4-hydroxyvaleric Acid Research Articles

Biotechnological Production and Characterization of Polyesters Containing 4-Hydroxyvaleric Acid and Medium-Chain-length Hydroxyalkanoic Acids

A two-stage aerobic fed-batch process for the biotechnological production of polyesters containing 4-hydroxyvaleric acid (4HV) and medium-chain-length hydroxyalkanoic acids by a recombinant strain of Pseudomonas putida was developed in mineral salts medium with octanoic and levulinic acid as carbon sources at a 15-L scale. The cells were first grown to high densities on octanoic acid at a pH between 7 and 8 and at a temperature of 30 °C. Accumulation conditions were induced in the second stage by nitrogen starvation at pH 7.0 and at 35 or 37 °C while levulinic acid was continuously supplied. At the end of the accumulation phase, 4HV-containing polyesters, contributing up to 50% (w/w) of the cellular dry weight, were cast into films after extraction with chloroform and precipitation with ethanol, and were spun to fibers. The unprocessed as well as the processed polyesters were characterized with respect to their molecular weight and their thermal, rheological, and mechanical properties. These polyesters sh...

Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli

The aim of this study was the production of the homopolyester poly(4-hydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichia coli. Wild-type strains and other widely used non-recombinant strains of E. coli are not able to produce polyhydroxyalkanoic acids (PHA) as storage compounds and cannot utilize 4-hydroxybutyric acid as sole carbon source. Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gene ( phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hydroxybutyric acid-coenzyme A transferase. A 3.5-kb genomic SmaI/ ApaI fragment from A. eutrophus, which comprises phaC, and a 1.8-kb genomic ApaI/ EcoRI fragment from C. kluyveri, which contained orfZ, were inserted into the SmaI and EcoRI sites of the vectors pKS − and pSK −, respectively. The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were transformed into E. coli XL1-Blue. Recombinant strains synthesized the homopolyester poly(4HB), when the cells were cultivated in Luria-Bertani broth and if glucose and 4-hydroxybutyric acid were provided as carbon sources. If glucose was omitted, a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid was accumulated. The homopolyester poly(4HB) was also accumulated during cultivation of these strains in M9 mineral salts medium containing glucose plus 4-hydroxybutyric acid as carbon sources. Poly(4HB) could amount up to approximately 80% (w/w) of the cell dry matter if E. coli XL1-Blue harboring pKSSE5.3 was cultivated in M9 mineral salts medium and if the cultures were not sufficiently supplied with oxygen. 4HB was also incorporated into PHA if γ-butyrolactone was used as carbon source. If levulinic acid, 4-hydroxyvaleric acid or γ-valerolactone were used as carbon sources, only very low amounts of PHA were accumulated which did not contain 4-hydroxyalkanoic acids as constituents.

Interaction between poly-3-hydroxybutyrate-co-3-hydroxyvalerate and a denitrifying Pseudomonas strain.

In a laboratory-scale system, dentrification activity of a heterotrophic microbial starter culture changed when different lots of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) were used as the solid carbon source in the heterotrophic denitrification reactor. In this study, possible influences of physical and chemical properties of commercially produced P(HB-co-HV) (Biopol) on biofilm formation and metabolic activity of a denitrifying starter culture were investigated. These parameters indicate the polymers' suitability for the application as the matrix substance in the bioreactor. No differences in microstructure were detected between the different lots of polymers. Growth inhibitory effects by chemical additives were found in the case of triacetine, which was included as a plasticizer in seven of eight tested lots. The amount of hydroxyvaleric acid in the polymer was not assumed to affect denitrification activity. Relevant differences could be detected regarding primary adhesion of the starter culture Pseudomonas sp. strain 2nIII. It showed good adsorption properties to hydrophobic substances with a dependence on precultivation conditions. Pseudomonas sp. strain 2nIII degraded poly-3-hydroxybutyrate acid homopolymer and P(HB-co-HV) copolymers but was unable to break up poly-3-hydroxyvaleric acid. A possible reason for these findings is the substrate specifity of the polyhydroxyalkanoate depolymerase.

Structural identification of polyhydroxyalkanoic acid (PHA) containing 4-hydroxyalkanoic acids by gas chromatography-mass spectrometry (GC-MS) and its application to bacteria screening

The methanolysis products of polyhydroxyalkanoic acids (PHAs) containing 4-hydroxybutyric acid (4HB), 4-hydroxyvaleric acid (4HV), and 4-hydroxyhexanoic acid (4HHx), when analyzed by GC-MS, showed two major chromatographic peaks with characteristic retention times of each methyl ester of 4-hydroxyalkanoic acid and the corresponding g-lactone (γ-butyrolactone, γ-valerolactone, γ-caprolactone, respectively). The method and results of GC-MS could be incorporated into an efficient screening procedure for isolation of bacterial strains which could accumulate a PHA containing 4-hydroxyalkanoic acid as the principal monomer from structurally related carbon substrates.

