Cloning and Sequencing of an Intracellular D(-)-3-Hydroxybutyrate Oligomer Hydrolase from Acidovorax sp. Strain SA1 and Purification of the Enzyme

Sugiyama, Akinori; Shiraki, Mari; Kobayashi, T; Morikawa, Gakushi; Yamamoto, Mizuho; Yamaoka, Michiyo; Saito, Terumi

doi:10.1007/s00284-001-0093-6

Cited by 6 publications

(5 citation statements)

References 19 publications

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“…Recently, several studies on intracellular PHB depolymerases, e.g. from R. eutropha , and on 3‐hydroxybutyrate oligomer hydrolases have been performed [6,12–15,26–28]. In this contribution we were able to purify a protein (activator) that was able to transform nPHB granules isolated from different sources in vitro into an activated form that is a substrate for the soluble PHB depolymerase of R. rubrum .…”

Section: Discussionmentioning

confidence: 99%

The activator of theRhodospirillum rubrumPHB depolymerase is a polypeptide that is extremely resistant to high temperature (121Â°C) and other physical or chemical stresses

Handrick

Technow

Reichart

et al. 2004

FEMS Microbiology Letters

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Hydrolysis of native (amorphous) polyhydroxybutyrate (nPHB) granules isolated from different sources by soluble PHB depolymerase of Rhodospirillum rubrum in vitro requires the presence of a heat-stable compound (activator). The activator was purified and was resistant against various physical and chemical stresses such as heat (up to 130 ‡C), pH 1^12, dryness, oxidation by H 2 O 2 , reducing and denaturing compounds (2-mercaptoethanol, 5 M guanidinium-HCl) and many solvents including phenol/chloroform. The activator coding gene was identified by N-terminal sequencing of the purified protein, and the deduced protein showed significant homology to magnetosome-associated protein (Mms16) of magnetotactic bacteria. Analysis of the activation process in vitro showed that the activator acts on nPHB granules but not on the depolymerase. The effect of the activator could be mimicked by pretreatment of nPHB granules with trypsin or other proteases but protease activity of the purified activator was not detected. Evidence is shown that different mechanisms were responsible for activation of nPHB by trypsin and activator, respectively. PHB granule-associated protein (PhaP) of Ralstonia eutropha nPHB granules were cleaved by trypsin but no cleavage occurred after activator treatment. Hydrolysis of artificial protein-free PHB granules coated with negatively charged detergents (sodium dodecyl sulfate (SDS), cholate but not cetyltrimethylammonium bromide (CTAB)) did not require activation and confirmed that surface layer proteins of nPHB granules are the targets of the activator rather than lipids. All experimental data are in agreement with the assumption that trypsin and the activator enable the PHB depolymerase to find and to bind to the polymer surface: trypsin by removing a portion of proteins from the polymer surface, the activator by modifying the surface structure in a not yet understood manner presumably by interaction with phasins of the proteinous surface layer of nPHB.

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Section: Discussionmentioning

confidence: 99%

The activator of theRhodospirillum rubrumPHB depolymerase is a polypeptide that is extremely resistant to high temperature (121Â°C) and other physical or chemical stresses

Handrick

Technow

Reichart

et al. 2004

FEMS Microbiology Letters

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“…Some enzymes (PhaZc in this study, PhaZc Weu ) hydrolyze the 3HB pentamer most efficiently and exhibit weak hydrolytic activity with amorphous PHB, but another PhaZc in Acidovorax sp. strain SA1 (PhaZc Asp ) (27) hydrolyzes the 3HB dimer at a high rate with no PHB-hydrolyzing activity. The type c enzyme was found in many other PHB-producing or PHB-degrading bacteria based on the BLAST search.…”

Section: Discussionmentioning

confidence: 99%

“…The molecular mass of PhaZc (32 kDa) is about one-half that of PhaZb, and the amino acid sequence of PhaZc resembles the amino acid sequence of an intracellular 3HB-oligomer hydrolase of Acidovorax sp. strain SA1 (27). There- 8.2).…”

Section: Discussionmentioning

confidence: 99%

“…In this study, a novel 3HB-oligomer hydrolase of W. eutropha was discovered using the amino acid sequence of an intracellular 3HB-oligomer hydrolase of Acidovorax sp. strain SA1 (27) as a probe. From a BLAST search using this probe, a candidate for an intracellular 3HB-oligomer hydrolase gene was identified in the genome sequence of Ralstonia solanacearum, a close relative of W. eutropha.…”

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confidence: 99%

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

et al. 2005

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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...

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“…Some enzymes (PhaZc in this study, PhaZc Weu ) hydrolyze the 3HB pentamer most efficiently and exhibit weak hydrolytic activity with amorphous PHB, but another PhaZc in Acidovorax sp. strain SA1 (PhaZc Asp ) (Sugiyama et al 2002) hydrolyzes the 3HB dimer at a high rate with no PHB-hydrolyzing activity. Some of the enzymatic properties of PhaZc in vitro clearly suggest that the enzyme is a specific 3HB oligomer hydrolase, not a lipase or a nonspecific esterase, and it may function partly as a PHB depolymerase.…”

Section: Short-chain-length Phbmentioning

confidence: 99%

Degradation of Natural and Artificial Poly[(R)-3-hydroxyalkanoate]s: From Biodegradation to Hydrolysis

Guérin

Renard

Langlois

2009

Microbiology Monographs

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Biodegradability of polymers has drawn much attention as a solution to problems concerning the global environment and biomedical technologies. Poly[(R)-3-hydroxybutyrate] and its copolymers are representative biodegradable polyesters which can be degraded in natural environments such as soil, sludge, freshwater, and seawater where many microorganisms utilize the degraded products as a carbon source. The ability to degrade poly[(R)-3-hydroxyalkanoate]s (PHAs) is widely distributed among fungi and bacteria and depends on the extracellular PHA depolymerases, which are carboxyesterases, and on the physical state of the polymer (amorphous or crystalline). Intracellular depolymerase systems lead to CO 2 and H 2 O when bacteria need energy or carbon sources. All polyesters are susceptible degradation by simple hydrolysis to some extent. The degradation rate is very dependent on the chemical structure and the crystallinity of the materials.

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Cloning and Sequencing of an Intracellular D(-)-3-Hydroxybutyrate Oligomer Hydrolase from Acidovorax sp. Strain SA1 and Purification of the Enzyme

Cited by 6 publications

References 19 publications

The activator of theRhodospirillum rubrumPHB depolymerase is a polypeptide that is extremely resistant to high temperature (121Â°C) and other physical or chemical stresses

The activator of theRhodospirillum rubrumPHB depolymerase is a polypeptide that is extremely resistant to high temperature (121Â°C) and other physical or chemical stresses

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

Degradation of Natural and Artificial Poly[(R)-3-hydroxyalkanoate]s: From Biodegradation to Hydrolysis

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