of enzyme. This phenomenon, already observed by Mukai et al. with poly(hydroxyalkanoates) depolymerases from Alcaligenes faecalis, Pseudomonas pickettii, and Comamonas testosteroni, was analyzed according to the kinetic model proposed by these authors. The experimental data evidenced a general agreement with the kinetic model, although higher initial degradation rates were found with A. saperdae depolymerase.