Identification of 5-hydroxyhexanoic acid, 4-hydroxyheptanoic acid and 4-hydroxyoctanoic acid as new constituents of bacterial polyhydroxyalkanoic acids

A recombinant strain of Pseudomonas putida GPp104 (pHP1014::E146), which expressed the polyhydroxyalkanoic acid (PHA) synthase of Thiocapsa pfennigii exhibiting an unusual substrate specificity at a high level was incubated in two-stage batch or fed-batch accumulation experiments with 5-hydroxyhexanoic acid (5HHx) as carbon source in the second cultivation phase, copolyesters of 3-hydroxybutyric acid (3HB) plus 5HHx, or of 3HB, 3-hydroxyhexanoic acid (3HHx) plus 5HHx were accumulated as revealed by gas-chromatographic and 13C-NMR spectroscopic analysis. When the recombinant P. putida GPp104 was incubated with 4-hydroxyheptanoic acid (4HHp) as carbon source in the second cultivation phase, a copolyester consisting of 3HB, 3-hydroxyvaleric acid and 3- and 4-hydroxyheptanoic acid accumulated. Providing 4-hydroxyoctanoic acid as carbon source in the second cultivation phase led to the accumulation of a polyester that contained 1–2 mol% 4-hydroxyoctanoic acid besides 3-hydroxyoctanoic acid, 3HHx, 3-hydroxyvaleric acid and 3HB. In addition to PHA containing these new constituents, PHA with 4-hydroxyvaleric acid was accumulated from laevulinic acid. Eleven strains from five genera have been also analysed for their ability to utilize different carbon sources for colony growth, which might serve as potential precursors for the biosynthesis of PHA with unusual constituents. Although most of the carbon sources were utilized by some strains for colony growth, accumulation experiments gave no evidence for the accumulation of new PHA by these wild-type strains.

Accumulation of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) by mutants and recombinant strains ofAlcaligenes eutrophus

Alcaligenes eutrophus accumulated a terpolyester of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), and 4-hydroxyvaleric acid (4HV) during cultivation with 4HV as carbon and energy source under nitrogen starvation. The polyester accumulated by wild-type strains under these conditions contained 4HV at a molar fraction of approximately 5 mol% only. A catabolic pathway of 4HV was postulated, which included the activation of 4HV to 4HV-CoA and a conversion of 4HV-CoA to 3HV-CoA. Tn5::mob-induced mutants were isolated fromA. eutrophus HF39, which were affected in 4HV and/or valeric acid catabolism. Among 83 mutants were 27 4HV-negative or 4HV-leaky mutants; two mutants were identified which accumulated a terpolyester with a molar fraction of 10.1 to 22.7 mol% 4HV. In addition, a further increase in the molar fraction of 4HV in poly(3HB-co-3HV-co-4HV) and a two- to fourfold increase in the PHA synthase activity were monitored in these mutants or others and also in HF39, if the cells were complemented with the hybrid plasmid pHP1014::PP1, which contained the PHA biosynthesis genes ofA. eutrophus H16. Application of mutagenesis plus recombinant DNA techniques resulted in the accumulation of a terpolyester with up to 30 mol% 4HV and with approximately equimolar fractions of 3HB, 3HV, and 4HV.

Identification of 4-hydroxyvaleric acid as a constituent of biosynthetic polyhydroxyalkanoic acids from bacteria

Twenty-four different strains of aerobic Gram-negative bacteria, mainly belonging to the genera Alcaligenes, Paracoccus, Pseudomonas and Methylobacterium, were examined with respect to their ability to utilize 4-hydroxyvaleric acid (4HV), 4-valerolactone (4VL) and 3-hydroxypropionic acid (3HP) as carbon sources for growth and for accumulation of polyhydroxyalkanoic acid (PHA). A gas chromatographic (GC) method for the detection of 3-hydroxyalkanoic acid methyl esters has been extended for the detection of derivatives obtained from the methanolysis of 4-hydroxybutyric acid (4HB) and 4HV. Most of the Alcaligenes species and P. oxalaticus Ox1 accumulated a terpolyester consisting of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV) and 4HV as constituents from 4HV or 4VL as sole carbon sources in batch, fed-batch or two-stage fed-batch cultures. Poly(3HB-co-3HV-co-4HV) accumulated from 4HV by A. eutrophus strain NCIB 11599 amounted to approximately 50% of the cell dry matter and was composed of 42.0 mol % 3HB, 52.2 mol % 3HV and 5.6 mol % 4HV, respectively. Pseudomonads, which belong to the rRNA homology group I, were not able to incorporate 4HV. With 3HP as carbon source, the GC analysis provided evidence for the presence of 3HP in the PHA of many bacteria. Nuclear magnetic resonance spectroscopic analysis confirmed that, for example, A. eutrophus strain TF93 accumulated poly(3HB-co-3HP) with 98 mol % 3HB and 2 mol % 3HP if the cells were cultivated in the presence of 0.5% (w/v) 3HP.

