Article

A preparation of D(-)-β-hydroxybutyric acid

Authors:
To read the full-text of this research, you can request a copy directly from the author.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the author.

... Methods are known for depolymerizing the bacterial lipid into D(-)-f3-hydroxybutyric acid, either enzymatically (10) or chemically (7). If these methods could be adapted for low-cost production, lipid-rich H. eutropha could be a useful feed supplement as a source of both protein and energy. ...
Article
Hydrogenomonas eutropha is known to accumulate lipid, comprised largely of polymerized β-hydroxybutyric acid, when maintained in nitrogen-deficient medium. This lipid was very poorly absorbed by mice from bacteria-containing diets, even though nitrogen absorption was adequate. The monomer, free β-hydroxybutyric acid, was well absorbed from purified diet. Rats fed the monomer or butyric acid ate less food and grew more slowly than rats fed corn oil.
... The combined extracts were washed successively with 1% aqueous Na 2 S 2 O 3 (100 mL) and water (100 mL), dried over anhydrous Na 2 SO 4 , and evaporated on a rotary evaporator to afford a residue, which was purified by column chromatography through a short silica gel column to produce 7l, 7m or 17 after trituration with EtOAc/n-hexane. 43,144.45,132.46,131.24,131.14,129.74,128.11,127.74,127.21,124.74,123.80,122.11,44.41,32.72,22.93, 29,153.65,144.38,132.46,131.30,131.17,129.72,128.10,127.74,127.23,124.99,123.82,122.18,44.44,33.28,25.96,24.01,methyl)-4H-1,2,4-triazole ...
Article
Full-text available
In order to systematically explore and understand the structure-activity relationship (SAR) of a lesinurad-based hit (1c) derived from the replacement of the S atom in lesinurad with CH2, 18 compounds (1a-1r) were designed, synthesized and subjected to in vitro URAT1 inhibitory assay. The SAR exploration led to the discovery of a highly potent flexible URAT1 inhibitor, 1q, which was 31-fold more potent than parent lesinurad (IC50 = 0.23 μM against human URAT1 for 1q vs. 7.18 μM for lesinurad). The present study discovered a flexible molecular scaffold, as represented by 1q, which might serve as a promising prototype scaffold for further development of potent URAT1 inhibitors, and also demonstrated that the S atom in lesinurad was not indispensable for its URAT1 inhibitory activity.
... However, alkaline hydrolysis of PHB to 3HB is either slow (under mild alkaline conditions) or results in crotonate formation due to dehydration ( Fig. 1) [15]. PHB is extremely resistant to acidic hydrolysis and crotonization takes place at harsh conditions [16]. Although acidic hydrolysis of PHB to 3HB was judged to be impossible at all [17], the literature is too sparse to draw such a final conclusion. ...
Article
Activated sludge communities which performed enhanced biological phosphate removal (EBPR) were phylogenetically analyzed by 16S rRNA-targeted molecular methods. Two anaerobic-aerobic sequencing batch reactors were operated with two different carbon sources (acetate vs. a complex mixture) for three years and showed anaerobic-aerobic cycles of polyhydroxybutyrate- (PHB) and phosphate-accumulation characteristic for EBPR-systems. In situ hybridization showed that the reactor fed with the acetate medium was dominated by bacteria phylogenetically related to the Rhodocyclus-group within the beta-Proteobacteria (81% of DAPI-stained cells). The reactor with the complex medium was also predominated by this phylogenetic group albeit at a lesser extent (23% of DAPI-stained cells). More detailed taxonomic information on the dominant bacteria in the acetate-reactor was obtained by constructing clone libraries of 16S rDNA fragments. Two different types of Rhodocyclus-like clones (R1 and R6) were retrieved. Type-specific in situ hybridization and direct rRNA-sequencing revealed that R6 was the type of the dominant bacteria. Staining of intracellular polyphosphate- and PHB-granules confirmed that the R6-type bacterium accumulates PHB and polyphosphate just as predicted by the metabolic models for EBPR. High similarities to 16S rDNA fragments from other EBPR-sludges suggest that R6-type organisms were present and may play an important role in EBPR in general. Although the R6-type bacterium is closely related to the genus Rhodocyclus, it did not grow phototrophically. Therefore, we propose a provisional new genus and species Candidatus Accumulibacter phosphatis.
... Methods are known for depolymerizing the bacterial lipid into D(-)-f3-hydroxybutyric acid, either enzymatically (10) or chemically (7). If these methods could be adapted for low-cost production, lipid-rich H. eutropha could be a useful feed supplement as a source of both protein and energy. ...
