Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Saito, Terumi; Saegusa, Haruhisa; Miyata, Yoshiaki; Fukui, Tetsuya

doi:10.1016/0378-1097(92)90327-k

Cited by 12 publications

(18 citation statements)

References 18 publications

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“…The soluble intracellular PHB depolymerase from R. rubrum is active at pH 8.0 toward the native PHB granules from B. megaterium (16). The intracellular PHB depolymerase in soluble fractions from Z. ramigera I-16-M (18) and R. eutropha (21) also has a high optimum pH. PHB depolymerase characterized in this study was found only as a PHB granule-bound form.…”

Section: Discussionmentioning

confidence: 58%

“…In intracellular degradation of PHB in Ralstonia eutropha, weak hydrolysis activity against [ 14 C]PHB granules independent of the harvest time of the cells was reported (5). We have detected intracellular PHB depolymerase activity in Zoogloea ramigera I-16-M (18) and R. eutropha H16 (21), using proteasetreated native PHB granules as a substrate.…”

Section: Poly[d(ϫ)-3-hydroxybutyrate] (Phb) a Homopolymer Of D(ϫ)-3-mentioning

confidence: 99%

“…It is not easy to measure activity of the intracellular PHB depolymerase, because the enzyme can digest only amorphous PHB (16). Protease-treated native PHB granules show activity against cell extract from Z. ramigera I-16-M and R. eutropha H16, but they still have some autodigestive activity (18,21). Therefore, they may not be suitable for measuring low-level activity.…”

Section: Poly[d(ϫ)-3-hydroxybutyrate] (Phb) a Homopolymer Of D(ϫ)-3-mentioning

confidence: 99%

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Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene fromRalstonia eutrophaH16 and Characterization of the Gene Product

et al. 2001

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An intracellular poly[D(؊)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned fromRalstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(؊)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence 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 compared 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 indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha., is a storage material produced by some bacteria in response to environmental stress. The PHB biosynthesis genes from such bacteria have been cloned in Escherichia coli and studied in detail (13,24,26). However, only a few studies on the intracellular degradation of PHB have been published. In an in vitro system consisting of native PHB granules from Bacillus megaterium and the soluble fraction from PHB-depleted cells of Rhodospirillum rubrum, the existence of a thermostable activator and a thermolabile depolymerase has been reported (16). Various chemical and physical treatments inactivate the native PHB granules. In intracellular degradation of PHB in Ralstonia eutropha, weak hydrolysis activity against [ 14 C]PHB granules independent of the harvest time of the cells was reported (5). We have detected intracellular PHB depolymerase activity in Zoogloea ramigera I-16-M (18) and R. eutropha H16 (21), using proteasetreated native PHB granules as a substrate.A few intracellular poly-3-hydroxyoctanoate (PHO) depolymerase genes have been cloned. Huisman et al. have cloned an intracellular PHO depolymerase gene from Pseudomonas oleovorans using a PHO degradation mutant that cannot degrade PHO (8). Timm and Steinbüchel have cloned a PHO depolymerase gene from Pseudomonas aeruginosa PAO1 by hybridization using information on the DNA sequence of P. oleovorans (29). In both cases, the gene products have yet to be characterized. Although many extracellular PHB depolymerase genes have been cloned (11), no intracellular PHB depolymerase gene has been cloned to date. We tried unsuccessfully to clone the intracellular PHB depolymerase gene (phaZ) in R. eutropha by Southern hybridization using an extracellular PHB depolymerase gene as a probe. Therefore, we performed shotgun gene cloning by assaying enzyme activity of clones expressed...

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Section: Discussionmentioning

confidence: 58%

Section: Poly[d(ϫ)-3-hydroxybutyrate] (Phb) a Homopolymer Of D(ϫ)-3-mentioning

confidence: 99%

Section: Poly[d(ϫ)-3-hydroxybutyrate] (Phb) a Homopolymer Of D(ϫ)-3-mentioning

confidence: 99%

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Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene fromRalstonia eutrophaH16 and Characterization of the Gene Product

et al. 2001

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“…The methods for nPHB and dPHB granule preparation differ highly among PHA research laboratories: while Merrick et al and our laboratory isolated nPHB granules by glycerol density gradient centrifugation (32,34), others centrifuged crude cell extracts without glycerol and incubated the obtained polymer fraction with hydrolytic enzymes (25). The Saito laboratory used artificial PHB granules prepared according to the procedure described previously by Horowitz and Sanders (20) and used different surfactants such as oleate or phosphatidylglycerol in early investigations of intracellular PHB degradation (40,42,43). In recent studies, they used native PHB granules obtained by density gradient centrifugation (glycerol and sucrose) as the PHB depolymerase substrate (1,(26)(27)(28).…”

Section: Discussionmentioning

confidence: 99%

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Gebauer

Jendrossek

2006

Appl Environ Microbiol

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Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a softwarecontrolled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by highperformance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.Polyhydroxyalkanoates (PHAs) are typical storage compounds of carbon and energy and are widely found in prokaryotes. The most common PHA is poly(3-hydroxybutyrate) (PHB), and this polymer can be accumulated at up to 90% of the cellular dry weight during unbalanced growth in some bacteria (2a, 37, 49). PHAs are thermoplasts, and despite the relatively high production costs, PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate are industrially produced on a scale of a few hundred tons per year.One reason for the interest in biologically produced PHAs lies in the environmentally friendly production from renewable resources and in the biodegradability of PHAs. The ester bonds of the PHAs are the Achilles' heel of the polymer that can be hydrolyzed by a large variety of hydrolytic enzymes (PHA depolymerases). In vivo, PHAs can be hydrolyzed by the accumulating strain itself during periods of starvation (intracellular PHA hydrolysis by intracellular PHA depolymerases) (41). PHAs that were produced by humans or that were released from PHA-accumulating microorganisms (e.g., after death) can be cleaved by extracellular PHB depolymerases, and many examples of extracellular PHA depolymerases were described during the last two decades (14, 21). A differentiation of extracellular degradation from intracellular degradation is necessary because PHA exists in two biophysical conformations: in vivo, PHB is completely amorphous (native) and is covered by a surface layer that is about half the size of a cytoplasmic membrane (4, 5, 31) but apparently consists mainly of proteins, so-called phasins (48). After the release of the polymer from the cell (e.g., after cell lysis or by solvent extraction) or after damage of the surface l...

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“…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).…”

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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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Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Cited by 12 publications

References 18 publications

Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene fromRalstonia eutrophaH16 and Characterization of the Gene Product

Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene fromRalstonia eutrophaH16 and Characterization of the Gene Product

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

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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