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PubMed2005

[Repairing segmental radial bone defect with poly (3-hydroxybutyrate-co-3-hydroxyvalerate)/sol-gel bioactive glass composite porous scaffold].

OBJECTIVE: To investigate the capability of the bone regeneration of poly (3-hydroxybutyrate-co-3-hydroxyvalerate/sol-gel bioactive glass (PHBV/SGBG) composite porous scaffold. METHODS: PHBV/ SGBG composite porous scaffold was implanted into the segmental radial bone defect of the New Zealand white rabbits, PHBV/hydroxylapatite (PHBV/HA) as experimental control. The degradability, biocompatibility, and bone regeneration capability of the implants were evaluated through radiological, histological, computerized graphic, and biomechanical analysis. RESULTS: The new bone formation occurred as early as 4 weeks after implantation of PHBV/SGBG composite porous scaffold. The defect was filled with new bone 8 weeks after the implantation, and was completely repaired 12 weeks after operation. The new bone had normal bone structure and the medullar cavity regenerated. The biomechanical study showed that the anti-compression force of radial specimen in PHBV/SGBG groups was significantly higher than in PHBV/ HA groups (P < 0.05), but no significant difference existed between PHBV/SGBG group and autograft bone group (P>0.05). The PHBV/SGBG composite porous scaffold degraded no sooner than 4 weeks after the implantation and most of scaffold was absorbed after 12 weeks. The proportion of the scaffold to new bone decreased from 60% by week 4 to 8% by week 12. CONCLUSIONS: PHBV/SGBG composite porous scaffold is a degradable bone substitute. It can achieve early bone generation and complete repair. It can be used as an ideal scaffold for tissue-engineering bone.

Animals↗
PubMed2003

[Fermentative production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) by recombinant Aeromonas hydrophila 4AK4 (pTG01)].

Copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) (PHBHHx), a new type of biodegradable material, are receiving considerable attentions recently. The material properties are strongly related to the 3HHx fraction of PHBHHx. As the 3HHx fraction increase, crystallinity and melting point of PHBHHx decrease, flexibility and tractility increase. PHBHHx of different 3HHx fraction can meet different demands of commercial application and research. Aeromonas are the best studied PHBHHx-producing strains. Recent studies have been focused on optimizations of fermentative culture media and culture conditions for low-cost and efficient fermentative production. Aliphatic substrates such as long-chain fatty acid and soybean oil were used in the PHBHHx fermentation as the sole carbon source and energy source. Two-stage fermentation method was also developed for more efficient PHBHHx production. While studies on Aeromonas hydrophila revealed that the monomer composition of PHBHHx could not easily be regulated by fermentative process engineering methods such as changing substrates and fermentative conditions because precursors involved in the PHBHHx synthesis were all from the beta-oxidation pathway. In this study, phbA gene encoding beta-ketothiolase and phbB gene encoding acetoacetyl-CoA reductase were introduced into a PHBHHx-producing strain Aeromonas hydrophila 4AK4 so as to provide a new 3HB precursors synthesis way. phbA gene encodes beta-ketothiolase which can catalyze two acetyl-CoA to form acetoacetyl-CoA; phbB gene encodes acetoacetyl-CoA reductase catalyzing acetoacetly-CoA into 3HB-CoA which is the precursor of 3HB. The introduced novel 3-hydroxybutyrate precursor synthesis pathway allowed the recombinant strain to use unrelated carbon source such as gluconate to provide 3HB precursors for PHBHHx synthesis. Shake-flask experiments were carried out to produce PHBHHx of controllable monomer composition and fermentations in 5 L fermentor were also proceeded for confirmation of these result in large-scale culture. In flask culture, it was possible to reduce the 3HHx mol fraction in PHBHHx from 15 % in the wild type to 3% - 12% in the recombinant by simply changing the ratio of gluconate to lauric acid in the culture media. When lauric acid was used as the sole carbon source, 51.5 g/L Cell Dry Weight (CDW) containing 62 % PHBHHx with 9.7 % 3HHx mol fraction was obtained in 56 hours of fermentation in a 5 liter fermentor. When co-substrates of sodium gluconate and lauric acid (1:1) were used as carbon sources, 32.8 g/L CDW containing 52 % PHBHHx with 6.7% 3HHx mol fraction was obtained in 48 hours of fermentation. These results showed the possibility for fermentative production of PHBHHx with controllable monomer composition.