Article
Hydrogenomonas eutropha is known to accumulate lipid, comprised largely of polymerized beta-hydroxybutyric acid, when maintained in nitrogen-deficient medium. This lipid was very poorly absorbed by mice from bacteria-containing diets, even though nitrogen absorption was adequate. The monomer, free beta-hydroxybutyric acid, was well absorbed from purified diet. Rats fed the monomer or butyric acid ate less food and grew more slowly than rats fed corn oil.
Article
Two new detection methods for the determination of poly-beta-hydroxybutyrate (PHB) and -valerate (PHV) are described. Both methods are based on depolymerization of PHB/PHV to 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Depolymerization was achieved by either propanolic or hydrolytic digestion. Propanolic digestion transformed commercial PHB/PHV stoichiometrically into 3HB/3HV and yielded apparently complete recoveries of bacterial PHB/PHV from activated sludge. Hydrolytic digestion was suitable only for PHB determination. For quantification of 3HB and 3HV directly from digested sludge, a method based on ion-exchange chromatography and conductivity detection was developed (IC-method). Alternatively, the total of 3HB and 3HV was quantified using a commercial enzymatic test kit and colorimetric detection (enzyme method). Both detection methods are easier to perform than previous methods and are suitable for complex matrices such as activated sludge. The IC-method is recommended for high sample throughputs or if distinction between PHB and PHV is essential. Enzymatic detection is recommended if a few samples per day have to be measured immediately or if an ion chromatograph is unavailable.
Article
D(—)β-hydroxybutyric acid-14C was synthesized from 14CO2 and acetate-1-14C using Hydrogenomonas H16. The labeled substrate is first incorporated into poly-β-hydroxybutyric acid, which is easily isolated, purified, and degraded to D(—)β-hydroxybutyric acid. Using acetate-1-14C as substrate, the overall conversion of substrate to the desired product is approxmately 60%. This method affords a simple, inexpensive means of synthesizing D(—)β-hydroxybutyrie acid labeled with carbon-14 in specific positions of the molecule. It is equally adaptable to the synthesis of 14C-crotonic acid (trans isomer).
Article
Ion-exchange resins and inorganic gels have only limited application in the fractionation of DNA. Although DEAE-cellulose has been used extensively, ECTEO-LA-cellulose has promise in the fractionation of DNA. Carboxymethylcellulose or nitrocellulose have limited capabilities as regards DNA fractionation. Gel filtration techniques employing agar gels, DEAE-Sephadex and DEAE-dextran are of more value in the fractionation of s-RNA and polynucleotides than for highly polymerised DNA. Amongst the kieselguhr columns, the methylated serum albumin—kieselguhr column found wide applicability and gives reproducible profiles. An Amberlite IR-120 Al3+ column also gives interesting results with various potential applications. Alternate methods effecting finer resolution are still a pressing need.
Article
A quantitative description of the relative importance of endogenous metabolism to overall ATP production has not been established for mammalian cells. We report herein results of experiments using sperm (selected because of their simple metabolic potential and absence of biosynthetic pathways) and calorimetry (chosen because it serves as a general monitor of metabolism) to assess the importance of endogenous metabolism to total ATP synthesis under several incubation conditions. In experiments in which sperm were incubated at different temperatures (20 degrees C, 25 degrees C, 30 degrees C or 35 degrees C) and with different substrates (glucose, fructose, lactate, or beta-hydroxybutyrate), endogenous metabolism occurred at a constant rate regardless of the rate of ATP turnover in the cells or the nature of the exogenous substrate available to them. Sperm incubated at 35 degrees C with glycolyzable substrates synthesized more ATP (9 mumol ATP . h-1/10(8) cells) than did sperm incubated with the nonglycolyzable substrate, lactate (6.2 mumol ATP . h-1/10(8) cells). To investigate this substrate-related difference in the rate of ATP synthesis, the motility of sperm incubated at 35 degrees C with glucose or with lactate was determined. The velocities of the sperm incubated with either substrate were identical, indicating that the rates of ATP consumption for support of motility were identical. Most of the additional ATP synthesized by cells with glycolyzable substrates was consumed in the process of substrate cycling of the metabolic intermediates of glucose.
