CLONING AND CHARACTERIZATION OF THE POLY(HYDROXYALKANOIC ACID)-DEPOLYMERASE GENE LOCUS, PHAZ1, OF PSEUDOMONAS-LEMOIGNEI AND ITS GENE-PRODUCT

Four different DNA fragments each coding for poly(hydroxyalkanoic acid ) depolymerase (phaZ1-phaZ4) were isolated in pUC plasmids from a geno mic library of Pseudomonas lemoignei in Escherichia coli. All recombin ant strains secreted a highly active poly(3-hydroxybutyric acid) depol ymerase and produced large translucent halos on an opaque medium conta ining poly(3-hydroxybutyric acid) granules. One DNA region (phaZ1) was present in seven independently isolated clones. Three other cloned DN A fragments were different from phaZ1 and from each other (phaZ2-phaZ4 ). In phaZ1, an open-reading frame of 1245 bp was identified from the nucleotide sequence of a 5435-bp MboI fragment (57 mol G+C/100 mol) of this region and encoded a novel poly(hydroxyalkanoic acid) depolymera se of P lemoignei, poly(3-hydroxybutyric acid) depolymerase C. A leade r-sequence peptidase-cleavage site was predicted from the deduced amin o acid sequence between Ala37 and Leu38. The calculated relative molec ular masses of the precursor and the putative mature protein were 4346 8 and 39581, respectively. The polypeptide contains a lipase consensus sequence (Gly-Xaa-Ser-Xaa-Gly) and an unusually high proportion of th reonine residues (22 of 36 amino acids) near the C-terminus. The N-ter minus of the deduced amino acid sequence of PhaZ1 differed from that o f the purified poly(3-hydroxybutyric acid) depolymerases A, B and the poly(3-hydroxyvaleric acid) depolymerase of P lemoignei. The phaZ1 gen e product, poly(3-hydroxybutyric acid) depolymerase C, was partially p urified from recombinant E. coli (pUC91=phaZ1). The purified protein w as specific for poly(hydroxyalkanoic acid) consisting of monomers of f our or five carbon atoms and for p-nithrophenylbutyrate as substrates. The polymer-hydrolyzing activity, but not the p-nitrophenylate estera se activity, was inhibited by complex media such as Luria-Bertani medi um and by soluble E. coli proteins. The enzyme protein did not cross-r eact with antibodies raised against purified poly(3-hydroxyvaleric aci d) depolymerase of P lemoignei.