Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids.

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.     

Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.     

Melt electrospinning of poly-(ethylene glycol-block-epsilon-caprolactone)

Various block copolymers of poly(ethylene glycol) and poly(epsilon-caprolactone) (PEG-b-PCL) with molecular weights between 7000 and 26,900 g/mol were synthesized, and melt electrospun at temperatures between 60 degrees C and 90 degrees C. Two types of fibers were collected, including excellent quality fibers - highly coiled and continuous, with a constant diameter and relatively defect free. Such fibers, termed "solid fibers", were sufficiently cooled during their path between the spinneret and the collector that the symmetric fiber shape is maintained after landing on the collector. The second type of melt electrospun fiber were poor quality, large diameter fibers, flattened on the collector - termed "molten fibers". The solid and molten fibers were morphologically distinct from each other as determined from scanning electron microscopy (SEM). Using an SEM imaging method to assess the regional variations of collected electrospun material, we found the spinneret pump rate largely influenced the fiber quality. The polymer flow rate to the spinneret and the molecular weight of PEG-b-PCL had the greatest effect on the electrospun fibers collected, with an optimum rate of 0.05-0.1 mL/h for the highest molecular weight copolymers. The lowest molecular weight PEG-b-PCL tended to electrospray, while the material collected from higher molecular weight copolymers were conducive to fiber formation. The highest quality fibers were PEG-b-PCL block copolymers (22,000 and 26,900 g/mol) melt electrospun at temperatures of 85 degrees C and 90 degrees C, corresponding to shear viscosities of the polymer of between 28.1 and 39.4 Pa.S.     

Model flavoproteins : Interaction of ribofIavin-5'-mono-phosphate with poly-(α -L-lysine) and poly-(α -L-histidine)

The interactions of flavin mononucleotide (riboflavin-5'-monophosphate) with two polypeptides, poly-(α-L-lysine) and poly-(α-L-histidine) in water and 0.05 M phosphate buffer were studied by measuring circular dichroism in the pH range 3 to 11. The interation of flavin mononucleotide with the two polypeptides was due to hydrophobic as well as ionic associations and was further influenced by the involvement of the ribityl side chain. The results of the present study have shown that small changes in the environmental conditions of the interacting molecules could modify their mode of interaction considerably. CDRI Communication No. 2816.     

A recombinant cyanobacterium that accumulates poly-(hydroxybutyrate)

Summary A cyanobacterium, Synechococcus sp. PCC 7942 was transformed with a recombinant plasmid harboring poly-(hydroxybutyrate) (PHB)-synthesizing genes from Alcaligenes eutrophus. The acquired transformant accumulated about 1% PHB of dry cell weight in nitrogen-starved conditions. The PHB content of the transformant was kept stable during a series of batch cultures.     

Isolation of a microorganism capable of degrading poly-(L-lactide)

The isolation of poly-(L-lactide) (PLA)-degrading microorganisms was investigated. A PLA-degrading actinomycete, strain No. 3118, was isolated and tentatively identified as a member of the genus Amycolatopsis. The optimum conditions for degradation of PLA were 43 degrees C at about pH 7 in a mineral salt medium with a low concentration of organic nutrients (0.002% yeast extract). The original shape of PLA film (Mw=2.3x10(5) after sterilization, 20 &mgr;m thick) disappeared within 2 weeks. Lactic acid was detected after the film was incubated with culture supernatant.     

Protein immobilization on poly-(N-substituted) acrylamides

A series of water-soluble polymers containing side chains derived from N-acryloyl-β-alanine, N-ethylacrylamide and N-[3-(N′,N′,N′-trimethylammonio)propyl] acylamide chloride has been prepared and characterized. A related series of insoluble gels was also prepared. Protein may be attached to these materials by means of amide bond formation between carboxyl groups on the polymers and amino groups of the protein; the preparation and characterization of conjugates formed with α-chymotrypsin are described. Polymers bearing negatively or positively charged side chains are attached to this enzyme at only a single amino acid and the integrity of the active site is largely preserved in these systems. The corresponding gels are not able to bind as much enzyme as are the soluble polymers and bound enzyme is less active in these cases.     

