Microsomal metabolism of N-nitrosodi-n-propylamine: formation of products resulting from α-and β-oxidation

We have identified propionaldehyde, n-propanol, isopropanol and N-nitroso-2-hydroxy-propylpropylamine following incubation of N-nitrosodi-n-propylamine with a microsomal fraction from rat liver. Based on the yields of the various products, we have shown that β-oxidation occurs at about 15% of the level of α-oxidation. β-as well as α-oxidation was shown to be carried out by the microsomal mixed function oxidase system. N-nitroso-2-hydroxy-propylpropylamine is further oxidized by the microsomal preparation to yield N-nitroso-2-oxopropylpropylamine.     

Effect of organic loading rate on VFA production, organic matter removal and microbial activity of a two-stage thermophilic anaerobic membrane bioreactor

This study focused on the VFA (volatile fatty acid) profile variation with organic loading rate (OLR) of a two stage thermophilic anaerobic membrane bioreactor (TAnMBR). The two stage TAnMBR treating high strength molasses-based synthetic wastewater was operated under a side-stream partial sedimentation mode at 55 °C. Reactor performances were studied at different OLR ranging from 5 to 12 kg COD m⁻³ d⁻¹. Operational performance of TAnMBR was monitored by assessing biological activity, organic removal efficiency, and VFA. The major intermediate products of anaerobic digestion were identified as acetate, propionate, iso-butyrate, n-butyrate and valerate. Among them acetate and n-butyrate were identified as the most abundant components. Increase of OLR changes the predominant VFA type from acetic acid to n-butyric acid and the total VFA concentration was increased with increased OLR. Moreover, increased OLR increased organic removal efficiency up to second loading rate and dropped in third loading rate while biological activity was increased continuously.     

Kinetic modelling of isomerization and anaerobic degradation of n- and i-butyrate

Very clear experimental evidence of isomerization between n- and i-butyrate during their anaerobic degradation was presented. A first experiment in the presence of bromoethane sulfonic acid (BESA), an inhibitor of methanogenesis, allowed the equilibrium distribution of n-, i-butyrate and acetate to be determined. To elucidate the mechanism of the isomerization process, a kinetic analysis was employed. The results suggested that i-butyrate was isomerized into n-butyrate prior to being oxidized to acetate. A mechanism for butyrate degradation, based on the values of the kinetic parameters obtained, was proposed.     

Ricinoleic Acid Catabolism in Peroxisomes

Peroxisomes from castor bean endosperm and mung bean hypocotyl completely degrade ricinoleic acid (12-D-hydroxy-9-cis-octadecenoic acid) to acetyl-CoA. Concomitant NADH formation occurred with a stoichiometry of 9 nmol NADH formed per 1 nmol ricinoleate degraded. At the C₈-intermediate level, where the hydroxy group of ricinoleic acid forms a barrier to β-oxidation, 2-hydroxyoctanoate and 2-oxooctanoate were detected as intermediates. 2-Hydroxyoctanoate was oxidized to 2-oxooctanoate with H₂O₂ producing a reaction exhibiting 1:1 stoichiometry of the products. The peroxisomes appeared to oxidize both isomers of racemic 2-hydroxyoctanoate. 2-Oxooctanoate was metabolized to heptanoyl-CoA (propionyl-CoA and acetyl-CoA) in a NAD-dependent, but ATP-independent, reaction. Heptanoate was not detected as an intermediate. Imidazole, an inhibitor of α-oxidation, did not effect the degradation of ricinoleate or 2-oxooctanoate. Arsenite, an inhibitor of oxidative decarboxylation, inhibited the metabolism of ricinoleate at the C₈-intermediate level, according to the accumulation of 2-oxooctanoate and the stoichiometry of concomitant NADH formation. Arsenite completely inhibited the metabolism of 2-oxooctanoate. It is concluded that the barrier caused by the hydroxy group of ricinoleic acid and prevention of β-oxidation at the C₈-intermediate level, is circumvented by an α-hydroxy acid oxidase reaction followed by an oxidative decarboxylation allowing return to the β-oxidation track.     

Peroxisomal Localization of Enzymes Related to Fatty Acid β-Oxidation in an n-Alkane-grown Yeast,Candida tropicalis

In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β—oxidation—acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase—were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.

The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.     

Metabolic engineering of Escherichia coli for the production of benzoic acid from glucose

Benzoic acid (BA) is an important platform aromatic compound in chemical industry and is widely used as food preservatives in its salt forms. Yet, current manufacture of BA is dependent on petrochemical processes under harsh conditions. Here we report the de novo production of BA from glucose using metabolically engineered Escherichia coli strains harboring a plant-like β-oxidation pathway or a newly designed synthetic pathway. First, three different natural BA biosynthetic pathways originated from plants and one synthetically designed pathway were systemically assessed for BA production from glucose by in silico flux response analyses. The selected plant-like β-oxidation pathway and the synthetic pathway were separately established in E. coli by expressing the genes encoding the necessary enzymes and screened heterologous enzymes under optimal plasmid configurations. BA production was further optimized by applying several metabolic engineering strategies to the engineered E. coli strains harboring each metabolic pathway, which included enhancement of the precursor availability, removal of competitive reactions, transporter engineering, and reduction of byproduct formation. Lastly, fed-batch fermentations of the final engineered strain harboring the β-oxidation pathway and the strain harboring the synthetic pathway were conducted, which resulted in the production of 2.37 ± 0.02 g/L and 181.0 ± 5.8 mg/L of BA from glucose, respectively; the former being the highest titer reported by microbial fermentation. The metabolic engineering strategies developed here will be useful for the production of related aromatics of high industrial interest.     