Article
Rates of hydroxylation of C-atoms in various positions [ω-, (ω-1)-, (ω-2)-] of an increasing chain length have been measured with the 4-chloroanilides of acetic, propionic, butyric, and valeric acid as substrates. The hydroxylation products were separated by t.l.c, quantified by u.v. spectroscopy and further characterized by n.m.r. and o.r.d. spectroscopy. The hydroxylation products in which an asymmetric centre had been introduced by oxygenation, were shown to be optically active, the sign of the optical rotation indicating an excess of the S-over the R-isomer. From the alteration of the hydroxylation pattern caused by previous treatment of rabbits with either phenobarbital or 3-methylcholanthrene it can be deduced that the ω, (ω-1), and (ω-2)-hydroxylation of the lower fatty acyl residues is not only catalysed by PB- and 3-MCh-inducible forms of cytochrome P-450 but also by the forms not inducible by either PB or 3-MCh.
Article
A method is described for preparing the physiologically active isomer d(−)-β-hydroxybutyrate in quantities of several millimoles by the reduction of acetoacetate, catalysed by β-hydroxybutyrate dehydrogenase at pH 8. Economy in the use of coenzyme is achieved by coupling the reaction to the oxidation of galactose, catalysed by d-galactose dehydrogenase. The progress of the reaction can be followed by decarboxylating the remaining acetoacetate with aniline citrate, trapping the evolved CO2 in Ba(OH)2 and back titrating with HCl. After incubation for 48 h at 35° the mixture is acidified and continuously extracted with ether for 13 h. The ether extract is evaporated to dryness and the residue is dissolved in water and neutralized to give a solution of sodium d(−)-β-hydroxybutyrate. In a typical preparation the final yield determined enzymically agreed with that determined by optical rotation and titration, and specific tests showed that the product was free of the following contaminants: acetoacetate, d(+)-galactose, phosphate and d(−)-galactono-γ-lactone.
Article
Investigations involving fractional precipitation of A. senegal gum by sodium sulphate lead to a discussion on the type of heterogeneity exhibited by the gum. Molecular-sieve chromatography is used to estimate number-average molecular weights. Results obtained using this chromatographic technique on the degraded gum produced on autohydrolysis indicate that such mild conditions of hydrolysis are not always very selective as a means of degradation. The degraded gum is shown to have galactose residues as reducing end-groups. There is no evidence for labile, internal, arabinofuranosyl linkages in the whole gum. In addition, chromatographic evidence is obtained for the presence of 6-O-(4-O-methyl-β-D-glucopyranosyluronic acid)-D-galactose residues in A. senegal gum.
Article
On the Depolymerization of Poly-(R)-3-hydroxy-butanoate (PHB) From cell-free PHB or from dried cells of Alcaligenes eutrophus H 16, which had been grown in an aqueous fructose solution, enantiomerically pure methyl, ethyl, butyl or β-methoxyethyl (R)-3-hydroxy-butanoates are obtained in yields ranging from 75–90% (0.1 to 70 g scale). The depolymerization is achieved by heating the PHB-containing materials to temperatures of 80–160° in the corresponding alcohol with or without the cosolvent dichloroethane in the presence of either sulfuric acid or tetraethoxytitanium catalyst. Since (S)-3-hydroxy-butanoates are also readily obtained (by yeast reduction of aceto-acetates), starting materials derived from 3-hydroxy-butyric acid now belong to the especially useful group of synthetic building blocks which are available in both enantiomeric forms.
Article
Gel chromatography is also commonly known as “gel filtration,” “gel-permeation,” or “molecular-sieve chromatography.” This technique is based on the decreasing permeability of the three-dimensional network of a swollen gel to molecules of increasing size. In specific, if a solution containing a mixture of solutes of different molecular sizes is passed through a column packed with a suitable gel, the smaller molecules penetrate farther into the gel pores than do the larger, and are therefore retained for a longer time on the column. The solutes are thus eluted in the order of decreasing molecular size. This chapter emphasizes on the fundamental aspects of gel chromatography technique that are most likely to arise in carbohydrate chemistry. It illustrates the applicability of gel chromatography to a wide variety of carbohydrates. This procedure is considered as a rapid and relatively simple method for separating substances that differ in molecular size, or for fractionating polymers, such as polysaccharides, having broad molecular weight distributions.
Article
In this review, the possibility of using activated sludges as animal feed is discussed. After a brief description of the activated sludge process fundamentals, the chemical composition of the sludges is given (CHON, protein, amino-acid, carbohydrates, lipids, vitamins, etc.). Feeding experiments using activated sludges from various sources indicate the suitability of the sludge as a feed supplement. A following section deals with the modifications of the conventional activated sludge process for protein production from wastewater. The factors maximizing the nutritional value of the sludges are also discussed.A few problems associated with the use of some activated sludges as a feed supplement are mentioned but these problems can be solved on the basis of p present technology.
