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At least 19 recordsLinked to original sources
PubMed1991

In vitro and in vivo evaluation of polyhydroxybutyrate and of polyhydroxybutyrate reinforced with hydroxyapatite.

Polyhydroxybutyrate (PHB) is a polyester made by many microorganisms under conditions of nitrogen deficiency, and is produced commercially in bulk by biotechnology. It has been suggested that PHB-based materials (copolymers and composites) could be suitable for medical applications and may be biodegradable. This paper presents some findings regarding the degradation and biological properties of polyhydroxybutyrate and composites reinforced with particulate hydroxyapatite. It has been established that the strength and stiffness of these materials reduce on in-vitro environment exposure in phosphate-buffered saline at 37 degrees C for periods up to 4 months, and that the degradation rate is a function of composition and processing conditions. It has also been demonstrated that materials based on PHB produce a consistent favourable bone tissue adaptation response with no evidence of an undesirable chronic inflammatory response after implantation periods up to 12 months. Bone is rapidly formed close to the material and subsequently becomes highly organized, with up to 80% of the implant surface lying in direct apposition to new bone. The materials showed no conclusive evidence of extensive structural breakdown in vivo during the implantation period of the study.

Animals↗
PubMed2001

Polyhydroxybutyrate biosynthesis in Caulobacter crescentus: molecular characterization of the polyhydroxybutyrate synthase.

Caulobacter crescentus was investigated with respect to polyhydroxybutyrate (PHB) biosynthesis. Polyhydroxyalkanoate (PHA) accumulation contributing to approximately 18% of the cell dry weight was obtained in the presence of glucose. Gas chromatography-mass spectrometry and gel permeation chromatography of the purified PHA showed that this polyester was solely composed of 3-hydroxybutyrate and had a weight average molar mass of 5.5 x 10(5) g mol(-1) and a polydispersity of 1.6. An ORF encoding a conserved, hypothetical protein which shared approximately 47% identity with the PHB synthase from Azorhizobium caulinodans was identified within the complete C. crescentus genomic sequence. This putative C. crescentus PHB synthase gene, phaC, consisted of a 2019 nt stretch of DNA (encoding 673 aa residues), which encoded a PHB synthase with a molecular mass of approximately 73 kDa. This is currently the largest PHA synthase identified. The phaC coding region was subcloned into vector pBBR1-JO2 under lac promoter control. The resulting plasmid, pQQ4, mediated PHB accumulation in the mutant Ralstonia eutropha PHB(-)4 and recombinant Escherichia coli JM109(pBHR69), which produced the beta-ketothiolase and acetoacetyl-CoA reductase from R. eutropha, contributing to approximately 62% and 6% of cell dry weight, respectively. Functional expression of the coding region of phaC was confirmed by immunoblotting and in vitro PHB synthase activity.

Acyltransferases↗
PubMed2002

The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production.

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.

Bacterial Proteins↗
PubMed2005

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids.

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. leguminosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutant, or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Carbon↗
PubMedDate not supplied

[Study of molecular structure of polyhydroxybutyrate-a termoplastic anddegradable biopolymer].

The molecular structure of polyhydroxybutyrate from hydrogen-oxidizing bacteria Alcaligenes eutrophus was studied by X-ray diffraction analysis. It was shown that the degree of crystallinity of various samples depends little on the conditions of their preparation and is equal to 0.62-0.76. The molecular structure of solid samples and solution of polyhydroxybutyrate in chloroform were studied by the NMR and EPR methods. The conclusion is made that the molecular structure of polyhydroxybutyrate does not depend on the features of the strain and conditions of carbon nutrition of microorganisms producing polyhydroxybutyrate. Defects induced by gamma-radiation in polyhydroxybutyrate were studied. Free radicals were isolated, and their structure was decoded.

Alcaligenes↗
PubMed2002

A polyhydroxybutyrate biodegradable stent: preliminary experience in the rabbit.

