MOLECULAR CHARACTERIZATION OF THE EXTRACELLULAR POLY(3-HYDROXYOCTANOIC ACID) [P(3HO)] DEPOLYMERASE GENE OF PSEUDOMONAS-FLUORESCENS GK13 AND OF ITS GENE-PRODUCT

phaZ(Pfl), the gene encoding the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, was cloned, sequenced, and characterized. It comprises 837 bp and is transcribed as a monocistronic message of about 950 bp from a putative sigma(70)-like promoter 32 bp upstream of the ATG start codon. The deduced protein of 278 amino acids reveals a typical leader peptide at its N terminus. When expressed in Escherichia coli, the mature depolymerase started with Ala-23, whereas the mature enzyme purified from P. fluorescens GK13 started with both Leu-34 and Arg-35 determining proteins of 26,687 and 26,573 Da, respectively. The depolymerase is a strongly hydrophobic protein and includes the lipase consensus sequence Gly-X-Ser-X-Gly, which is known for serine hydrolases. Replacement of the central residue, Ser-172, in the corresponding sequence (Gly-Ile-Ser-Ser-Gly) of PhaZ(Pfl) with alanine resulted in complete loss of enzyme activity, indicating that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.