Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.Polyhydroxyalkanoates, which are bacterial polyesters, are a group of storage materials that are used as sources of carbon and energy (1). One of the most abundant polyhydroxyalkanoates is poly(3-hydroxybutyrate) (PHB), a homopolymer of D-(Ϫ)-3-hydroxybutyrate (3HB). PHB is synthesized from D-(Ϫ)-3-hydroxybutyryl-coenzyme A by PHB synthase (16,18) and is mobilized by intracellular PHB depolymerases (15,19).PHB-producing bacteria contain intracellular PHB depolymerases. Intracellular PHB depolymerases have been found in Rhodospirillum rubrum (15), Paracoccus denitrificans (4), and Wautersia eutropha (19) (formerly Ralstonia eutropha). In W. eutropha H16, a gene encoding an intracellular PHB depolymerases, phaZ1 (19), and a gene encoding a 3HB-oligomer hydrolase, phaZb (formerly phaZ2) (10,20), have been cloned, and some properties of their products have been reported. Both hydrolases showed hydrolytic activity for amorphous PHB. PhaZb hydrolyzed 3HB oligomers at a high specific activity and degraded PHB into 3HB monomers at a lower specific activity.The function of PhaZ1 or PhaZb in vivo has been examined by constructing a phaZ1 or phaZb deletion strain (5, 10, 19). The deletion mutants exhibit considerably less degradation of PHB in vivo in some conditions. However, even a PhaZ1-PhaZb double-deletion mutation did not completely inhibit the mobilization of PHB (10). Therefore, it has been suggested that there are other depolymerases or related hydrolases (10).Since the amino acid sequence of the first intracellular PHB depolymerase, PhaZ1, was reported for W. eutropha H16, several isoenzymes have been found. Recently, the genome sequences of W. eutropha and related bacteria have become available. Four putative PHB depolymerase genes homologous to phaZ1 have been identified using the genome sequence of Wautersia metallidurans (32), the sequence of the megaplasmid of W. eutropha (25), and the incomplete gen...