Mechanism of the interaction of hydrophobically-modified poly-(N-isopropylacrylamides) with liposomes

Interactions of hydrophobically-modified poly-(N-isopropylacrylamides) (HM PNIPAM) with phospholipid liposomes were studied as a function of the lipid type, the lipid bilayer fluidity, and the polymer conformation. Fluorescence experiments monitoring non-radiative energy transfer (NRET), between naphthalene attached to the HM PNIPAM and 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the lipid bilayer, confirmed the direct penetration of hydrophobic anchor groups linked to the polymer into the liposome hydrophobic core. Contraction of the polymer backbone above the lower critical solution temperature (LCST) resulted in a partial withdrawal of the anchor groups from the lipid bilayer. Analysis of polymer/lipid mixtures by centrifugation and quasi-elastic light scattering (QELS) revealed the polymer-induced fission of liposomes in the liquid-crystalline state, resulting in the formation of vesicles 150–230 nm in diameter. The process is reversible and upon transition of the bilayer into the gel state these vesicles are converted into larger aggregates. According to the results of gel-filtration experiments the HM PNIPAM is in dynamic exchange between the liquid-crystalline lipid bilayer and the water solution, while the binding to the bilayer in the gel state is more static in nature. The binding constant for mixture of HM PNIPAM with DMPC liposomes, evaluated from the centrifugation experiments, was found to be 120 M⁻¹.     

Biodegradability and mechanical properties of poly-(beta-hydroxybutyrate-co-beta-hydroxyvalerate)-starch blends.

Wheat starch granules and poly-(beta-hydroxybutyrate-co-beta-hydroxyvalerate) [P(HB-co-HV), (19.1 mol% HV)] were blended at 160 degrees C. Increasing the starch content from 0 to 50% (wt/wt) decreased the tensile strength of P(HB-co-HV) from 18 MPa to 8 MPa and diminished flexibility as Young's modulus increased from 1,525 MPa to 2,498 MPa, but overall mechanical properties of the polymer remained in a useful range. A mixed microbial culture required more than 20 days to degrade 150-microns-thick samples of 100% P(HB-co-HV), whereas samples containing 50% (wt/wt) starch disappeared in fewer than 8 days. Starch granules degraded before P(HB-co-HV) did. Aerobic degradation proceeded more rapidly than anaerobic degradation.     

Blends of poly-(epsilon-caprolactone) and polysaccharides in tissue engineering applications

Bioartificial blends of poly-(epsilon-caprolactone) (PCL) with a polysaccharide (starch, S; dextran, D; or gellan, G) (PCL/S, PCL/D, PCL/G 90.9/9.1 wt ratio) were prepared by a solution-precipitation technique and widely characterized by differential scanning calorimetry analysis (DSC), Fourier transform infrared-attenuated total reflectance spectroscopy (FTIR-ATR), optical microscopy (OM), wide-angle X-ray diffraction analysis (WAXD), and thermogravimetry (TGA). DSC showed that the polysaccharide reduced the crystallinity of PCL and had a nucleation effect, which was also confirmed by OM analysis. Hoffman-Weeks analysis was performed on PCL and blend samples allowing calculation of their equilibrium melting temperatures (). WAXD showed that the crystalline unit cell type was the same for PCL and blends. FTIR-ATR did not evidence interactions between blend components. Thermal stability was affected by the type of polysaccharide. Microparticles (<125 microm) were produced from blends by cryogenical milling and characterized by scanning electron microscopy analysis (SEM). Selective laser sintering (SLS), a new rapid prototyping technology for scaffold fabrication, was applied to sinter blend microparticles according to a PC-designed two-dimensional geometry (strips and 2 x 2 mm(2) square-meshed grids). The optimal experimental conditions for sintering were established and laser beam parameters (beam speed, BS, and power, P) were found for each blend composition. Morphology of sintered objects was analyzed by SEM and found to be dependent on the morphology of the sintered powders. Sintered samples were analyzed by chemical imaging (CI), FTIR-ATR, DSC, and contact angle analysis. No evidence of the occurrence of degradation phenomena was found by FTIR-ATR for sintered samples, whereas DSC parameters of PCL and blends showed changes which could be attributed to some molecular weight decrease of PCL during sintering. CI of sintered samples showed that the polysaccharide phase was homogeneously dispersed within the PCL matrix, with the only exception being the PCL/D blend. The contact angle analysis showed that all samples were hydrophilic. Fibroblasts were then seeded on scaffolds to evaluate the rate and the extent of cell adhesion and the effect of the polysaccharides (S, D, G) on the bioactivity of the PCL-based blends.     