3-Hydroxybutyric Acid↗
PubMed2005

[Effects of SRT and carbon concentration on the PHB in the anaerobic/aerobic alternative operating SBR process].

Effects of SRT and carbon concentration on the PHB of the anaerobic/aerobic SBR process are studied. The results show that by the order of SBR51,SBR10 and SBR52,the maximum PHB content of MLVSS were 9.8%, 5.72% and 18.89%, respectively, while carbon translating ratios were 46%, 34% and 36.3% during the first 20min of aerobic phase and PHB forming rate were 196.6mg/(L x h), 140mg/(L x h) and 295.35mg/(L x h). PHB degradation rate can be described with a first order degradation rate with respect to the PHB content of the cells. Reclamation of PHB should consider from several factors. Carbon concentration gradient is the main factor influencing PHB formation under batch experiment.

Bacteria, Aerobic↗
PubMedDate not supplied

Induction of mutation in Azotobacter chroococcum MAL-201 for improvement of P(3HB) production.

Azotobacter chroococcum MAL-201 (MTCC 3853), a poly-3-hydroxybutyric acid [P(3HB)] producing organism was subjected to mutagenesis by UV-irradiation and N'-methyl-N'-nitro-N'-nitrosoguanidine (MNNG). A sharp decline in survival percentage of treated cells both in UV (1.2% at 20 sec. exposure) and in MNNG ((0.26% at 30 microg/ml) suggest high degree of sensitivity of the isolate to these mutagens. A total of 124 mutant colonies were isolated from viable population based on their morphological features, antibiotic resistance and dependence on exogenous organic nitrogenous substances. Nature of mutants were confirmed by replica plating and growth on antibiotic and organic nitrogen supplemented Norris agar medium. Majority of mutants were devoid of exopolysaccharide, dependent on organic nitrogen and with weak P(3HB) content. The mutant strain, UC-220, a nitrogen-dependent, chloramphenicol-resistant mutant showed an enhancement of more than 10% (w/w) P(3HB) production compared to that of wild type strain when grown under identical conditions.

Azotobacter↗
PubMed2005

Poly-(R)-3-hydroxybutyrate and the pioneering work of Rosetta Natoli Reusch.

In investigating genetic competence, Reusch and collaborators have found that the concentration of short chain poly-(R)-3-hydroxybutyrate (PHB) and polyphosphate (polyP) complexes increases with genetic transformability and that interrupting DNA uptake yields single-stranded donor DNA complexed with short chain PHB. This would be consistent with the organic polyphosphate, DNA, replacing the inorganic polyphosphate, polyP, in the PHB pore so allowing the DNA to be drawn into the cell. Reusch has gone on to show that PHB and polyphosphate, extracted from membranes or synthesized chemically, together form a voltage-activated calcium-selective channel. One may wonder whether the classical proteinaceous calcium channels have a short chain PHB/polyP core--and whether other ion channels have this core too. It is therefore significant that in Streptomyces lividans the potassium channel KcsA, which resembles that of eukaryotes, forms tetramers that contain polyP whilst both monomers and tetramers are covalently linked to short chain PHB. Pumps are the counterparts of channels. Reusch has also shown that a model pump, the calcium ATPase pump of human erythrocytes, contains both cPHB and polyP and has strongly implicated these polymers in its functioning. Again, one may wonder whether these polymers are essential constituents of other pumps. Reusch has gone on to show that a wide range of proteins are modified post-translationally by covalent addition of short chain PHB in both prokaryotes and eukaryotes including DNA-binding proteins such as histones. Finally, Reusch has extended the importance of short chain PHB to medicine by showing its likely involvement in atherogenic plaques and diabetes. And yet this opus has gone largely unnoticed.

3-Hydroxybutyric Acid↗
PubMed2006

Identification of two gene loci involved in poly-beta-hydroxybutyrate production in Rhodobacter sphaeroides FJ1.