PURPOSE: The lifelong persistence of foreign bodies within the arteries may contribute to restenosis. Thus, biodegradable devices might decrease recurrence rates. METHODS: Eleven polyhydroxybutyrate biodegradable stents and 13 tantalum stents were implanted into the iliac arteries of New Zealand white rabbits for up to 30 weeks. After killing the animals, the specimens were harvested, fixed in formalin, processed in paraffin, and stained. RESULTS: Polyhydroxybutyrate instigated intense inflammatory and proliferative reactions with an increase in collagen (2.4- to 8-fold vs native segments), thrombosis and in-stent lumen narrowing (375.5-606.6 mm vs 655.6 +/- 268.8 mm in native segments). The elastic membranes were destroyed in all specimens. The tantalum stents increased the in-stent lumen progressively (769.7 +/- 366.6 mm vs 1309.9 +/- 695.3 mm), penetrated the external elastic membrane, and increased mural collagen content (6- to 8.6-fold vs native segments). Neither restenoses nor thromboses occurred. CONCLUSIONS: In the rabbit iliac artery, polyhydroxybutyrate stents caused intensive inflammatory vascular reactions which ban them from clinical use.

Animals↗
PubMed1989

Controlled release of a GnRH agonist from a polyhydroxybutyric acid implant: reversible suppression of the menstrual cycle in the macaque.

The use of polyhydroxybutyric acid as a matrix for delivery of the GnRH agonist buserelin was studied in adult stumptailed macaques with regular menstrual cycles. This material contained 5.0 mg buserelin in a disk of 0.6 cm diameter and 0.15 cm thickness. The effects on pituitary-ovarian function were examined and buserelin release profiles from 30,000 and 150,000 molecular weight polyhydroxybutyric acid matrices were compared. In an attempt to reduce the high initial release rate of buserelin from these disks, and to prolong the effective period of release, implants of 30,000 molecular weight were coated with cyanoacrylate. The implants were removed after 28, 35 or 42 days for the 30,000, the 150,000 and the coated implants, respectively. The urinary excretion profile for the agonist revealed a high initial release of buserelin from these implants followed by a rapid decline. The coated disks released significantly less (p less than 0.05) buserelin over the first 3 days than did either of the uncoated implants, but maintained release of the agonist over a longer period. Inhibition of ovulation and suppression of estradiol secretion lasted for the duration of treatment in 3 of 5 macaques receiving the 30,000 implant, and in 5 of 5 receiving the 150,000 or the coated implant. In 3 macaques in whom the coated implant was left in situ, ovulation was suppressed for 104 +/- 3.7 days. These results demonstrate that a polyhydroxybutyric acid matrix can be used as a depot formulation for a GnRH agonist and that improved control of release of the agonist can be achieved by coating such implants with cyanoacrylate. This approach may be useful in experimental and clinical situations and may be applicable for delivery of other peptide hormones.

Animals↗
PubMed2005

The crystal structure of polyhydroxybutyrate depolymerase from Penicillium funiculosum provides insights into the recognition and degradation of biopolyesters.

Polyhydroxybutyrate is a microbial polyester that can be produced from renewable resources, and is degraded by the enzyme polyhydroxybutyrate depolymerase. The crystal structures of polyhydroxybutyrate depolymerase from Penicillium funiculosum and its S39 A mutant complexed with the methyl ester of a trimer substrate of (R)-3-hydroxybutyrate have been determined at resolutions of 1.71 A and 1.66 A, respectively. The enzyme is comprised of a single domain, which represents a circularly permuted variant of the alpha/beta hydrolase fold. The catalytic residues Ser39, Asp121, and His155 are located at topologically conserved positions. The main chain amide groups of Ser40 and Cys250 form an oxyanion hole. A crevice is formed on the surface of the enzyme, to which a single polymer chain can be bound by predominantly hydrophobic interactions with several hydrophobic residues. The structure of the S39A mutant-trimeric substrate complex reveals that Trp307 is responsible for the recognition of the ester group adjacent to the scissile group. It is also revealed that the substrate-binding site includes at least three, and possibly four, subsites for binding monomer units of polyester substrates. Thirteen hydrophobic residues, which are exposed to solvent, are aligned around the mouth of the crevice, forming a putative adsorption site for the polymer surface. These residues may contribute to the sufficient binding affinity of the enzyme for PHB granules without a distinct substrate-binding domain.

