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PubMed2006

Modulation of talA gene in pentose phosphate pathway for overproduction of poly-beta-hydroxybutyrate in transformant Escherichia coli harboring phbCAB operon.

The talA gene encoding transaldolase, the key enzyme in the nonoxidative pentose phosphate pathway, was amplified in a transformant Escherichia coli harboring the phbCAB operon to shift the metabolic flux of the hexose mono-phosphate shunt to the odd-ball biosynthesis pathway for poly-beta-hydroxybutyrate overproduction. The PHB content in the transformant E. coli coharboring the phbCAB operon and talA gene increased from 28.2% to 52.3%, and the retarded cell growth was overcome. This increase seems to be mainly due to the concomitant supplies of the intermediates NADPH and acetyl-CoA, which are from the activated pentose phosphate pathway through the modulation of the talA gene and from the Embden-Meyerhof pathway.

Bacterial Proteins↗
PubMed2001

Isolation of poly(3-hydroxybutyrate) (PHB)-degrading microorganisms and characterization of PHB-depolymerase from Arthrobacter sp. strain W6.

Microbial degraders of poly(3-hydroxybutyrate) (PHB) were isolated from soil. Arthrobacter sp. strain W6 used not only PHB as a carbon source, but also PHAs such as poly(3-hydroxybutyrate-co-[5%]3-hydroxyvalerate), poly(3-hydroxybutyrate-co-[14%]3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-[22%]3-hydroxyvalerate). PHB-depolymerase was purified to homogeneity from the culture broth of Arthrobacter sp. strain W6 by a procedure involving DEAE- and butyl-Toyopearl column chromatographies. The Mr of the enzyme was estimated to be about 47,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 8.5 and 50 degrees C, and was inhibited by phenylmethylsulfonyl fluoride, Hg2+, Ag+, and Pb2+.

Arthrobacter↗
PubMed2002

Enzyme inhibitors to increase poly-3-hydroxybutyrate production by transgenic tobacco.

Chemical regulation of secondary-metabolite synthesis was investigated through the improvement of poly-3-hydroxybutyrate (PHB) production in transgenic tobacco plants by the use of enzyme inhibitors. Two tobacco lines, BC3 and rCAB8, that produce PHB in both the cytosol and plastids were used. An acetyl-CoA carboxylase inhibitor, D-(+)-Quizalofop-ethyl, increased PHB accumulation in both lines 2-fold. The accumulation rate of plastidial PHB in the rCAB8 line was 2.5-fold higher than that of cytosolic PHB in the BC3 line. A specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, mevastatin, also increased PHB accumulation but only in the BC3 line. These results indicated that chemical regulation of the native metabolic flows by the specific enzyme inhibitors increased secondary-metabolite production in the transgenic tobacco plants we used.

Acetyl-CoA Carboxylase↗
PubMedDate not supplied

Aerobic and anaerobic microbial degradation of poly-beta-hydroxybutyrate produced by Azotobacter chroococcum.

Food industry wastewater served as a carbon source for the synthesis of poly-beta-hydroxybutyrate (PHB) by Azotobacter chroococcum. The content of polymer in bacterial cells grown on the raw materials reached 75%. PHB films were degraded under aerobic, microaerobic, and anaerobic conditions in the presence and absence of nitrate by microbial populations of soil, sludges from anaerobic and nitrifying/denitrifying reactors, and sediment from a sludge deposit site. Changes in molecular mass, crystallinity, and mechanical properties of PHB were studied. Anaerobic degradation was accompanied by acetate formation, which was the main intermediate utilized by denitrifying bacteria or methanogenic archaea. On a decrease in temperature from 20 to 5 degrees C in the presence of nitrate, the rate of PHB degradation was 7.3 times lower. Under anaerobic conditions and in the absence of nitrate, no PHB degradation was observed, even at 11 degrees C. The enrichment cultures of denitrifying bacteria obtained from soil and anaerobic sludge degraded PHB films for a short time (3-7 d). The dominant species in the enrichment culture from soil were Pseudomonas fluorescens and Pseudomonas stutzeri. The rate of PHB degradation by the enrichment cultures depended on the polymer molecular weight, which reduced with time during biodegradation.

Aerobiosis↗
PubMed2005

Synthesis of polyhydroxyalkanoate (PHA) from excess activated sludge under various oxidation-reduction potentials (ORP) by using acetate and propionate as carbon sources.