BACKGROUND AND PURPOSE: Polyhydroxyalkanoates (PHAs), biopolyesters of hydroxyl fatty acids, are synthesized and deposited as cytoplasmic inclusions in many bacteria. We isolated a poly-beta-hydroxybutyrate (PHB)-producing bacterium designated Rhodobacter sphaeroides FJ1. To characterize PHB biosynthesis in this organism, we isolated the genes encoding proteins involved in PHB metabolism. METHODS: The genes responsible for the synthesis, accumulation, and degradation of PHB in R. sphaeroides FJ1 were cloned and characterized. RESULTS: Genes involved in the biosynthesis and metabolism of PHB were found to be located in 2 different loci in the genome of R. sphaeroides FJ1. One locus contained genes encoding PHB depolymerase (phbZ), PHB synthase (phbC), phasin (phbP) and the regulator protein (phbR). The other locus contained the beta-ketothiolase gene (phbA) and the acetoactyl-CoA reductase gene (phbB). The phbZ gene was orientated in an opposite direction to that of phbC, phbP and phbR genes that were located in the same cluster. R. sphaeroides FJ1 was able to grow in wastewater released from the human waste treatment plant of Fu-Jen University. Optimal growth and PHB production were achieved when R. sphaeroides FJ1 was grown in tryptic soy broth containing 50% wastewater. PHB production by R. sphaeroides FJ1 varied in media with different carbon to nitrogen ratios, but the level of PHB synthase was constant, suggesting that PHB production depends mainly on substrate supply. CONCLUSIONS: Six genes encoding proteins related to PHB metabolism are clustered in 2 separate loci, phbZCPR and phbAB, in a PHB-producing bacterium R. sphaeroides FJ1 isolated from wastewater. PHB synthase, the key enzyme for PHB synthesis, is constitutively expressed, and its expression level is not affected by different growth conditions.

Acyltransferases↗
PubMed2006

Thalassobius aestuarii sp. nov., isolated from tidal flat sediment.

A strictly aerobic, non-motile, ovoid-shaped Alphaproteobacteria, designated strain JC2049(T), was isolated from a tidal flat sediment sample. The results of 16S rRNA gene sequence analysis indicated that this isolate belonged to the genus Thalassobius, with a sequence similarity of 96.9-97.3% to other valid Thalassobius spp. The cells required 1-7% NaCl for growth (optimum 2%) and accumulated poly-beta-hydroxybutyrate. Nitrite was reduced to nitrogen, but nitrate was not reduced to nitrite. No genetic potential for aerobic anoxygenic photosynthesis was detected. The primary isoprenoid quinone (Ubiquinone-10), predominant cellular fatty acids (C(18:1)omega7c, 11 methyl C(18:1)omega7c and C(16:0)) and DNA G+C content (61 mol%) were all consistent with the assignment of this isolate to the genus Thalassobius. Several phenotypic characteristics clearly distinguished our isolate from other Thalassobius species. The degree of genomic relatedness between strain JC2049(T) and other Thalassobius species was in a range of 20-43%. The polyphasic data presented in this study indicates that our isolate should be classified as a novel species within the genus Thalassobius. The name Thalassobius aestuarii sp. nov. is therefore proposed for this isolate; the type strain is JC2049(T) (= IMSNU 14011(T) = KCTC 12049(T) = DSM 15283(T)).

Fatty Acids↗
PubMed2006

[Development of an anti-infection nano-hydroxypatite drug delivery microsphere and its drug-release in vitro].

OBJECTIVE: To develop an anti-infection nano-hydroxypatite (nano-HA) microsphere for local drug delivery for treating osteomyelitis. METHODS: The nano-HA was used as the core carrier to load gentamicin (GM) and coated with poly(-hydroxybutyrate-co- hydroxyvalerate)/polyethylene glycol (PHBV/PEG), which was degradable and biocompatible, to prepare nano-HA-PHBV/PEG-GM microsphere. The surface structure and in vitro drug-release of the microsphere were studied. RESULTS: The microsphere had good drug delivery capability. The samples weighing 90 mg each were soaked in PBS and gentamicin release within the first day was 165.2 microg/ml, which maintained a low release rate in the following days. After 28 days, gentamicin release declined to 8.5 microg/ml, which was higher than the minimal inhibitory concentration of gentamicin (2 microg/ml). CONCLUSION: The local drug delivery system has good drug-release performance in vitro and may possess potential value in clinical management of osteomyelitis.

Anti-Bacterial Agents↗
PubMed2006

[Research on polyhydroxyalkanoate form a key aspect to enhanced biological phosphorus transformation].