Amino Acid Sequence↗
PubMed1994

Immediate implant placement using a biodegradable barrier, polyhydroxybutyrate-hydroxyvalerate reinforced with polyglactin 910. An experimental study in dogs.

The purpose of this study was to evaluate the use of a biodegradable membrane of polyhydroxybutyrate-hydroxyvalerate copolymer reinforced with polyglactin 910 fibers, as an occlusive barrier over implants placed into fresh extraction sockets. Ten dogs had the 3rd and 4th mandibular premolars extracted bilaterally. Each dog had 4 Astra Dental Implants placed directly into the fresh extraction sockets. The top of the fixtures was placed at the same level as the top of the buccal cortical bone. The two implants in the right side were covered with the hydrolyzable polyester material (polyhydroxybutyrate-hydroxyvalerate reinforced with polyglactin 910 fibers; PHB-HV/PG), and the 2 implants in the left side were controls without occlusive membranes. Soft tissue dehiscences were registered for half of the implants in the test side but were not noted in the control side. The histomorphological measurements after 12 weeks showed that the mean distance from the top of the fixtures to the first bone-to-implant contact was significantly greater than for the control side. The membrane-covered implants without dehiscences showed also significantly less bone fill compared with the control side. Inflammatory cell infiltrates were seen adjacent to all PHB-HV/PG membranes, and frequently the membrane material was surrounded by a fibrous tissue capsule. The polyester membranes used in this study interfered with the marginal bone healing adjacent to the immediately placed implants. An increased inflammatory reaction and significantly less marginal bone healing was registered in the membrane side compared with the control side.

Animals↗
PubMed1999

Polyhydroxybutyrate: plastic made and degraded by microorganisms.

Polyhydroxybutyrate (PHB) offers many advantages over traditional petrochemically derived plastics. In addition to its complete biodegradability, PHB is formed from renewable resources. It possesses better physical properties than polypropylene for food packaging applications and is completely nontoxic. The poor low-impact strength of PHB is solved by incorporation of hydroxyvalerate monomers into the polymer to produce polyhydroxybutyrate-co-valerate (PHBV), which is commercially marketed under the trade name Biopol. Like PHB, PHBV completely degrades into carbon dioxide and water under aerobic conditions. Microbial synthesis of PHB is the best method for industrial production because it ensures the proper stereochemistry for biodegradation. Microorganisms synthesize and store PHB under nutrient-limited conditions and degrade and metabolize it when the limitation is removed. Current production employs Alcaligenes eutrophus because it grows efficiently on glucose as a carbon source, accumulates PHB up to 80% of its dry weight, and is able to synthesize PHBV when propionic acid is added to the feedstock. PHBV is currently 16 times the price of polypropylene. However, the development of transgenic PHA-producing organisms is expected to greatly reduce its cost. Benefits of using transgenic systems include lack of a depolymerase system, ability to use faster-growing organisms, production of highly purified polymers, and ability to utilize inexpensive carbon sources. Because transgenic plants may someday result in the evolution of plastic crops that could lower the price of PHA to a competitive level, future research will surely focus on such recombinant DNA techniques.

Bacteria↗
PubMed2000

Kinetic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha.

Purified Ralstonia eutropha polyhydroxybutyrate (PHB) synthase from recombinant cells can exist as monomer and dimer. The polymerization reaction catalyzed by this enzyme displays a lag phase, which causes difficulties for kinetic and mechanistic characterization of the enzymatic polymerization reaction. In this study, we developed a method to eliminate the lag phase of PHB synthase by physical means, i.e., adding multihydroxyl compounds to the enzyme solution. This method allows us to recognize the nature of the lag phase as a physical rather than a chemical process. With such lag-phase-free-enzyme, the kinetic properties of the enzyme were investigated. The results indicate that 3-hydroxybutyryl-CoA (3HBCoA) is the optimal substrate for the enzyme. A slower catalytic rate and lower binding ability account for a lower reactivity of 3-hydroxyvaleryl-CoA (3HVCoA) compared to that of 3HBCoA. The change of hydroxyl group from the beta to the gamma position causes dramatic decreases in the binding ability of 4-hydroxybutyryl-CoA (4HBCoA). By using a dilution strategy and size exclusion chromatographic technique, the active form of the enzyme was identified to be the dimeric form. The number of catalytic sites in the dimeric form of the enzyme was examined by comparing the molecular weight of polyhydroxybutyrate as a function of substrate-to-enzyme ratio. The results suggest that the dimeric enzyme has only one catalytic site. A revised model of polymerization reaction catalyzed by R. eutropha PHB synthase is described.