Physico-mechanical properties of degradable polymers used in medical applications: A comparative study

The physico-mechanical properties of degradable polymers used for medical applications have been characterized. The following polymers were included in this study: three samples of poly(ortho esters) derived from 3,9-bis(ethylidene 2,4,8,10-tetraoxaspiro[5,5]undecane) and various ratios of 1,6-hexanediol and trans-cyclohexane dimethanol, poly(glycolic acid), six samples of poly( l-lactic acid) and poly( d,l-lactic acid) with mol wt from 21 000 to 550 000, poly(ε-caprolactone), poly(β-hydroxybutyrate) and three copolymers of β-hydroxybutyric acid and various amounts of hydroxyvaleric acid, one sample each of two different types of poly(anhydrides), poly(trimethylene carbonate) and two different poly(iminocarbonates). For each polymer, the thermal properties (glass transition temperature, crystallization, melting and decomposition points) were determined by differential scanning calorimetry and by thermogravimetric analysis. The tensile properties (Young's modulus, tensile strength and elongation at yield and break) were determined by tensile testing on an Instron stress-strain tester. The flexural storage modulus as a function of temperature was determined by dynamic mechanical analysis.

Direct copolymerization of glycolic acid with lactones in the absence of catalysts

Glycolic acid (GA) was copolymerized with various lactones such as γ-butyrolactone (BL), δ-valerolactone (VL) and ϵ-caprolactone (CL) in the presence of water without catalysts at 200°C under N 2, giving relatively low molecular weight polyesters. This reaction is characterized by direct condensation between linear GA and linear ω-hydroxy acids produced by hydrolysis of the cyclic lactones, e.g. 4-hydroxybutyric acid (HBA) for the cyclic BL, 5-hydroxyvaleric acid (HVA) for the cyclic VL, and 6-hydroxycaproic acid (HCA) for the cyclic CL. 1H-NMR spectroscopy showed that GA reacts quantitatively with HVA and HCA, in contrast to low reactivity for HBA.

Direct copolycondensation of ϵ-caprolactone with δ-valerolactone in the absence of catalysts

Waxy and pasty copolymers of ϵ-caprolactone (CL) and δ-valerolactone (VL) with relatively low molecular weights were synthesized by direct copolycondensation without catalysts in the presence of water at 200° under nitrogen. This reaction proceeds by direct condensation between two linear monomers, i.e. linear 6-hydroxycaproic acid and linear 5-hydroxyvaleric acid produced by hydrolysis of the cyclic CL and VL, respectively. It was found by 1H-NMR that the molar ratio in the copolymer is markedly different from that in the initial monomer mixture; the CL contents in the copolymers are ca 92, 78, 71, 53 and 19 mol% for initial CL contents in the feed of 85, 70, 50, 30 and 15 mol%, respectively. The crystallinities of the copolymers were determined by differential scanning calorimetry and X-ray diffraction. Each homopolymer had relatively high melting point but it showed a marked decrease by copolycondensation, reaching a minimum for equimolar composition of copoly(CL/VL). This effect is closely related to the distortion of crystal structure because of a rapid decrease in block lengths of ϵ-oxycaproyl and δ-oxyvaleryl units in the copolymer, the so-called sequence effects.

Isolation and properties of dihydrodipicolinate synthase-defective threonine-producing mutants from a Brevibacterium flavum strain with feedback-sensitive aspartokinase.

An efficient method for the isolation of dihydrodipicolinate synthase (DPS)-defective threonine producers from a Br evibacterium strain with feedback-sensitive aspartokinase (AK, Aks) was established. After mutagenesis of a strain with AK, No. 70, mutants resistant to α-amino-β- hydroxyvaleric acid were isolated and then selected as to threonine productivity in the presence of diaminopimelic acid. DPS activity in the strains in which the threonine production was inhibited by lysine was found to be absent or reduced to less than 10 % of the level in the parent. On the other hand, the strains in which the production was not inhibited by lysine were conventional threonine producers with feedback-resistant homoserine dehydrogenases (HDs and HDRs) and wild type DPS. The HD activities of most of the threonine mutants were also markedly reduced. However, only one mutant lacking DPS, DK330, exhibited an HD level comparable to that in the parent and produced the largest amount of threonine among the threonine prod...

