Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16

Abstract

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...

Identification and Characterization of the Bacillus thuringiensi s phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

“…As far as is known, the intracellular PHB depolymerase PhaZa1 of Ralstonia eutropha H16 has no classical lipase box sequence and its PHB-hydrolyzing activity is relatively weak (1, 33). The intracellular PhaZb and PhaZc of Ralstonia eutropha H16 are actually 3HB oligomer hydrolases (20,21). The intracellular PhaZd of Ralstonia eutropha H16 shows similarity with the type I catalytic domain of extracellular PHB depolymerases from bacteria such as R. pickettii T1 and Paucimonas lemoignei (17) and produces various 3HB oligomers from amorphous PHB as hydrolytic products.…”

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

“…PhaZb Weu probably participates also in the degradation of PHB, because a synergistic effect for PhaZa1 Weu has been reported (20). Although PhaZc Weu also has weak PHB-hydrolyzing activity, most PhaZc Weu occurs in the cytosolic fraction (19). Therefore, PhaZc Weu probably mainly hydrolyzes 3HB oligomers produced by other depolymerases during the mobilization of PHB together with PhaZb Weu .…”

“…In Wautersia eutropha (formerly Ralstonia eutropha) H16, three types of intracellular PHB depolymerase or 3HB oligomer hydrolase genes have been cloned and some features of the gene products have been reported (19,20,21,31,32). PhaZa1 Weu (formerly PhaZ1 Reu ) exists in PHB inclusion bodies and degrades artificial amorphous PHB (20,30).…”

“…Additionally, enzymes with properties similar to PhaZa1 Weu have been studied in other bacteria (6,18). PhaZb Weu (formerly PhaZ2 Reu ) (20,32) and PhaZc Weu (19) mainly degrade 3HB oligomers and the degradation product is 3HB monomer. Another PHB depolymerase or related hydrolase in W. eutropha H16 with different properties from these enzymes has been reported (35), but it has not been fully characterized.…”

Degradation of Polyhydroxyalkanoate (PHA): a Review

“…Accumulated PHA granules in the cells are hydrolyzed as carbon and energy sources (Madison and Huisman, 1999;Luengo et al, 2003). From there, PHA is broken down to 3-hydroxyalkanoic acid, a monomeric component by PHA depolymerase and oligomer hydrolase (Kobayashi et al, 2005). If the PHA is comprised of only one type of monomer, for example, 3-hydroxybutyrate, the resulting PHA is called poly(3-hydroxybutyrate) [P(3HB)] homopolymer.…”