Cloning and expression of the polyhydroxyalkanote depolymerase gene from Pseudomonas putida, and characterization of the gene product

A polyhydroxyalkanote (PHA) depolymerase gene (phaZ) was cloned by PCR from Pseudomonas putida and overexpressed in Escherichia coli as inclusion bodies. Nucleotide sequence analysis predicted an 852 by open reading frame encoding a protein of 283 amino acids with a predicted molecular weight of 31283 Da. The deduced amino acid sequence had at least 80% homology to the PHA depolymerase from other Pseudomonas strains and consisted a conserved lipase box-like sequence (G-X-S-102-X-G). The inclusion bodies were refolded and biochemically characterized. The depolymerase activity was optimal at 40 degreesC and pH 8.