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PubMed2002

Posttranslational modification of E. coli histone-like protein H-NS and bovine histones by short-chain poly-(R)-3-hydroxybutyrate (cPHB).

Short-chain poly-(R)-3-hydroxybutyrate (cPHB), a highly flexible, amphiphilic molecule with salt-solvating properties, is a ubiquitous constituent of prokaryotic and eukaryotic cells, wherein it is mainly conjugated to proteins. The solvating properties and cellular distribution of cPHB suggest it may be associated with proteins that bind and/or transfer DNA. Here we examine Escherichia coli protein H-NS and calf thymus histones, H1, H2A, H2B, H3, and H4, for the presence of cPHB. The proteins are related in that all bind to DNA and are implicated in the compact organization of the chromosome. The presence of cPHB in E. coli H-NS was first detected in Western blots of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of total cell proteins, probed with anti-cPHB IgG, and then by Western blot analysis of the purified protein. Western blot analysis of the calf thymus histones indicated that each contained cPHB. The presence of cPHB in H-NS and histones was confirmed by chemical assay. The in vivo size of conjugated cPHB could not be established due to the lack of standards and degradation of cPHB during protein purification and storage. The molecular characteristics of cPHB and its presence in histone-like and histone proteins of diverse organisms suggest it may play a role in DNA binding and/or DNA organization.

Animals↗
PubMed1997

Novel components and enzymatic activities of the human erythrocyte plasma membrane calcium pump.

The plasma membrane Ca2+ pump is essential for the maintenance of cystolic calcium ion concentration levels in eukaryotes. Here we show that the Ca2+-ATPase, purified from human erythrocytes, contains two homopolymers, poly(3-hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), which form voltage-activated calcium channels in the plasma membranes of Escherichia coli and other bacteria. Furthermore, we demonstrate that the plasma membrane Ca2+-ATPase may function as a polyphosphate kinase, i.e. it exhibits ATP-polyphosphate transferase and polyphosphate-ADP transferase activities. These findings suggest a novel supramolecular structure for the functional Ca2+-ATPase, and a new mechanism of uphill Ca2+ extrusion coupled to ATP hydrolysis.

Adenosine Triphosphate↗
PubMed2002

Determination of poly-beta-hydroxybutyric acid in Bacillus thuringiensis by capillary zone electrophoresis with indirect ultraviolet absorbance detection.

A new capillary electrophoresis method for determining poly-beta-hydroxybutyric acid (PHB) in Bacillus thuringiensis was established. Poly-beta-hydroxybutyric acid in samples was hydrolyzed by sulphuric acid and neutralized by Ba(OH)2. The content of produced beta-hydroxybutyrate was then determined by capillary zone electrophoresis (CZE) with indirect UV detection at 254 nm. With 5 mM p-hydroxybenzoate and 0.5 mM tetradecyltrimethylammonium bromide (TTAB) at pH 8.0 as carrier electrolyte, beta-hydroxybutyrate can be determined within 6 min. Standard regression equation was made by beta-hydroxybutyrate, and the linear range was 2-1000 microg/ml. The relative standard deviations (RSDs) for migration time and peak area are both less than 1.0%. The detection limit for beta-hydroxybutyrate was 0.2 microg/ml, which is two to three orders of magnitude lower than that of the gas chromatography (GC) method. The capillary electrophoresis method was successfully applied to determine poly-beta-hydroxybutyric acid in fermentation broth and single colony. The added standard recovery was 96%.

Bacillus thuringiensis↗
PubMed2001

Storage and degradation of poly-beta-hydroxybutyrate in activated sludge under aerobic conditions.

This research analyses the accumulation and degradation of poly-beta-hydroxybutyrate (PHB) in experiments with pulse addition of acetate to samples of activated sludge from pilot-plant and full-scale wastewater treatment plants. The experiments are divided into two periods: a feast period defined as the time when acetate is consumed and a famine period when the added acetate has been exhausted. In the feast period the significant process occurring is the production of PHB from acetate. The produced PHB is utilised in the famine period for production of glycogen and biomass. According to modelling results approximately 90% of the total potential growth occurs in the famine period utilising the stored PHB. The degradation rate for PHB in the famine period is found to be dependent on the level of PHB obtained at the end of the feast period. It was found that multiple order kinetics gives a good description of the rate of PHB degradation. The examined sludge of low SRT origin is found to degrade PHB faster than long SRT sludge at high fractions of PHB. The observed yield of glycogen on PHB in the famine period is in the range of 0.22-0.33 g COD/g COD depending on the SRT. The storage pool of glycogen in the examined sludge is more slowly degraded than PHB (COD/COD/h).

Acetates↗
PubMed2001

The storage of acetate under anoxic conditions.