Accumulation of poly hydroxyalkanoate (PHA) from excess activated sludge (EAS) was monitored and controlled via the oxidation-reduction potential (ORP) adjusting process. The ORP was adjusted and controlled by only regulating the gas-flow rate pumped into the cultural broth in which sodium acetate (C2) and propionate (C3) were used as carbon sources. Productivity of PHA and the PHA compositions at various C2 to C3 ratios were also investigated. When ORP was maintained at +30 mV, 35% (w/w) of PHA of cell dry weight obtained when C2 was used as sole carbon source. The PHA copolymer, poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), accumulated by EAS with different 3-hydroxyvalarate (3HV) molar fractions ranged from 8% to 78.0% when C2 and C3 was used as sole carbon source, By using ORP to monitor and control the fermentation process instead DO meter, the ORP system provided more precise control to the PHA accumulation process from EAS under low dissolved oxygen (DO) concentrations. Adjusting the C2 to C3 ratios in the media could control the composition such as the 3HV/3HB ratios of the PHBV. Furthermore, it might be an effective way to adjust the 3HV molar fractions in PHBV by controlling the DO concentration via the ORP monitoring system. The 3HV molar fractions in the PHBV declined with increasing ORP from -30 mV to +100 mV by adjusting the gas-flow rate (i.e. the DO concentration). It is concluded that the DO plays a very important role in the synthesis of 3HV subunits in PHBV co-polymer from the EAS. Therefore, a hypothetic metabolic model for PHA synthesis from EAS was proposed to try to explain the results in this study.

Acetates↗
PubMed2005

Effects of fatty acids on growth and poly-3-hydroxybutyrate production in bacteria.

The effects of saturated and unsaturated fatty acids (lauric acid, palmitic acid, steric acid, oleic acid, linoleic acid, soybean oil) on Sphaerotilus natans, 0B17 (Pseudomonas sp.), and recombinant Escherichia coli DH5(/pUC19/CAB were studied. Oleic acid enhances Poly-3-hydroxybutyrate (PHB) production in these three bacterial strains, suggesting that the single double bond of the acid activates the polyhydroxylkanoate accumulation enzymatic reaction. Under the effect of lauric acid and linoleic acid, the growth of S. natans and 0B17 were totally inhibited. However, the enhanced PHB accumulation in recombinant E. coli was observed.

Bioreactors↗
PubMed1999

A high-copy-number plasmid capable of replication in thermophilic cyanobacteria.

A 2.5 kb high-copy-number plasmid, pMA4 in thermophilic cyanobacterium Synechococcus sp. MA4 was isolated and characterized to develop a genetic engineering system for thermophilic cyanobacteria. The copy number of pMA4 was determined to be by densitometry about 350/cell. The pMA4 may be a type of rolling-circle plasmid, because a possible rep gene encoding 34 kD-protein and a consensus sequence of a double-stranded origin nick site of rolling circle plasmids were found in the pMA4 sequence. The pMA4 was electro-introduced into another thermophile, Synechococcus sp. MA19, which is the strongest poly-beta-hydroxybutyrate (PHB) accumulator in photoautotrophic organisms. The pMA4 was incorporated and retained in MA19. These results indicate that pMA4 could be developed as a useful vector for thermophilic cyanobacteria.

Amino Acid Sequence↗
PubMed2002

Optimization of the neck region of dental implants with a bioactive, resorbable coating.

With the goal of shortening the postoperative healing phase and the minimization of the risk of peri-implantitis around dental implants, a polymer coating, with improved surface biocompatibility in the region of soft tissue penetration by the implant, was investigated. The polymer used was the relatively slowly resorbable poly-beta-hydroxybuterate (PHB) whose surface was further activated in a NH3--plasma. The influence of surface roughness of the substrate (commercially pure titanium) as well as the influence of the edge radii on the test samples was determined in prior studies. These studies formed the basis for an optimised coating process. In-vitro biocompatibility was determined using a human gingival primary cell culture. Surface morphology was determined with SEM and AFM to complement the cell culture studies.

Cell Division↗
PubMed2005

A large, voltage-dependent channel, isolated from mitochondria by water-free chloroform extraction.

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >+/-60 mV, conductance fluctuated in the range of approximately 50- approximately 200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (V(rev) approximately -15 mV) to ideally cation-selective (V(rev) approximately +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.

Animals↗
PubMed2006

Determination of poly(3-hydroxybutyrate) using a combination of enzyme-based biosensor and alkaline hydrolysis.

The combination of an enzyme-based biosensor and alkaline hydrolysis was developed for the measurement of poly(3-hydroxybutyrate) (PHB). The principle of the determination is based on that the alkaline condition converts PHB to produce its monomer, 3-hydroxybutyrate (3-HB), which generates a detectable current signal by an amperometric biosensor through coupled two-enzyme reactions on an electrode. This method takes less than 40 min, and results in a linear detection range of 0.5-110 mg L-1 PHB with a detection limit of 0.3 mg L-1 by the saturated production of 3-HB; it can also take less than 15 min and result in a linear detection range of 1.0-160 mg L-1 PHB with a detection limit of 0.5 mg L-1 by a part production of 3-HB. The method also shows simple operation and high reproducibility.

Biosensing Techniques↗
PubMed2006

Multistage wastewater treatment using separated storage driven denitrification and nitrification biofilms.

