Sequence distribution of β-hydroxyalkanoate units with higher alkyl groups in bacterial copolyesters (original) (raw)
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Rapid Communications in Mass Spectrometry, 1992
Two different samples of copolyesters produced by Pseudomonas oleouorans when grown either on nonanoic acid alone or on an equimolar mixture of nonanoic and octanoic acids were evaluated for sequence distribution by controlled methanolysis to their oligomers, separation of the oligomers by HPLC, and analysis of the fractions so obtained by fast atom bombardment mass spectrometry (FAB-MS). The copolyesters contained primarily 0-hydroxyheptanoate and 0-hydroxynonanoate units, which showed no observable differences in their l H and *3C NMR spectra with changes in composition, so those analyses could not be applied. The observed oligomer composition and that calculated on the basis of Bernoullian statistics for sequence distributions in random copolymers of the compositions studied were in good agreement.
Macromolecules, 2000
Two novel bacterial poly(3-hydroxyalkanoates) (PHAs) with either 3-hydroxy-7-oxooctanoate (HOO) and 3-hydroxy-5-oxohexanoate (HOH) or 8-acetoxy-3-hydroxyoctanoate (AHO), 6-acetoxy-3hydroxyhexanoate (AHH), and 4-acetoxy-3-hydroxybutyrate (AHB) monomer units were produced at pilot scale. For the biosynthesis of these PHAs Pseudomonas oleovorans was cultivated at a 24 L scale in two-liquid-phase fed-batch processes using mineral salts medium and mixtures of 2-octanone/octane or n-octylacetate/octane as carbon sources. The bacterial accumulation of the polyesters was induced by nitrogen starvation and the addition of the substrate mixtures. Under these conditions, 26 and 45 g of PHA were isolated. The PHAs contained 10.3 and 3.3 mol % of oxo and acetoxy group monomers. Physical characterization was done with respect to their molecular weights, and thermal properties and similar results were found as for octane-based PHA. All monomer units have been identified by 2D 1 H and 13 C heteronuclear correlated NMR spectroscopy (HSQC, HMBC, and HSQC-TOCSY), and the composition of the copolyesters was quantified from 1 H NMR spectra.
Polymers, 2020
Aneurinibacillus sp. H1 is a promising, moderately thermophilic, novel Gram-positive bacterium capable of the biosynthesis of polyhydroxyalkanoates (PHA) with tunable monomer composition. In particular, the strain is able to synthesize copolymers of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV) with remarkably high 4HB and 3HV fractions. In this study we performed an in-depth material analysis of PHA polymers produced by Aneurinibacillus sp. H1 in order to describe how the monomer composition affects fundamental structural and physicochemical parameters of the materials in the form of solvent-casted films. Results of infrared spectroscopy, X-ray diffractometry and thermal analysis clearly show that controlling the monomer composition enables optimization of PHA crystallinity both qualitatively (the type of the crystalline lattice) and quantitatively (the overall degree of crystallinity). Furthermore, resistance of the films against thermal and/or enzym...
We synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3-HB-co-3-HV)] copolymer having different contents of 3- hydroxyvalerate (3-HV) units (16.04, 16.3, 24.95, 25.62, and 16.52 mol % 3-HV) with different yields of polyhydroxyalkanoates (PHAs) by feeding with different cooking oils and with Alkaliphilus oremlandii OhILAs strain. The PHA production efficiency of the Alkaiphilus strain was compared with that of the control strain, Bacillus cereus. The synthesis of each PHA biopolymer was performed with different toxic spent oils as the sole carbon source in an oil-in-water-based microemulsion medium. We observed that the productivity of the poly(3- hydroxybutyrate) [P(3-HB)] copolymer from the Alkaliphilus strain was higher than those of the PHAs isolated from B. cereus and the Escherichia coli XL1B strain. The synthesized PHA copolymers were characterized by 1H-NMR and Fourier transform infrared (FTIR) spectroscopy. In the 1H-NMR spectra, a doublet resonance peak at 1.253 ppm of the/ methyl protons of the 3-hydroxybutyrate (3-HB) side group and one at 0.894 ppm due to the methyl protons of the 3-HV side group indicated the presence of 3-HB and 3-HV units in the copolymer. The chemical shift values at 1.25 and 2.2 ppm, due to the resonance absorption peaks of the methyl protons and methylene protons, confirmed the synthesis of the P(3-HB) homopolymer. From the FTIR spectra, a strong C@O stretching frequency in the range of 1745–1727 cm21, together with strong CAO stretching bands near 1200 cm21 and a strong band near 3400 cm21, confirmed the synthesis of P(3-HB-co-3-HV) and P(3-HB). Thus, waste cooking oil as a substrate provided an alternate route for the formation of P(3-HB-co-3- HV) and P(3-HB) by Alkaliphilus and E. coli strains, respectively
Magnetic Resonance in Chemistry, 2009
NMR spectroscopy was applied for quantitative and qualitative characterization of the chemical composition and microstructure of a series of poly(3-hydroxybutyrate-co-3-hydoxyvalerate) copolymers, P(3HB-co-3HV), synthesizedbymixedmicrobial cultures at several different feeding strategies. The monomer sequence distribution of the bacterially synthesized P(3HB-co-3HV) was defined by analysis of their high-resolution 1D 13 C NMR and 2D 1 H/ 13 C HSQC and 1 H/ 13 C HMBC NMR spectra. The results were verified by employment of statistical methods and suggest a block copolymer microstructure of the P(3HB-co-3HV) copolymers studied. Definitive distinction between block copolymers or a mixture of random copolymers could not be achieved. NMR spectral analysis indicates that the chemical composition and microstructure of the copolymers can be tuned by choosing a correct feeding strategy.
Journal of Separation Science, 2008
A simple HPLC-MS method for the quantitative determination of the composition of bacterial medium chain-length polyhydroxyalkanoates Bacterial poly(hydroxyalkanoates) (PHAs) vary in the composition of their monomeric units. Besides saturated side-chains, unsaturated ones can also be found. The latter leads to unwanted by-products (THF ester, secondary alcohols) during acidic cleavage of the polymer backbone in the conventional analytical assays. To prevent these problems, we developed a new method for the reductive depolymerization of medium chain-length PHAs, leading to monomeric diols that can be separated and quantified by HPLC/MS. Reduction is performed at room temperature with lithium aluminum hydride within 5 -15 min. The new method is faster and simpler than the previous ones and is quantitative. The results are consistent with the ones obtained by quantitative 1 H NMR.
Applied microbiology and biotechnology, 2016
Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.
Indian Journal of Microbiology, 2009
Rhizobium meliloti produced a copolymer of short chain length polyhydroxyalkanoate (scl-PHA) on sucrose and rice bran oil as carbon substrates. Recombinant Escherichia coli (JC7623ABC1J4), bearing PHA synthesis genes, was used to synthesize short chain length-co-medium chain length PHA (scl-co-mcl-PHA) on glucose and decanoic acid. Fourier transform infrared spectroscopy (FTIR) spectra of the PHAs indicated strong characteristic bands at 1282, 1723, and 2934 cm−1 for scl-PHA and at 2933 and 2976 cm−1 for scl-co-mcl-PHA polymer. Differentiation of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate-P(HB-co-HV) copolymer was obseverd using FTIR, with absorption bands at 1723 and 1281 for PHB, and at 1738, 1134, 1215 cm−1 for HV-copolymer. The copolymers were analyzed by GC and 1H NMR spectroscopy. Films of polymer blends of PHA produced by R. meliloti and recombinant E. coli were prepared using glycerol, polyethylene glycol, polyvinyl acetate, individually (1:1 ratio), to modify the mechanical properties of the films and these films were evaluated by FTIR and scanning electron microscopy.