Article
Poly-β-hydroxybutyric acid has been isolated in quantities sufficient for characterization and evaluation of mechanical properties from both Bacillus megaterium and a special strain of Rhizobium. Its presence in a larger variety of bacteria than previously suspected has been demonstrated by a screening method based on density-gradient centrifugation. The intrinsic viscosity of the isolated polymer has been shown to depend on the technique of isolation; basic solvents facilitate hydrolytic cleavage of the polyester. Mechanical properties of the polymer are based on the high crystallinity and polarity of the material; in a highly crystallized state, it is quite brittle, but its rigidity exceeds that of polypropylene. Thermal degradation of molecular weight, due to beta-eliminatin of the carboxyl group from the polyester structure, severely limits processing of the polymer in the melt.
Article
Poly(hydroxyalkanoates) (PHAs) are polyesters formed by saturated short chain hydroxyacids, among which 3-hydroxybutanoic (HB) and 3-hydroxypentanoic (3-hydroxyvalerate, HV) are the most common monomers of homopolymers (e.g. poly(3-hydroxybutyrate), PHB) and copolymers (e.g. poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHB-HC). The most widely used approach for their determination is the polymer methanolysis followed by gas chromatography-mass spectrometry (GC-MS) analysis of the methylated monomers; this procedure generally requires the use of additional reagents (e.g. sulfuric acid) and is performed with harmful chlorinated solvents, such as chloroform. The development of fast routine solventless methods for the quantitative determination of PHAs and their monomeric composition is highly desirable to reduce sample pretreatment, speed up the analysis and decrease overall costs. It has been reported that under thermal treatment (e.g. pyrolysis, Py), PHAs are degraded in high yield (>40%, w/wPHA) into the corresponding 2-alkenoic acid (e.g. crotonic acid from PHB). This work aimed at investigating this reaction for direct analysis of PHAs in bacterial cells. The sample was directly subjected to pyrolysis and trapped pyrolysis products were analyzed by GC-FID. Off-line Py/GC-FID was first optimized on pure polymers with different monomer composition (PHB, PHB-HV, PHB-HC) and then applied to bacterial samples deriving from both mixed microbial cultures or selected strains, containing various types and amounts of PHAs. The Py/GC-FID method provided RSD <15% range, limit of detection of 100μg (1% PHAs in biomass), and results comparable to that of methanolysis (R(2)=0.9855), but with minimal sample pretreatment.
Chapter
Polyhydroxyalkanoic acids (PHA) are water-insoluble polyesters of alkanoic acids containing a hydroxyl group as at least one functional group in addition to the carboxy group, and possess the general formula shown in Fig. 1. Although some of these polymers are also available from chemical synthesis, many of them are synthesized by bacteria and are deposited in abundant amounts in the cytoplasm of the cells. The variability of the position of the hydroxyl group and of the type of the R-pendant group of the constituents (see below), a large variety of different constituent monomers in copolyesters, as well as the varying degree of polymerization, allow the biosynthesis of many different polymers exhibiting different physical properties.
Article
Soybean (Glycine max) nodule bacteroids contain high concentrations of poly-beta-hydroxybutyrate and possess a depolymerase system that catalyzes the hydrolysis of the polymer. Changes in poly-beta-hydroxybutyrate content and in activities of nitrogenase, beta-hydroxybutyrate dehydrogenase, and isocitrate lyase in nodule bacteroids were investigated under conditions in which the supply of carbohydrate from the soybean plants was interrupted. The poly-beta-hydroxybutyrate content of bacteroids did not decrease appreciably until the carbohydrate supply from the host plants was limited by incubation of excised nodules, incubation of plants in the dark, or by senescence of the host plant. Isocitrate lyase activity in bacteroids was not detected until poly-beta-hydroxybutyrate utilization appeared to begin. The presence of a supply of poly-beta-hydroxybutyrate in nodule bacteroids was not sufficient for maintenance of high nitrogenase activity under conditions of limited carbohydrate supply from the host plant. An unusually high activity of beta-hydroxybutyrate dehydrogenase was observed in bacteroid extracts but no significant change in the activity of this enzyme was observed as a result of apparent utilization of poly-beta-hydroxybutyrate by nodule bacteroids.
