Purification and Characterization of Extracellular Poly(3-hydroxybutyrate) Depolymerase from Penicillium simplicissimum LAR13

An extracellular PHB depolymerase was purified from P. simplicissimum LAR13 cultural medium by Sepharose CL-6B chromatography. When the fungus was grown in a basal salt medium with poly(3-hydroxybutyrate) (PHB) as the sole carbon source, PHB depolymerase production reached maximum at its stationary phase. The mycelial growth rate was higher at 37$^{\circ}C$ than at 30$^{\circ}C$ and even higher than at 25$^{\circ}C$, However, the enzyme production was lower at 37$^{\circ}C$ than 30$^{\circ}C$ or 25$^{\circ}C$. The isolated enzyme is composed of a single polypeptide chain with a molecular mass of about 36 kDa as determined by SDS-PAGE. The optimum conditions for the enzyme activity are pH 5.0 and 45$^{\circ}C$. The enzyme was stable for 30 min at a temperature lower than 50$^{\circ}C$, and stable at pH higher than 2.0 but it was unstable at pH 1.0.1 mM Fe$\^$2+/ reduced the enzyme activity by 56% and the enzyme was inhibited almost completely by 4 mM Fe$\^$2+/ . The enzyme was partially inhibited by phenylmethylsulfonyl fluoride and was very sensitive to diazo-DL-norleucine methyl esters dithiothreitol and mercuric ion. However, N-p - tosyl - L - Iysinechloromethyl ketone, p -hydroxymercuricbenzoate and N- acetylimidazole had no influence upon its activity.