Purification and Properties of <scp>d</scp>(−)‐3‐Hydroxybutyrate‐Dimer Hydrolase from <i>Zoogloea ramigera</i> I‐16‐M

Tanaka, Yoshimasa; Saito, Terumi; Fukui, Tetsuya; Tanio, Tomoharu; Tomita, Kazuyoshi

doi:10.1111/j.1432-1033.1981.tb05502.x

Cited by 51 publications

(20 citation statements)

References 28 publications

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“…Although the physiological role of the 3HB-oligomer hydrolase is not clear, it may be important to study this hydrolase in relation to extracellular degradation of PHB. Similar enzymes were found within the bacterial cells (8,16,25). Pseudomonas sp.…”

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Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

1997

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An extracellular D-(؊)-3-hydroxybutyrate oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Pseudomonas sp. strain A1. The purified enzyme hydrolyzed the D-(؊)-3-hydroxybutyrate dimer and trimer at similar rates. The enzyme activity was inhibited by a low concentration of diisopropylfluorophosphate. The molecular weight of the hydrolase was estimated to be about 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 10-kbp DNA fragment of A1 was detected by hybridization with the gene (2 kbp) of an extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Subsequent subcloning showed that a SmaI-KpnI fragment (2.8 kbp) was responsible for expression of the hydrolase in Escherichia coli and an in vitro transcription-translation system. The expressed protein detected by immunostaining had the same molecular weight as the purified enzyme from A1. The protein band detected in the in vitro transcription-translation system had a molecular size of 72 kDa. The nucleotide sequence of the SmaI-KpnI fragment was determined, and one open reading frame (2,112 nucleotides) was found. It specifies a protein with a deduced molecular weight of 72,876 (704 amino acids). In this sequence, the consensus sequence of serine-dependent hydrolysis, G-X-S-X-G, did not exist.Poly-3-hydroxybutyrate (PHB) is a unique intracellular reserve of organic carbon and/or chemical energy found in a wide variety of bacteria (6, 9) and is regarded as a potential biodegradable thermoplastic not derived from petroleum (12). Recently similar bacterial polyesters have been found, and these polyesters including PHB were called "polyhydroxyalkanoates" (PHAs) (7). Extracellular PHB metabolism is important in the application of these bacterial polyesters as biodegradable plastics. Many microorganisms that are capable of degrading PHB exist in the environment, and they secrete PHB depolymerases to degrade this polymer. There have been many studies on the extracellular PHB depolymerases from several bacteria and fungi (2,3,4,11,15,22,26,29). Genes for some enzymes were also cloned and sequenced (10,19,21). Alcaligenes faecalis T1, a PHB-degrading bacterium, secretes a D-(Ϫ)-3-hydroxybutyric acid oligomer hydrolase (3HB-oligomer hydrolase) (EC 3. 1.1.22) (23) besides a PHB depolymerase. Although the physiological role of the 3HB-oligomer hydrolase is not clear, it may be important to study this hydrolase in relation to extracellular degradation of PHB. Similar enzymes were found within the bacterial cells (8,16,25). Pseudomonas sp. strain A1 was isolated from activated sludge as a PHB-degrading bacterium (22) and was found to have an extracellular 3HB-oligomer hydrolase besides a PHB depolymerase. In this communication, we describe the purification and characterization of the 3HB-oligomer hydrolase and cloning and sequencing of its gene. MATERIALS AND METHODSBacterial strains, plasmids, and culture. Pseudomonas sp. strain A1 was isolated from activated sludge of the sewage treatment facility of...

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“…Chemicals were obtained as follows: butyl-Toyopearl, Tosoh (Tokyo; Japan) TEAE-cellulose, Serva Feinbiochemica. Dimeric and trimeric esters of D-(Ϫ)-3-hydroxybutyric acid were prepared as described previously (25). Purified PHB granules for the assay of PHB depolymerase were prepared as described by Smibert and Krieg (24).…”

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confidence: 99%

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

1997

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“…The type c enzyme was found in many other PHB-producing or PHB-degrading bacteria based on the BLAST search. In addition, three proteins whose molecular masses are about 30 kDa were reported to be 3HB-oligomer hydrolases in Pseudomonas lemoignei (3), Zoogloea ramigera (28), and Pseudomonas denitrificans (30). These enzymes also could be homologous to PhaZc.…”

Section: Discussionmentioning

confidence: 96%

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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“…The released free acid was titrated with 5 mM NaOH (pH 8.5) at 30°C under a nitrogen atmosphere. The linear 3HB-oligomers were prepared as described previously (29). The cyclic 3HB-oligomers were obtained from D. Seebach (21).…”

Section: Methodsmentioning

confidence: 99%

Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase

Kobayashi

Shiraki²,

Abe³

et al. 2003

J Bacteriol

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An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2 Reu ) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2 Reu . The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1 Reu ). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.Poly(3-hydroxybutyrate) (PHB), a homopolymer of R(Ϫ)-3-hydroxybutyrate (3HB), is a storage material produced by some bacteria under certain conditions (1). In the past few decades, the application of this biopolymer to biodegradable polymers or plastics has been studied extensively (12). In these studies, the extracellular metabolism of PHB has been clarified in many bacteria and some fungi (6, 7). However, only a few studies on the intracellular degradation of PHB have been published (13,14,17,19,20). An intracellular PHB (iPHB) depolymerase system in Rhodospirillum rubrum was first reported in 1964 and consisted of a thermostable activator and a thermolabile esterase (13). This system is still not well understood in spite of a recent reinvestigation (14). The molecular cloning of an iPHB depolymerase from Ralstonia eutropha H16 has been also reported (17). This enzyme (PhaZ1 Reu ) degraded artificial amorphous PHB granules but not crystalline PHB. A mutant lacking PhaZ1 Reu showed a higher PHB content compared to the wild-type in a nutrient-rich medium, but in this mutant the mobilization of PHB was not inhibited completely, suggesting that the cloned depolymerase gene is not the only gene responsible for the biodegradation of PHB in this bacterium (5, 17). In regard to this point, recently we found another esterase (PhaZ2 Reu ) that hydrolyzes 3HB-oligomers and cloned its gene (18). We examined the properties of the ...

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Purification and Properties of d(−)‐3‐Hydroxybutyrate‐Dimer Hydrolase from Zoogloea ramigera I‐16‐M

Cited by 51 publications

References 28 publications

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

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

Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase

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