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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↗

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↗

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↗

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↗

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↗

PubMed1999

Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases.

The substrate specificities of three extracellular polyhydroxybutyrate (PHB) depolymerases from Alcaligenes faecalis (PhaZ Afa), Pseudomonas stutzeri (PhaZ Pst), and Comamonas acidovorans (PhaZ Cac), which are grouped into types A and B based on the position of a lipase box sequence in the catalytic domain, were examined for films of 12 different aliphatic polyesters. Each of these PHB depolymerases used was capable of hydrolyzing poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxypropionate) (P(3HP)), poly(4-hydroxybutyrate) (P(4HB)), poly(ethylene succinate) (PESU), and poly(ethylene adipate) (PEA) but could not hydrolyze another seven polyesters. In addition, the binding characteristics of substrate binding domains from PhaZ Afa, PhaZ Cac, and PHB depolymerase from Comamonas testosteroni (PhaZ Cte) were studied by using fusions with glutathione S-transferase (GST). All of fusion proteins adsorbed strongly on the surfaces of polyester granules of P(3HB), P(3HP), and poly(2-hydroxypropionate) (P(2HP)) which was not hydrolyzed by the PHB depolymerases used in this study, while they did not bind on Avicel and chitin granules. The adsorption kinetics of the fusion proteins to the surface of P(3HB) and P(2HP) granules were found to obey the Langmuir isotherm. The cross-area per molecule of fusion protein bound to P(3HB) granules was estimated to be 12+/-4 nm2/molecule. It has been suggested that the active sites in catalytic domains of PHB depolymerases have a similar conformational structure, and that several amino acids in substrate-binding domains of PHB depolymerases interact specifically with the surface of polyesters.

Adsorption↗

PubMed1998

Synthesis and mechanical properties of interpenetrating networks of polyhydroxybutyrate-co-hydroxyvalerate and polyhydroxyethyl methacrylate.

Naturally occurring, biocompatible, and biodegradable polyhydroxybutyrate-co-hydroxyvalerate (PHBV), and synthetic, non-degrading polyhydroxyethylmethacrylate (PHEMA) membranes were prepared and their mechanical properties were studied. Their performances were compared with the interpenetrating networks (IPN) prepared by photopolymerization of HEMA in the presence of PHBV. The modulus of elasticity, failure stress and failure strain indicated that the IPNs are viscoelastic with properties closer to PHEMA but much stronger than PHEMA homopolymers. Incorporation of PHBV (7, 14 and 22% HV) affected the mechanical properties positively. Increasing the PHBV content increased the modulus of elasticity and failure stress nearly in all samples tested. PHBV (7, 14, and 22% HV, 300 mg) samples showed an approximately 17-30 fold increase in terms of modulus of elasticity and 7-10 fold increase in terms of failure stress. The scanning electron micrographs of the membranes showed that the PHEMA membranes are more porous than the PHBV membranes but the IPN structure displayed channels on the membrane surface indicating that HEMA polymerization was achieved by using the PHBV as a scaffold. With the use of the present technique, it is possible to synthesize supramolecular structures from molecules that are not compatible and miscible with each other.

Biocompatible Materials↗

PubMed1998

The LuxR regulator protein controls synthesis of polyhydroxybutyrate in Vibrio harveyi.

The LuxR regulatory protein of Vibrio harveyi has been shown to control synthesis of polyhydroxybutyrate (PHB) as well as luminescence so as to occur at high cell density, suggesting that it is a general regulatory protein. Mutants defective in the production of LuxR (D1, D34, and MR1130) were found to be missing PHB, whose synthesis could be restored by complementation with luxR. Triparental mating with a V. harveyi genomic library revealed the presence of three genomic clones (G1, G2 and G3) that could also restore PHB synthesis and luminescence to cells which express low levels of luxR (D1 and D34) but not to luxR- cells (MR1130) suggesting that luxR expression was being stimulated. Analyses of luxR mRNA levels by mRNA dot blot hybridization and by primer extension confirmed that luxR mRNA levels were increased 4 to 7-fold in the D1 and D34 cells by the G1, G2 and G3 fragments and show that expression of a single genomic copy of luxR is sufficient to restore synthesis of PHB. The results demonstrate that V. harveyi LuxR controls the induction of a process not intimately involved in the bioluminescence system and clearly distinguishes its role in V. harveyi from that of LuxR from Vibrio (Photobacterium) fischeri, which has only been associated with regulation of light emission.

Hydroxybutyrates↗

PubMed1997

PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo.

A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding beta-ketothiolase and acetoacetyl-CoA reductase behind a modified synthase gene containing an Escherichia coli promoter and ribosome binding site. Plasmids containing the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E. coli DH5 alpha and analyzed for polyhydroxybutyrate production. The molecular weight of polymer isolated from recombinant E. coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E. coli containing the native A. eutrophus operon. A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.

Acyltransferases↗

PubMed1996

Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate.

