Cloning of an intracellular poly[D(-)-3-hydroxybutyrate] depolymerase genefrom Ralstonia eutropha H16 and characterization of the gene product

An intracellular poly[D(-)3-hydrolrybutyrate] (PHB) depolymerase gene (phaZ ) has been cloned from Ralstonia eutropha H16 by the shotgun method, sequen ced, and characterized. Nucleotide sequence analysis of a 2,3-kbp DNA fragm ent revealed an open reading frame of 1,260 bp, encoding a protein of 419 a mino acids with a predicted molecular mass of 47,316 Da, The crude extract of Escherichia coli containing the PWB depolymerase gene digested artificia l amorphous PHB granules and released mainly oligomeric D(-)3-hydroxybutyra te, with some monomer. The gene product did not hydrolyze crystalline PHB o r freeze-dried artificial amorphous PHB granules. The deduced amino acid se quence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X -Gly, The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene was disrupted showed a higher PHB content compare d to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results in dicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.