Direct copolymerization of l-lactic acid with δ-valerolactone in the absence of catalysts

The copolymerization of l-lactic acid (LA) and δ-valerolactone (VL) was performed in the presence of water without catalysts at 200° under nitrogen. The reaction proceeds by direct condensation between linear LA and linear 5-hydroxyvaleric acid formed by hydrolysis of the cyclic VL. The copolymers were characterized with respect to composition by means of 1H-NMR spectra, their sequences by means of 13C-NMR spectra, and their crystallinities by means of DSC and X-ray measurements. The LA/VL molar ratios in the copolymers agree well with the average lengths of the homogeneous lactyl/δ-oxyvaleryl blocks. From the result of 13C-NMR spectroscopy, it was found that sequential copolymers with suitable block lengths of lactyl and δ-oxyvaleryl units can be obtained by controlling the molar ratio. The DSC measurements show a close relationship between the nature of sequences and the crystallinity.

Reduction of the beta-Cys-gamma-Glu thiol ester bond of human C3 with sodium borohydride.

Further chemical evidence has been obtained using NaB3H4 to support our previous assignment of a thiol ester bond in human C3 (Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., and Prahl, J. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5764-5768). Following trypsin activation of human C3 in the presence of NaB3H4, 3H was shown to have incorporated specifically into the alpha'-chain of C3b. Subsequent fragmentation of [3H]C3b with porcine elastase further localized the label to the C3d subdomain. Under identical conditions, native C3 or C3 pretreated with trypsin (C3b) showed low reactivity with NaB3H4. A tryptic peptide containing the 3H label was isolated following digestion of [3H]C3b on activated thiol-Sepharose. After hydrolysis and saponification of the peptide hydrolysate, amino acid analysis indicated that the 3H had been incorporated into alpha-amino-delta-hydroxyvaleric acid, the product expected from reduction of an ester bond involving a glutamyl residue. On sequence analysis of the labeled peptide, the 3H was shown to reside at the position of the glutamyl residue previously proposed to be involved in the thiol ester bond. The residue at this position was confirmed as alpha-amino-delta-[3H] hydroxyvaleric acid by high performance liquid chromatography analysis and, after back hydrolysis, by amino acid analysis. These data significantly strengthen earlier studies which indicated the presence of a beta-Cys-gamma-Glu thiol ester bond in human C3.

Isolation and characterization of hydroxyvaleric acid derivatives from tobacco leaves and the hydrolyzate of tobacco extract.

Isolation and characterization of hydroxyvaleric acid derivatives from tobacco leaves and the hydrolyzate of tobacco extract.

Hydroxy-group participation in ester hydrolysis

Aryl esters of 4-hydroxybutyric acid, 5-hydroxyvaleric acid, 2-hydroxyphenylacetic acid, and 3-(2-hydroxyphenyl)propionic acid are lactonised in reactions whose rates are proportional to 10pH – pKW. The anchimeric assistance corresponds to rate enhancements of 103–106 over the rates of hydrolysis of analogous esters without the hydroxy-groups. The reaction of phenyl 4-hydroxybutyrate shows buffer catalysis, possibly general base catalysis, in acetate and phosphate buffers. The esters of 4-hydroxybutyric acid and 5-hydroxyvaleric acid also react with participation by the hydroxy-groups in the acid-catalysed hydrolysis but those of 2-hydroxyphenylacetic acid and 3-(2-hydroxyphenyl)propionic acid do not. The rate of hydrolysis of phenyl 4-hydroxybutyrate increases more rapidly with acid concentration than that of phenyl butyrate does. The ϕ values are 0·59 and 0·99 respectively. The hydrolyses of the lactones of 2-hydroxyphenylacetic acid and 3-(2-hydroxyphenyl)propionic acid were also studied.

A comparison of neighbouring group participation by phenolic and alcoholic hydroxy-groups in ester hydrolysis

Aryl esters of 2-hydroxyphenylacetic acid, 3-(2-hydroxyphenyl)propionic acid, 4-hydroxybutyric acid, and 5-hydroxyvaleric acid react with neighbouring group participation in aqueous solution.

310. Effects of γ-radiation. Part VII. Irradiation of 2,3-dihydro-4H-pyran and related compounds

Irradiation of aqueous solutions of 2,3-dihydro-4H-pyran in the presence of oxygen and in vacuo gave mainly tetrahydropyran-2,3-diol and tetrahydropyran-2- ol if barium carbonate was added to retain neutrality. The production of delta - hydroxyvaleric acid is explained by scission of the ether linkage, and supporting evidence is produced by studying the radiation products from tetrahydropyran. (auth)