Till now the role of storage in activated sludge processes under transient conditions has been deeply investigated under anaerobic (EBPR processes) or aerobic (bulking control) environments. Little attention has been given to the role of storage in processes including anoxic environments. Hence, the aim of the present work was to investigate the anoxic storage along with other substrate removal mechanisms under transient conditions. Several mixed culture were ad hoc selected under anoxic environment and periodic feeding (acetate as carbon source) at different organic load rate (OLR) and feed length; then their transient response to substrate spike was investigated by batch tests under both anoxic and aerobic conditions. The relative role of different mechanisms in the substrate removal was established on the basis of COD balance assuming that the acetate COD removed from the liquid phase could be oxidised for energy needs or recovered into solids as poly-3-hydroxybutyric acid (PHB) (storage), other internal precursors or intermediates (accumulation) and active biomass (growth, as estimated by ammonium uptake). In all tested conditions, growth response was very little while PHB storage was prevailing. In some operating conditions, indirect evidence of accumulation (in forms still to be identified) was also found. The transient response was not affected by the presence of free amino acids, at least for the unacclimated mixed culture under observation. Transient response under aerobic condition was quite similar to the anoxic one.

Acetates↗
PubMed2002

Poly-beta-hydroxybutyrate metabolism in dynamically fed mixed microbial cultures.

The kinetics of production and degradation of poly-beta-hydroxybutyrate (PHB) by a mixed activated sludge culture growing on acetate was studied in a sequencing batch reactor (SBR). Occasionally a very high amount of acetate was added to the steady state system in order to obtain high PHB concentrations in the cells (fPHB). This made it possible to follow PHB production and degradation over a wide range of fPHB-data (between 0 and 0.8 Cmol/Cmol). The results were compared with data available in literature and with equations derived by metabolic modeling. This led to some remarkable observations. For the feast period, the ratio q(feast)PHB/-q(feast)Ac (specific PHB production rate over specific acetate uptake rate) was used to indicate which fraction of the substrate is stored. Experimentally and theoretically it was shown that this ratio has a constant value for dynamically fed systems operated at a sludge retention time (SRT) > 2d. This value is 0.6 Cmol/Cmol under aerobic conditions and 0.4-0.5 Cmol/Cmol under anoxic conditions, irrespective of the specific growth rate of the biomass and the specific acetate uptake rate in the feast period. Degradation of internal stored PHB could be described with a first order degradation rate with respect to the PHB content of the cells. Degradation of PHB appeared to be independent of the type of electron acceptor present in the system and independent of the SRT of the system. The kinetic descriptions can be used to predict PHB production and consumption in general in dynamic fed wastewater treatment systems, and they provide some trends for modeling purposes.

Acetates↗
PubMed2001

Effect of dissolved oxygen concentration in the fermentation medium on transformation of the carbon sources during the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxypropionate) by Alcaligenes latus.

Effects of fermentation conditions on the comonomer composition and its distribution of poly(3-hydroxybutyrate-co-3-hydroxypropionate) [P(3HB-co-3HP)] have been investigated for bacterial synthesis of P(3HB-co-3HP)s by Alcaligenes latus from sucrose and 3-hydroxypropionate (3HPA) mixed carbon sources. Comparison of the microstructures of these samples drew a conclusion that when the concentration of oxygen dissolved (DO) in the fermentation medium was controlled between 5 and 20% (based on the concentration at saturation), the 3HP content and the comonomer compositional distribution (CCD) of the copolymer would not be influenced by the DO values. The concentration of the carbon sources was monitored during the fermentation. The results indicated that the comonomer composition and its distribution of P(3HB-co-3HP)s were interrelated to the amounts of carbon sources transported into the bacterial cells. When the bacteria consumed more sucrose, the more 3HPA they would utilize, and the broader the CCD of the copolymer would be. Furthermore, the efficiencies of the transformation of the two carbon sources to the copolymer constituents were found to be similar.

Alcaligenes↗
PubMed2002

Identification and characterization of poly-3-hydroxybutyrate granule-associated protein, PGA12 and PGA16 in Zoogloea ramigera I-16-M.

Poly-3-hydroxybutyrate (PHB) granules of Zoogloea ramigera I-16-M contained two major PHB granule-associated proteins (PGA12 and PGA16) as revealed by sodium dodecyl sulfate-polyacrylamide gel elecrophoresis. N-terminal amino acid sequences of these proteins were determined. The genes encoding these proteins were cloned and sequenced. The structural genes of PGA12 and PGA16 were 351 and 447 bp long, which encode polypeptides with deduced molecular masses of 12.3 and 16.0 kDa, respectively. PGA12 and PGA16 were expressed in Escherichia coli. PHB granules were isolated from cells of recombinant strains of E. coli JM109, which harbored and expressed the PHB-synthetic genes of Ralstonia eutropha H16 and PGA12 or PGA16. These PHB granules contained PGA12 or PGA16 as a major protein. The presence of pga12 or pga16 did not affect the amount of PHB synthesized in E. coli. PGA12 and PGA16 bound to crystalline and amorphous PHB granules.