Electromigration behavior of poly-(L-glutamate) conformers in concentrated polyacrylamide gels

During electrophoretic separation of anionic polyamino acids, resolution according to the number of peptide units can be achieved in capillaries filled with hydrophilic gels. While polyaspartate preparations yield single peaks for the individual oligomers at pH above 8.0, polyglutamates exhibit an anomalous behavior of peak splitting, which is attributed here to the separation of the oligopeptide conformers. An Asp-Glu (1:1) copolymer yields single peaks under similar conditions. At pH near 4.5, where polyglutamate is expected to exist in its α-helix form, peak splitting disappears. Upon heating to 95°C for at least 120 h (procedure described to transform the α-helix into a β form), peak splitting disappeared, but could be reestablished after cooling for several days. When a highly charged cation spermine was added to the operational electrolyte, triple peaks appeared in the electropherogram due to the ion-pair formation. The largest peak in every triplet has been tentatively assigned to the α-helix form. The electrophoretic results described have been largely supported by CD spectra. © 1993 John Wiley & Sons, Inc.     

Production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei using rice-based ethanol stillage with simultaneous recovery and re-use of medium salts

Haloferax mediterranei holds promise for competitive industrial-scale production of polyhydroxyalkanoate (PHA) because cheap carbon sources can be used thus lowering production costs. Although high salt concentration in production medium permits a non-sterile, low-cost process, salt disposal after process completion is a problem as current environmental standards do not allow total dissolved solids (TDS) above 2000 mg/l in discharge water. As the first objective of this work, the waste product of rice-based ethanol industry, stillage, was used for the production of PHA by H. mediterranei in shake flasks. Utilization of raw stillage led to 71 ± 2 % (of dry cell weight) PHA accumulation and 16.42 ± 0.02 g/l PHA production. The product yield coefficient was 0.35 while 0.17 g/l h volumetric productivity was attained. Simultaneous reduction of BOD₅ and COD values of stillage by 83 % was accomplished. The PHA was isolated by osmotic lysis of cells, purification by sodium dodecyl sulfate and organic solvents. The biopolymer was identified as poly-3-(hydroxybutyrate-co-15.4 mol%-hydroxyvalerate) (PHBV). This first report on utilization of rice-based ethanol stillage for PHBV production by H. mediterranei is currently the most cost effective. As the second objective, directional properties of decanoic acid together with temperature dependence of water solubility in decanoic acid were applied for two-stage desalination of the spent stillage medium. We report for the first time, recovery and re-use of 96 % of the medium salts for PHA production thus removing the major bottleneck in the potential application of H. mediterranei for industrial production of PHBV. Final discharge water had TDS content of 670 mg/l.     

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens

B. FONNESBECH, H. FRØKIAER, L. GRAM AND C. MOSBY JESPERSEN. 1993. Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria ( Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum ). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.     

Highly effective DNA in stimulating poly-(ADP-ribose) polymerase reaction.

The principle and the experimental realization of spin echo correlated spectroscopy (SECSY) are described and its use for studies of proteins is illustrated with ¹H n.m.r. spectra of the basic pancreatic trypsin inhibitor. This technique yields two-dimensional homonuclear correlated spectra which manifest connectivities between spin coupled nuclei and thus provide first level assignments of individual spin systems in biopolymers. Compared to previously described two-dimensional correlated n.m.r. techniques, which were applied exclusively to small molecules, SECSY uses a smaller data size both in the time domain and the frequency domain, which makes it particularly suitable for studies of macromolecules.     

Synthesis of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by recombinant Escherichia coli

Several recombinant Escherichia coli strains, including XL1-Blue, JM109, HB101, and DH5alpha harboring a stable high-copynumber plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were constructed. These recombinant strains were examined for their ability to synthesize and accumulate poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer from glucose and either propionate or valerate. All recombinant E. coli strains could synthesize the P(3HB-co-3HV) copolymer in the medium containing glucose and propionate. However, only the homopolymer poly-(3-hydroxybutyrate) [P(3HB)] was synthesized from glucose and valerate. The PHA concentration and the 3HV fraction could be increased by inducing with acetate and/or oleate. When supplemented with oleate, the 3HV fraction increased by fourfold compared with that obtained without induction. Induction with propionate resulted in lower PHA concentration due to the inhibitory effect, but an 3HV fraction of as high as 33.0% could be obtained. These results suggest that P(3HB-co-3HV) can be efficiently produced from propionate by recombinant E. coli by inducing with acetate, propionate, or oleate. (c) 1996 John Wiley & Sons, Inc.