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

A polyhydroxyalkanote (PHA) depolymerase gene ( pha Z) was cloned by PCR from Pseudomonas putida and over-expressed in Escherichia coli as inclusion bodies. Nucleotide sequence analysis predicted an 852 bp 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 degrees C and pH 8.