Article
Poly-#?-hydroxybutyrate has been previously shown to be a major component of bacterial 'lipid' granules. In the present study, the conditions under which it was formed and degraded by Bacillus cereus and B. megaterium were studied in washed suspensions. Suitable substrates for synthesis were glucose, pyruvate or /3-hydroxybutyrate. Acetate, although alone unable to induce synthesis, greatly enhanced formation in presence of these substrates, Under optimal conditions, suspensions synthesized up to eight times their original content of poly-/3-hydroxy- butyrate in 4 hr. Formation was inhibited by high concentrations of oxygen, d- though no synthesis occurred anerobically in nitrogen. The optimal concentration of oxygen was about 5 yo. B. cereus only was able to synthesize poly-p-hydroxybuty- rate in an atmosphere of hydrogen. In the absence of an external carbon and energy source, degradation occurred rapidly aerobically, to carbon dioxide and water, and more slowly anaerobically to p-hydroxybutyrate and acetoacetate. The evidence that poly-#?-hydmxybutyrate is a reserve carbon and energy source is discussed. Lemoigne (1923) showed that an aerobic spore-forming bacillus, designated Bacillus ' M ' formed quantities of p-hydroxybutyric acid in anaerobic sus- pensions in the absence of an external carbon and energy source. In sub- sequent investigations (Lemoigne, 1925) he made quantitative estimations of this acid formed and concluded that it accounted for the greater part of the acidic substances produced under the conditions of his experiments. He then demonstrated (Lemoigne, 1927) that a substance having the empirical formula (C,H,O,), could be extracted from the bacilli by chloroform and he was able to show that the material was a polymer of P-hydroxybutyric acid. Subse- quently it became clear (Lemoigne, Delaporte & Croson, 1944) that there was a correlation between the amount of this polymer which could be extracted and the amount of refractile ' fatty ' cytoplasmic granular material exhibited by the bacilli. Confirmation that the polymer was a major constituent of these 'lipid' granules was obtained by Weibull (1953) in his observations on the nature of the granules isolated after dissolution of the cell wall of Bacillus megaterium by lysozyme. Williamson & Wilkinson (1958) showed that an alkaline hypochlorite solution would liberate the granules which upon recovery and analysis were shown to consist largely of poly-p-hydroxybutyrate. A rapid quantitative method for the estimation of this substance was also described. Lemoigne, Grelet & Croson (1950) drew attention to the different amounts of poly-p-hydroxybutyrate obtained by growing B, meguterium on different media, and Macrae & Wilkinson (1958) showed that more of this substance was formed as the glucose concentration of the growth medium was increased; the subsequent depletion of the product during the later stages of
Article
Both dextrorotatory and levorotatory forms of β-hydroxybutyric acid are oxidized aerobically by suspensions of rat liver and kidney particles (mitochondria). However the data clearly indicate that the initial stages of oxidation of the two isomers are quite different in liver, although ultimately both isomers are oxidized via the tricarboxylic acid cycle. The enzymic mechanisms involved in the primary dehydrogenation of the two isomers were examined in clear extracts of acetone-dried mitochondria. It was found that the l-isomer causes the reduction of DPN+, presumably by action of the already known l-specific DPN-linked β-hydroxybutyric dyhydrogenase, which of course does not attack the d-isomer. The d-isomer also causes reduction of DPN+ but only if the extracts are supplemented with ATP, Coenzyme A, and Mg++. Evidence is presented that d-BOH is capable of forming a CoA derivative at the expense of ATP: d-β-hydroxybutyrate + CoA {S-E arrow with hook} ATP d-β-hydroxybutyryl-CoA The extracts contain a dehydrogenase catalyzing reversibly the reduction of DPN+ by d-β-hydroxybutyryl-CoAd-β-hydroxybutyryl-CoA + DPN+ {S-E arrow with hook} acetoacetyl-CoA + DPNH + H+. Reaction (1) is not stereochemically specific; both isomers form a CoA derivative. However the dehydrogenase catalyzing reaction (2) appears to be specific for d-β-hydroxybutyryl-CoA. The metabolic significance of the pathways taken by the two isomers of β-hydroxybutyrate are briefly discussed.
  • A Magnus-Levy
Magnus-Levy, A. (1901). Arch. exp. Path. Pharmakol. 45, 389.
  • A Mckenzie
McKenzie, A. (1902). J. chem. Soc. 81, 1402.
  • R A Reed
Reed, R. A. (1957). Roy. Inst. Chem. Lect., Monogr., Rep. no. 5.
  • R D Hartley
  • G J Lawson
Hartley, R. D. & Lawson. G. J. (1960). J. Chromat. 4,410. Lehninger, A. L. & Greville, G. D. (1953). Biochim. biophys. Acta, 12, 188.
  • R D Hartley
  • G J Lawson
Hartley, R. D. & Lawson. G. J. (1960). J. Chromat. 4,410.