Amino Acid Sequence↗
PubMed1992

Biodeuteration of poly(beta-hydroxybutyrate).

The formation of poly(beta-hydroxybutyrate), PHB, by Rhodobacter sphaeroides and Alcaligenes eutrophus was studied using the following carbon sources and solvents: (1), acetate in H2O; (2), D3-acetate in H2O; (3), acetate in 90 to 92% D2O; and (4), D3-acetate in 90 to 92% D2O. The growth of Rb. sphaeroides cultured under condition (2) showed no apparent deuterium isotope effect, while considerably slowed growth in the presence of D2O was observed under conditions (3) and (4). In all cases, the PHB produced under deuterium enriched conditions was of high molecular weight. Interestingly, comparatively high volumetric formation of partially deuterated PHB was obtained using culture condition (4) for A. eutrophus. Fourier transform infrared spectroscopy (FT-i.r.), pyrolysis gas chromatography mass spectrometry (PGC-m.s.), and nuclear magnetic resonance (n.m.r.) were used to establish the extent and distribution of deuterium in the PHB samples produced. Partially deuterated PHB was obtained in each case, using a deuterium enriched culture. Considerable differences in the extent and distribution of deuterium were found between micro-organisms and culture conditions.

Acetates↗
PubMed1992

Plasticization of poly(hydroxybutyrate) in vivo.

The influence of a variety of treatments on the mobility and crystallinity of poly(hydroxybutyrate) (PHB) in whole cells and native granules has been proved using 13C-n.m.r. spectroscopy and X-ray powder diffraction, and correlated with the known biological effects of these treatments. It was concluded that at least water is responsible for PHB plasticization in vivo, and that only native mobile PHB is susceptible to depolymerases. Another, probably hydrophobic, component appears to be involved either as plasticizer or nucleation inhibitor. Three states of the granule are identified in addition to the native, biologically-competent state: freeze-drying of whole cells leads to a partially-immobilized amorphous state which can be restored virtually to native mobility by rehydration; extended centrifugation of native granules in aqueous suspension, or treatment with hydrophobic detergents under certain conditions, leads to a crystalline state that is less susceptible to exogenous depolymerase; and heating to 95 degrees C or refrigeration has no detectable effect on mobility but leads to inactivation of the granule, presumably via damage to superficial membrane or protein.

Alcaligenes↗
PubMed1992

Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei.

Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source.

Carboxylic Ester Hydrolases↗
PubMed1992

Biosynthesis of poly(3-hydroxyalkanoate) from amino acids.

It was found that an optically active copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), denoted as P(3HB-co-3HV), is synthesized by Alcaligenes eutrophus H16 from several amino acids under various fermentation conditions. The optimum condition for the biosynthesis from one amino acid, threonine, was investigated and its biosynthetic pathway was discussed on the basis of the relation between the fermentation condition and the co-monomer composition of the produced polyesters.

Alcaligenes↗
PubMed1998

The adsorption of substrate-binding domain of PHB depolymerases to the surface of poly(3-hydroxybutyric acid).

The binding characteristic of PHB depolymerase has been studied by using glutathione S-transferase (GST) fusion proteins with substrate-binding domain of three bacterial PHB depolymerases, Alcaligenes faecalis, Comamonas acidovorans and Comamonas testosteroni. Analysis using immuno-gold labeling technique and transmission electron microscopy indicated that a novel GST fusion protein derived from A. Faecalis enzyme adsorbed to the surface of poly(3-hydroxybutyric acid) (P(3HB)) single crystals like other fusion proteins. Comparison of inhibiting degree of P(3HB) powder hydrolysis activity of PHB depolymerase by fusion proteins indicated that three fusion proteins bind to P(3HB) powder in the same degree. The measurement of the surface hydrophobicity of proteins suggests that the interaction of the substrate-binding domain with insoluble P(3HB) may include not only a hydrophobic effect but also molecule-specific contacts.

Acyltransferases↗
PubMed1999

Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Burkholderia cepacia D1.

Copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced by Burkholderia cepacia D1 at 30 degrees C in nitrogen-free culture solutions containing n-butyric acid and/or n-valeric acid. When n-valeric acid was used as the sole carbon source, the 3HV fraction in copolyester increased from 36 to 90 mol% as the concentration of n-valeric acid in the culture solution increased from 1 to 20 g/l. The addition of n-butyric acid to the culture solution resulted in a decrease in the 3HV fraction in copolyester. The copolymers biosynthesized by this method were mixtures of random copolymers having a wide variety of composition of the 3HV component. The melting points of the fractionated copolymers show a concave curve with the minimum at the 3HV content of approximately 40 mol%. The alpha-parameter of lattice indices of the P(3HB) crystal for the fractionated copolymers largely increased as the 3HV composition increased. Biodegradability of the copolymer increased with the lower content of 3HV composition and/or the lower crystallinity.

Burkholderia cepacia↗
PubMedDate not supplied

Recent advances in polyhydroxyalkanoate production by bacterial fermentation: mini-review.

Poly(3-hydroxybutyrate) [P(3HB)] and other polyhydroxyalkanoates (PHAs) have been drawing much attention as biodegradable substitutes for conventional nondegradable plastics. For the economical production of P(3HB), various bacterial strains, either wild-type or recombinant, and new fermentation strategies were developed for the production of P(3HB) with high concentration and productivity. To reduce the cost of carbon substrate, several processes for P(3HB) production from cheap carbon sources were also developed. P(3HB) can now be produced to a content of 80% of cell dry weight with the productivity greater than 4 g/l per h. Fermentation strategy was also developed for the efficient production of medium chain length PHA by high cell density culture. With all these advances, P(3HB) and PHAs can be produced by bacterial fermentation at a cost (ca. $2/kg) similar to that of other biodegradable polymers under development.

Bacteria↗
PubMedDate not supplied

Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria--the metabolism and potential for CO2 recycling.

Regulatory mechanism in PHB [poly-(hydroxybutyrate)] accumulation by cyanobacteria, especially by a thermophilic isolate, Synechococcus MA19 was reviewed in comparison with a genetically engineered strain. The strain, MA19 accumulates PHB under nitrogen starved and photoautotrophic conditions (MA19-N). Little PHB synthase activity was detected in crude extracts from the cells grown in nitrogen sufficient conditions (MA19 + N). The activity was detected exclusively in membrane fractions from MA19 + N. The change of the enzyme activity was insensitive to chloramphenicol, which suggests post-translational activation. In vitro, acetyl phosphate activated PHB synthase in membrane fractions from MA19 + N, and the extent of activation depended on the concentration of acetyl phosphate. Phosphotransacetylase which catalyzes the conversion of acetyl-CoA to acetyl phosphate was detected in crude extracts from MA19-N but not in those from MA19 + N. These results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C-N balance and intracellular acetyl-CoA concentration. On the contrary, in genetically-engineered cyanobacterium (transformant with PHB synthesizing genes from Ralstonia eutropha), it did not seem to be PHB synthase but acetyl-CoA flux that limits PHB synthesis. The closer association of PHB granules with thylakoid membranes in MA19 is suggested than that in the genetically-engineered cyanobacterium, which may reflect the difference of distribution of PHB synthase. Transposon-mutagenesis was used to acquire mutants of its altered PHB regulatory mechanism. PHA production by cyanobacteria was considered from the aspects of photobioreactors.

Acyltransferases↗
PubMedDate not supplied

Chain termination in polyhydroxyalkanoate synthesis: involvement of exogenous hydroxy-compounds as chain transfer agents.

We have identified a range of compounds which, when present during poly(3-hydroxybutyrate) [P(3HB)] accumulation by Ralstonia eutropha (reclassified from Alcaligenes eutrophus), can act as chain transfer agents in the chain termination step of polymerization. End-group analysis by 31P NMR of polymer derivatized with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane revealed that all these compounds were covalently linked to P(3HB) at the carboxyl terminus. All chain transfer agents possessed one or more hydroxyl groups, and glycerol was selected for further investigation. The number-average molecular mass (Mn) of P(3HB) produced by R. eutropha from glycerol was substantially lower than for polymer produced from glucose, and we identified two new end-group structures. These were attributed to a glycerol molecule bound to the P(3HB) chain via the primary or secondary hydroxyl groups. When a primary hydroxyl group of glycerol is involved in chain transfer, the end-group structure is in both [R] and [S] configurations, implying that chain transfer to glycerol is a random transesterification and that PHA synthase does not catalyse chain transfer. 3-Hydroxybutyric acid is the most probable chain transfer agent in vivo, with propagation and termination reactions involving transfer of the P(3HB) chain to enzyme-bound and free 3-hydroxybutyrate, respectively. Only carboxyl end-groups were detected in P(3HB) extracted from exponentially growing bacteria. It is proposed that a compound other than 3-hydroxybutyryl-CoA acts as a primer in the initiation of polymer synthesis.

Alcaligenes↗