The polyhydroxybutyrate (PHB) synthase gene of the bacterium Alcaligenes eutrophus was used to construct a yeast plasmid which enabled expression of the functional synthase enzyme in Saccharomyces cerevisiae. Cells transformed with the synthase plasmid accumulated up to 0.5% of cell dry weight as PHB, with accumulation occurring in the stationary phase of batch growth. The identity of PHB in recombinant yeast cells was confirmed with 1H-NMR spectra of chloroform-extracted cell material. In addition, freeze-fracture electron microscopy revealed cytoplasmic granules exhibiting plastic deformations characteristic for PHB. GC results indicated a low background level of PHB in the wild-type strain, but intact polymer could not be detected by 1H-NMR. Formation of PHB in the recombinant strain implies the participation of native yeast enzymes in the synthesis of D-3-hydroxybutyryl-CoA (3-HB-CoA). Inhibition studies with cerulenin indicated that the fatty acid synthesis pathway is not involved in PHB precursor formation. Wild-type cell-free extracts showed D-3-HB-CoA dehydrogenase activity [150-200 nmol min-1 (mg protein)-1] and acetoacetyl-CoA thiolase activity [10-20 nmol min-1 (mg protein)-1], which together could synthesize monomer from acetyl-CoA. PHB accumulation was simultaneous with ethanol production, suggesting that PHB can act as an alternate electron sink in fermentative metabolism. We propose that PHB synthesis in recombinant yeast is catalysed by native cytoplasmic acetoacetyl-CoA thiolase, a native beta-oxidation protein possessing D-3-HB-CoA dehydrogenase activity and heterologous PHB synthase.

Acyltransferases↗

PubMed1989

Polyhydroxybutyrate as a drug carrier.

Polyhydroxybutyrate (PHB) can be used as an alternative polymer to polylactide-glycolides for drug carrier production. It is a linear homopolymer biosynthesized by various strains of bacteria by condensation of D(-)-B-hydroxybutyric acid and used as an energy and carbon source. PHB can be obtained by extraction from bacteria or by chemical synthesis. To be suitable as drug carrier the polymer has to be biocompatible, biodegradable in certain applications, and nontoxic. PHB seems to be biocompatible and biodegrades readily to carbon dioxide in bacteria; however, in humans, the reports are few and contradictory. The polymer is nontoxic and its monomer seems to be tolerated well in relatively high concentrations. Possible applications include implants and i.m.-administered particulates for controlled release and i.v.-injected colloidal carriers for drug targeting.

Animals↗

PubMed1992

Development of a degradable composite for orthopaedic use: in vivo biomechanical and histological evaluation of two bioactive degradable composites based on the polyhydroxybutyrate polymer.

As a direct method for the evaluation of tissue bonding to two polyhydroxybutyrate (PHB) based composites, a mechanical push-out test was performed on implants in the femur of mature Japanese White rabbits. Three composites were tested. The first, a hydroxyapatite/PHB (HA/PHB) composite showed an increase in interfacial shear strength (ISS) up to 8 wk, after which the ISS decreased due to degradation of the implant. The second composite was an HA/glass/PHB (HGP) composite and this gave lower values for the ISS attributed to ion release from the glass causing a soft tissue reaction at the interface. The third composite was a carbon fibre reinforced polysulfone (CFRP) and this showed high interfacial shear strength values, which continued to increase with time. These conclusions were supported by contact microradiography (CMR) and histology which showed enhanced endosteal bone growth for the HA/PHB but for the HGP, no periosteal or endosteal activity was detected. Interposed soft tissue for the HGP composite was difficult to discern, histologically, but it was proposed that this was the reason for the low ISS values. It was concluded that the high ISS values for the carbon fibre control were due to surface morphology allowing deep ingrowth of soft tissue and this was confirmed by SEM.

Animals↗

PubMed1993

Development of a degradable composite for orthopaedic use: mechanical evaluation of an hydroxyapatite-polyhydroxybutyrate composite material.

This study evaluates the mechanical properties of a composite material comprising polyhydroxybutyrate with hydroxyapatite added in proportions varying from 0 to 50%. Among the three methods of production, injection moulding was found to result in the most satisfactory mechanical properties. The tensile and compressive strength and the modulus of elasticity of composite produced in this way fell within the range for fresh human bone from different anatomical sites. With the additional advantages of biocompatibility, biodegradability and the potential for piezoelectric stimulation of new local bone formation, it was concluded that the injection-moulded composite material has considerable potential for use in orthopaedic surgery, both as a material to construct certain orthopaedic implants and as an alternative to corticocancellous bone graft.

Biocompatible Materials↗

PubMed2000

Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase.

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.

Acyltransferases↗

PubMed1997

Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater.

Comamonas acidovorans YM1609 secreted a polyhydroxybutyrate (PHB) depolymerase into the culture supernatant when it was cultivated on poly(3-hydroxybutyrate) [P(3HB)] or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] as the sole carbon source. The PHB depolymerase was purified from culture supernatant of C. acidovorans by two chromatographic methods, and its molecular mass was determined as 45,000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures below 37 degrees C and at pH values of 6 to 10, and its activity was inhibited by diisopropyl fluorophosphonate. The liquid chromatography analysis of water-soluble products revealed that the primary product of enzymatic hydrolysis of P(3HB) was a dimer of 3-hydroxybutyric acid. Kinetics of enzymatic hydrolysis of P(3HB) film were studied. In addition, a gene encoding the PHB depolymerase was cloned from the C. acidovorans genomic library. The nucleotide sequence of this gene was found to encode a protein of 494 amino acids (M(r), 51,018 Da). Furthermore, by analysis of the N-terminal amino acid sequence of the purified enzyme, the molecular mass of the mature enzyme was calculated to be 48,628 Da. Analysis of the deduced amino acid sequence suggested a domain structure of the protein containing a catalytic domain, fibronectin type III module as linker, and a putative substrate-binding domain. Electron microscopic visualization of the mixture of P(3HB) single crystals and a fusion protein of putative substrate-binding domain with glutathione S-transferase demonstrated that the fusion protein adsorbed strongly and homogeneously to the surfaces of P(3HB) single crystals.

3-Hydroxybutyric Acid↗

PubMed1999

Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains.

The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZPst) of Pseudomonas stutzeri was cloned and sequenced. phaZPst was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B from Clostridium paraputrificum. The binding characteristics of SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S-transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.

Amino Acid Sequence↗