To investigate the influence of PHB and PHV formed on phosphorus (P) release, uptake and removal during enhanced biological phosphorus removal (EBPR), anaerobic/aerobic batch experiments were conducted with biomass acclimated with propionic to acetic acid carbon molar ratios of 0.5 and 2 on two sequencing batch reactors (SBR1 and SBR2). Statistically significant correlations between polyhydroxyalkanoate (PHA) quantity and form and P release/uptake and removal were observed (R2 >0.90). The regression coefficients showed that for biomass cultured with customizing wastewater P release and uptake were both a function of PHB but not of PHV, but higher P removal was largely because of PHV as the predominant type rather than PHV. For biomass cultured with different ratios of propionic to acetic acid, the SBR2 biomass synthesized and utilized more PHB and less PHV and showed higher net P removal (average increase of 16.69%) than SBR1. Thus acetate/propionate content of influent had a major influenceon PHA type and quantity and determine phosphorus (P) release, uptake and removal. Accordingly, PHB and PHV transformations should be taken into account as key aspect for optimizing EBPR.

Bacteria, Aerobic↗
PubMed2005

[Rubomycin microincapsulation with biodegradable polymer matrix].

A procedure for preparation of microspheres from biodegradable linear polyether of microbiological origin (polyhydroxybutirate, PHB) with using the technology of solvent evaporation was developed considering a specific example of two- and three-component emulsions. The procedure provided permanent preparation of the microspheres of high quality. The influence of the procedure (emulsion type, dispersion process and medium temperature) on the yield of the microspheres, their structure and size was shown. The temperature had a significant impact on incorporation of the antitumor anthracycline antibiotic daunorubicin (rubomycin) to the polymer matrix. The microspheres with various levels of the drug load (29 and 90% of the initial content in the emulsion) were prepared and the kinetics of the in vitro rubomycin release was studied. The dynamics of the highly toxic rubomycin release from the microspheres was on the whole even with the curve profile reaching the plateau in 20-22 hours of the observation period. The rate of the rubomycin release to the medium depended on the value of the antibiotic incorporation and was maximum within the first two hours (3.3 and 13.0 mcg/ml.h) that corresponded to the release of 0.97 and 3.89 of the incorporated antibiotic. The average rate of rubomycin release during 300 hours was 0.81 x 10(-4) and 2.3 x 10(-4) mcg/ml x h. The release constituted respectively 3.9 and 13.11% of the antibiotic incorporated to the microspheres.

Antibiotics, Antineoplastic↗
PubMedDate not supplied

[New poly-(3-hydroxybutyrate)-based systems for controlled release of dipyridamole and indomethacin].

New poly-(3-hydroxybutyrate)-based systems for controlled release of anti-inflammatory and antithrombogenic drugs have been studied. The release occurs via two mechanisms (diffusion and degradation) operating simultaneously. Dipyridamole and indomethacin diffusion processes determine the rate of the release at the early stages of the contact of the system with the environment (the first 6-8 days). The coefficient of the release diffusion of a drug depends on its nature, the thickness of the poly-(3-hydroxybutyrate) films containing the drug, the concentrations of dipyridamole and indomethacin, and the molecular weight of the poly-(3-hydroxybutyrate). The results obtained are critical for developing systems of release of diverse drugs, thus, enabling the attainment of the requisite physiological effects on tissues and organs of humans.

Azotobacter↗
PubMedDate not supplied

[Preparation of biodegradable porous films for use as wound coverings].

We studied the preparation of polymeric films formed from solutions of poly-3-hydroxybutyrate and poly-epsilon-caprolactone in chloroform and methylene chloride. A morphological study of film chips (electron microscopy) showed that solvent evaporation results in the formation of a heterogeneous structure with interpenetrating pores (1-20 microm). We proposed a new method for introducing the proteolytic enzyme and the aminopolysaccharide chitosan into the composition of polyester films. Composite films possessed necrolytic activity and were characterized by increased hydrophilicity. Properties of enzyme-containing films from a mixture of polymers (proteolytic activity, porous structure, and increased hydrophilicity) account for their use in the preparation of biodegradable wound coverings.

Bandages↗
PubMed1989

The poly-beta-hydroxybutyrate granule in vivo. A new insight based on NMR spectroscopy of whole cells.

High resolution 13C NMR spectroscopy of live cells has been used to show that poly-beta-hydroxybutyrate (PHB) is predominantly in a mobile state within the storage granules of Alcaligenes eutrophus, Methylobacterium extorquens, and Methylobacterium AM1. Comparison of chemical and NMR analysis of PHB indicates that about 70% of the polymer in A. eutrophus gives sharp observable resonances. Temperature-dependent line widths and relaxation rates together with nuclear Overhauser effect measurements demonstrate that the observed material is effectively a mobile amorphous elastomer that is well above its glass transition temperature. The hydroxyvalerate-hydroxybutyrate copolymer produced by propionate-fed A. eutrophus has virtually the same mobility as the homopolymer. Evidence is presented indicating that water is an integral component of the PHB granule and that this component acts as a plasticizer for the polymer. These observations strongly suggest that the enzyme(s) responsible for PHB biosynthesis and consumption operate only on mobile hydrated material and that the solid granules characteristic of dried cells are partially artifactual. This model is supported by a reinterpretation of previously inexplicable biochemical results.