The feasibility of combining a previously reported storage driven denitrification biofilm, where 80% of influent acetate can be converted to poly-beta-hydroxybutyrate (PHB), with a suitable nitrification reactor, either submerged or trickling filter design, to achieve complete biological nitrogen removal was tested. The reactor system showed the potential of complete biological nitrogen removal of waste streams with a C/N ratio as low as 3.93 kg COD/kg N-NH3 at an overall nitrogen removal rate of 1.1 mmole NH3/L/h. While the efficiency and the rates of nitrogen removal were higher than what is observed in traditional or simultaneous nitrification and denitrification (SND) systems, there were two problems that require further development: (a) the incomplete draining of the reactor caused ammonia retention and release in the effluent, limiting the overall N-removal and (b) pH drifts in the nitrification step slowed down the rate of nitrification if not corrected by appropriate pH adjustment or buffering.

Ammonia↗
PubMed2004

Pain control via opioid analgesic-local anesthetic loaded IPNs.

Relief of chronic pain is an important clinical problem requiring special care and approaches. The present study was designed for the construction of a controlled release system for local application of analgesics (hydromorphone (HM), morphine (M), and codeine (C)) and a local anesthetic, bupivacaine (BP). An interpenetrating network (IPN) drug release system was prepared by using a biocompatible, biodegradable copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and another biocompatible but synthetic, nondegradable polymer, poly (2- hydroxyethyl methacrylate), (PHEMA). In situ release kinetics of the IPN system was first order for BP but could not be fitted to any known equation for the other drugs. Complete release from the IPNs occurred within a considerably short time (24 h for 80 % of the drugs) most probably due to the significant hydrophilicity of PHEMA. In order to slow down the release rate these IPNs were coated with PHBV. Release from these coated IPNs (cIPN) resulted in rates that could be described by Higuchi's equations. In vivo measurement of antinociceptive efficacy was carried out in rats with tail flick and paw-withdrawal tests after inducing chronic pain created by sciatic nerve ligation at the right side. Control groups received placebo implants. In vivo studies showed potent, prolonged (2-3 days) antinociception at the site of injury (right paw) for strong opioids (HM and M) and about 2 days for the weak opioid (C) and local anesthetic (BP). In all cases the release rate was found to be as important as the antinociceptive potency. The weakest opioid analgesic of those evaluated (C) had a higher first day antinociception than its stronger counterpart M, probably due to its higher initial concentration that was expected from its faster release rate in the in situ experiments.

Analgesics, Opioid↗
PubMedDate not supplied

Comparison of fatty acid composition and kinetics of phosphorus-accumulating organisms and glycogen-accumulating organisms.

It was demonstrated that glycogen-accumulating organisms (GAOs) were able to compete with phosphorus-accumulating organisms (PAOs) for acetate in a biological phosphorus removal (BPR) process, leading to a loss of BPR capability. Cellular fatty acid composition, which serves as a fingerprint for microbial identification, was used to determine microbial population change and to investigate the competition mechanisms of PAOs and GAOs. Analysis of cellular fatty acid composition indicated that PAOs grown with acetate and glucose were different species and that GAOs and PAOs grown with the same substrate were also different species. Glycogen-accumulating organisms seemed to coexist with PAOs even in a well-developed BPR process. The GAOs were able to accumulate more poly-beta-hydroxybutyrate (PHB) and glycogen than PAOs during the anaerobic stage of the BPR process. The GAOs synthesized more in-cell glycogen than PAOs. The growth rate for PAOs was always greater than that for GAOs at various acetate or glucose concentrations, while GAOs had higher acetate uptake and PHB synthesis rates than PAOs. Therefore, GAOs are thought to compete with PAOs only at long solids retention times (> or = 20 days).

Bacteria↗
PubMedDate not supplied

The role of poly-hydroxy-alkanoate form in determining the response of enhanced biological phosphorus removal biomass to volatile fatty acids.

Anaerobic-aerobic batch experiments indicated that poly-hydroxy-alkanoate (PHA) form was important in determining the net phosphorus removal resulting from different volatile fatty acids (VFAs). Poly-3-hydroxy-butyrate (3HB) content was found to correlate fairly well with higher observed aerobic phosphorus uptake per unit PHA carbon degraded. Poly-3-hydroxy-valerate (3HV) correlated with lower aerobic phosphorus uptakes per unit PHA carbon degraded. These experiments, conducted with synthetic wastewater, imply that VFA speciation might have a significant effect on aerobic phosphorus uptakes and net phosphorus removal. In addition, the model parameter fP.UPT (Barker and Dold, 1997) could vary with the proportion of acetic to propionic acid received (i.e., the acetic/propionic acid ratio may be an important parameter for these systems). Carbohydrate data implied that the lower aerobic phosphorus uptake resulting from 3HV might have been caused by a greater fraction of PHA carbon shunting to carbohydrate biosynthesis during aerobiosis.

Acetic Acid↗