Purification and Properties of an Extracellular Polyhydroxybutyrate Depolymerase from Pseudomonas mendocina DSWY0601

An extracellular polyhydroxybutyrate(PHB) depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was approximately 59800 at the optimal temperature and pH value being 50 degrees C and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20-50 degrees C and sensitive to pH value within a pH range of 8.0-9.5. PHB depolymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate(P3/4HB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate(PHBV) but did not degrade poly(lactic acid)(PLA), poly(butylene succinate)(PBS) or poly(caprolactone)(PCL). PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride(PMSF), H2O2 and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer(3HB) by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis of phaZ(pm) gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA(dPHA(SCL), PHA polyhydroxyalkanoate) depolymerase containing catalytic domain, linker and substrate-binding domain.