Alcaligenes↗
PubMed1989

[Preparation of preoral sustained-release preparations with a base of biodegradable polymers. 3. Preparation of matrix tablets with a base of poly-3-hydroxybutyric acid].

Poly-3-hydroxybutyric acid belongs to the biological polymers, which are produced by bacterials. The determination of the grain size, moisture content, flowability and the parameters for the direct compression was performed in regard of their use as auxillary substance for the preparation of solid sustained release dosage forms. The production of the matrix tablets was performed on the basis of a factorial design. The content of substance, an addition of Heweten 12 and the compression power served as factors. Caffeine was used as model drug. The in vitro release values show, that all three factors have an influence of the drug release. Optimized matrix tablets were produced on the basis of this result.

Chemistry, Pharmaceutical↗
PubMed1989

Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase.

The poly-beta-hydroxybutyrate biosynthetic thiolase gene from Zoogloea ramigera was used as a hybridization probe to screen restriction digests of Alcaligenes eutrophus H16 DNA. Specific hybridization signals were obtained and two fragments (a 2.3-kilobase PstI fragment and a 15-kilobase EcoRI fragment) cloned in the Escherichia coli vector pUC8 (plasmids pAeT3/pAeT10 and pAeT29, respectively). Biochemical analysis of lysates of E. coli cells containing each plasmid identified significant levels of beta-ketothiolase and acetoacetyl-CoA reductase enzyme activities in lysates of E. coli cells containing plasmids pAeT10 or pAeT29. Nucleotide sequence analysis of the pAeT10 insert identified two open reading frames which encode polypeptides of Mr = 40,500 and Mr = 26,300 corresponding to the structural genes for beta-ketothiolase (phbA) and acetoacetyl-CoA reductase (phbB), respectively. Amino acid sequence homologies between the two bacterial and two mammalian thiolases are discussed with respect to the chain length specificity exhibited by the different thiolase enzymes.

Acetyl-CoA C-Acyltransferase↗
PubMed1989

Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC).

The phbC gene encoding the third enzyme of the poly-beta-hydroxybutyrate biosynthetic pathway, poly-beta-hydroxybutyrate polymerase, in Alcaligenes eutrophus H16 has been identified by the complementation of poly-beta-hydroxybutyrate negative mutants of A. eutrophus H16. These results demonstrate that the three enzymes of the poly-beta-hydroxybutyrate biosynthetic pathway are organized phbC-phbA-phbB. Expression of all three genes in Escherichia coli results in a significant level (50% dry cell weight) of poly-beta-hydroxybutyrate production. phbC encodes a polypeptide of Mr = 63,900 which has a hydropathy profile distinct from typical membrane proteins indicating that poly-beta-hydroxybutyrate biosynthesis probably does not involve a membrane complex.

Alcaligenes↗
PubMed1987

Purification and characterization of NADP-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M.

An NADP-linked acetoacetyl-CoA reductase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium. The purified enzyme showed specific activity of 412 mumol acetoacetyl-CoA reduced per min per mg protein, which constituted an 880-fold purification compared to the crude extract, with a 32% yield. Electrophoretic analysis of the purified enzyme which had been cross-linked with dimethylsuberimidate showed that the native enzyme (Mr 92,000) is a tetramer of four identical subunits (Mr 25,500). Among the various D-(-)- and L-(+)-3-hydroxyacyl-CoAs tested, the purified enzyme oxidized only D-(-)-3-hydroxybutyryl-CoA and to a lesser extent D-(-)-3-hydroxyvaleryl-CoA in the presence of NADP+. The antiserum prepared against the purified enzyme completely inhibited poly(3-hydroxybutyrate) synthesis from acetyl-CoA by a crude extract of Z. ramigera I-16-M cells. These findings indicate that this enzyme plays an indispensable role as the supplier of D-(-)-3-hydroxybutyryl-CoA in poly(3-hydroxybutyrate) synthesis in this bacterium.

Alcohol Oxidoreductases↗