Acyltransferases↗
PubMed1998

Metabolic modeling of polyhydroxybutyrate biosynthesis.

A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium.

Acetyl-CoA C-Acetyltransferase↗
PubMed1999

Sulbactam-cefoperazone polyhydroxybutyrate-co-hydroxyvalerate (PHBV) local antibiotic delivery system: in vivo effectiveness and biocompatibility in the treatment of implant-related experimental osteomyelitis.

In this study, a novel antibiotic carrier system for use in the treatment of implant-related and chronic osteomyelitis was developed. Sulbactam-cefoperazone was introduced to rods of polyhydroxybutyrate-co-hydroxyvalerate (22 mol % HV, w/w), a member of a family of microbial-origin polymer that is biodegradable, biocompatible, and osteoconductive due to its piezoelectric property. The antibiotic-loaded carrier was implanted into the infection site that was induced by Staphylococcus aureus inoculation into the rabbit tibia. The effectiveness of this was assessed macroscopically, radiographically, bacteriologically, and histopathologically. Findings of infection subsided on day 15 and almost complete remission was observed on day 30. The control side that contained antibiotic-free rods, however, worsened. These findings prompted us to conclude that the novel biodegradable antibiotic carrier developed in the present study seems to be a promising candidate for use in the treatment of severe bone infection.

Animals↗
PubMed2004

Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions.

Activated sludge submitted to aerobic dynamic feeding conditions showed a good and stable capacity to store polyhydroxybutyrate (PHB). The system, working for 2 years, selected a microbial population with a high PHB storage capacity. The influence of carbon and nitrogen concentrations on the PHB accumulation yield was studied in a range of 15-180 Cmmol/l for acetate and 0-2.8 Nmmol/l for ammonia. Low ammonia concentrations favored PHB accumulation. The maximum PHB content, 67.5%, was obtained for 180 Cmmol/l of acetate supplied in one pulse. However, such high substrate concentration proved to be inhibitory for the storage mechanism, causing a slowdown of the specific PHB storage rate. In order to avoid substrate inhibition, 180 Cmmol/l of acetate was supplied in different ways: continuously fed and in three pulses of 60 Cmmol/l each. In both cases the specific PHB storage rate increased and the PHB content obtained were 56.2% and 78.5%, respectively. The latter value of PHB content is similar to that obtained by pure cultures and was never reported for mixed cultures. Addition of acetate by pulses controlled by the oxygen concentration was kept for 16 days, the PHB content being always above 70% of cell dry weight.

Acetates↗
PubMed2005

Mathematical modelling of a mixed culture cultivation process for the production of polyhydroxybutyrate.

Mixed cultures submitted to acetate "feast" and "famine" cycles are able to store intracellularly high quantities of polyhydroxybutyrate (PHB). It was demonstrated in a previous study that the intracellular PHB content can be increased up to 78.5% (g HB/gVSS) of cell dry weight in a sequencing batch reactor (SBR) with optimised operating conditions. The specific PHB formation rate was also shown to be higher for mixed cultures than for pure cultures. Such high intracellular PHB contents and specific productivity open new perspectives for the industrial production of polyhydroxyalkanoates (PHA) using mixed cultures instead of pure cultures. The main goal in this work was to develop a mathematical model of mixed cultures envisaging the optimisation of PHB production. A relatively simple two-compartments cell model was developed based on experimental observations and other models proposed in the literature. A convenient experimental planing allowed to identify the kinetic parameters and yield coefficients. Experiments were performed with and without ammonia limitation enabling the analysis of PHB formation independently of the cell growth process. The experimental true yields partially confirm the theoretical values proposed in the literature. The final model exhibited high accuracy in describing the process state of most experiments performed, thus opening good perspectives for future model-based optimisation studies.

Bacteria↗
PubMed2006

Mutation on N-terminus of polyhydroxybutyrate synthase of Ralstonia eutropha enhanced PHB accumulation.

Polyhydroxyalkanoate (PHA) synthase is the central enzyme involved in the biosynthesis of PHA, a family of bacterial biodegradable polyesters. Due to its high variability, the N-terminal fragment of this enzyme was previously considered as unnecessary for a functionally active enzyme. In this study, polyhydroxybutyrate synthase from Ralstonia eutropha (PhbC(Re)) with a deletion on N-terminal 88 amino acid residues showed a significant reduced activity, as reflected by only 1.5% PHB accumulation compared with the wild type which produced 58.4% PHB of the cell dry weight. Whilst several site-specific mutagenesis results revealed the amphiphilic alpha-helix assembled by the amino acid region, D70-E88 played an important role in both maintaining the PHB synthase activity and regulating molecular weight and polydispersity of accumulated PHB homopolymer.

Acyltransferases↗
PubMed2004

A polyhydroxybutyrate-producing Pseudomonas sp. isolated from Antarctic environments with high stress resistance.

Pseudomonas sp. 14-3, a strain that accumulates large quantities of polyhydroxybutyrate (PHB) when grown on octanoate, was isolated from Antarctic environments. This isolate was characterized on the basis of phenotypic features and partial sequencing of its 16S ribosomal RNA gene. Pseudomonas sp. 14-3 showed increased tolerance to both thermal and oxidative stress compared with three other Pseudomonas species. Stress tolerance of Pseudomonas sp. 14-3 was analyzed in polyhydroxyalkanoate accumulating and non-accumulating conditions, and increased levels of stress resistance were observed when PHB was produced. Pseudomonas sp. 14-3 was isolated from Antarctic regions, a habitat normally exposed to extreme conditions. An association between high PHB accumulation and high stress resistance in bacteria adapted to extreme environments is suggested.

Antarctic Regions↗
PubMed2004

Polyhydroxybutyrate synthesis in transgenic flax.

Flax (Linum usitatissimum L.) is an annual plant species widely cultivated in temperate climates for bast fibres and linseed oil. Apart from traditional textile use, the fibres are fast becoming an integral part of new composite materials utilized in automobile and constructive industry. Especially attractive for environmental safety demands are biodegradable and renewable biocomposities based on polyhydroxybutyrate (PHB) polymer as a matrix and reinforced with the flax fibres. Manufacturing of PHB by bacteria fermentation is however substantially more expansive as compared to technologies producing conventional plastics. We report for the first time generation of transgenic plants which produce both components of flax/PHB composites, i.e. the fibres and the thermoplastic matrix in the same plant organ of a crop. The flax (cv. Nike) plants were transformed using constructs bearing either single cDNA, encoding the beta-ketothiolase enzyme (C plants), or all three of the genes necessary for poly-beta-hydroxybutyrate (PHB) synthesis (M plants). Both constructs contained a plastidial targeting sequence. The amount of PHB produced by the transgenic plants was up to over 70-fold higher than in wild-type plants, when analysed using the gas chromatography/mass spectrometry (GC-MS method). The PHB accumulation in plastids caused change both in their shape and size. The use of a stem-specific promoter for transgene expression protected the transgenic plant from growth retardation and also provided higher PHB synthesis than in the case of constructs governed by the 35S CaMV constitutive promoter. None toxic effects that could lead to stunted growth or the loss of fertility were observed, when 14-3-3 promoter was used as the stem-specific. Significant modifications in stem mechanical properties were accompanied to the PHB accumulation in growing cell of fibres in the transgenic plants. The Young's modulus E, the average measure of stem tissues resistance to tensile loads increased up to twice in M plants as compared to a single gene transformed ones. However, a wide range of E values, from 24.1 to 54.4 MPa, was observed in dependence of tested strain. Potential commercial significance of the genetic manipulation approach enabling synthesis of thermoplastic in crops cultivated for fibres is discussed.

Carbohydrate Metabolism↗