Lipid catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

All of the enzymes of the β-oxidation sequence have been demonstrated in the plerocercoids ofS. solidus. However, the plerocercoids could not oxidize exogenous [¹⁴C-U-]palmitate although labelled palmitate was readily taken up and encorporated into the neutral and phospholipid fractions. This suggests that despite the presence of all of the enzymes of β-oxidation, the pathway is not functional in S. solidus plerocercoids; possible roles of the β-oxidation enzymes in the metabolism of S. solidus are discussed.     

Immobilization of Pseudomonas cepacia lipase onto electrospun polyacrylonitrile fibers through physical adsorption and application to transesterification in nonaqueous solvent

The lipase of Pseudomonas cepacia was immobilized onto electrospun polyacrylonitrile (PAN) fibers and used for the conversion of (S)-glycidol with vinyl n-butyrate to glycidyl n-butyrate in isooctane. The rate of reaction with the adsorbed lipase was 23-fold higher than the initial material. After 10 recyclings, the initial reaction rate was 80% of the original rate. This system of enzyme immobilization is therefore suitable for carrying out transesterification reactions in nonaqueous solvents.     

Effect of n-butyrate on cell cycle progression and in situ chromatin structure of L1210 cells

In the presence of 1–5 mM n-butyrate, murine leukemic L1210 cells cease proliferation and become arrested in the G1A compartment of the G1 phase. Cells in this compartment, in comparison with the remaining cells of the G1 phase (G1B), are characterized by low RNA content and more condensed chromatin. During unperturbed growth the cell residence times in G1A are of indeterminate duration (exponentially distributed); the half-time of L1210 cell residence in G1A is about 1.4 h. The effect of n-butyrate in arresting cells in G1A was concentration-dependent. However, the sensitivity of L1210 cells to this drug was markedly enhanced when cells were treated for longer than one generation (12 h). Cells arrested in G1A remained viable and when n-butyrate was removed, after a lag period, they resumed progression through the cycle.The effect of n-butyrate on cell progression through various parts of the cycle was studied in a stathmokinetic experiment. The rate of cell entrance into mitosis was decreased by 30, 60 and 110%, in the presence of 1, 2.5 and 5 mM n-butyrate respectively, thus indicating a slowdown in cell progression through G2 and S. The duration of G2 was prolonged by 20, 70 and 140% at 1, 2.5 and 5 mM n-butyrate respectively. The half-time of cell residence in G1A was increased by as much as 1.5-, 6.3- and 15.6-fold by 1, 2.5 and 5 mM n-butyrate. Progression through late G1 (G1B) was not affected at 1 mM, and could not be estimated at higher drug concentrations. The effects on cell cycle progression were evident 1 h after addition of n-butyrate.DNA in situ in nuclei of n-butyrate-treated cells had lowered (by 2–8 °C) stability to thermal denaturation and increased (by 15%) accessibility to DNase I. The decrease in DNA stability to heat was more pronounced when permealized cells were heated in the presence of 1 mM MgCl₂ rather than EDTA. DNA in situ in the nuclei of n-butyrate-treated cells also showed decreased sensitivity to acid-induced denaturation. Changes in chromatin were seen in all cells, regardless of cell cycle phase, within the first hours after addition of n-butyrate. Mitotic cells, however, reacted to n-butyrate more rapidly than interphase cells. The observed changes in L1210 cells are most likely a consequence of histone modifications (acetylation of inner histones, dephosphorylation of histone H1) induced by n-butyrate.     

Studies on the Decarboxylation of Amino Acids with Glyoxal

Decarboxylation of about twenty kinds of α, β and γ-amino acids in the reaction with glyoxal or ninhydrin was investigated. The decarboxylation rate of amino acids proved that steric and polar effects had important roles in the reaction.

From the data of pK₂ values and decarboxylation rates of amino acids, it can be concluded that under a similar steric environment, the decarboxylation rate depends on the anion concentration of amino acids.

Besides carbon dioxide, acetaldehyde, 2-propanone and propionaldehyde were respectively detected from the reaction of β-alanine, β and γ-amino-n-butyric acids with glyoxal or ninhydrin. The decarboxylation mechanism of these amino acids seemed to take place through the corresponding β- or γ-keto acid.

Oxygen absorption was also observed from the reaction of amino acids with dicarbonyl compounds.     

Regulation of palmitoylcarnitine oxidation in isolated rat liver mitochondria. Role of the redox state of NAD(H)

The redox-mediated regulation of palmitoylcarnitine oxidation was studied in isolated rat liver mitochondria in which the mitochondrial free NADH/NAD⁺ ratio was controlled by graded concentrations of acetoacetate and ketomalonate in a rotenone and malonate-inhibited system in the presence of ADP. The NADH/ NAD⁺ ratio was buffered kinetically by adjusting the concentrations of the hydrogen acceptor substances and determined by calibrated NAD(P)H fluorometry of the mitochondrial suspension. A two-fold variation in the β-oxidation rate and a five-fold variation in the free NADH/NAD⁺ ratio was obtained in the presence of rotenone. A non-linear negative correlation was found between the acetyl-CoA concentration and the β-oxidation rate and a negative correlation between the long-chain acyl-CoA concentration and the β-oxidation rate. The data indicate that the redox state is a partial controller of the β-oxidation rate in liver mitochondria. The contribution of acetyl-CoA, a putative regulator of β-oxidation at the acyl-CoA thiolase step is small under the conditions used.     

The large subunit of the pig heart mitochondrial membrane-bound β-oxidation complex is a long-chain enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme

The subunit locations of the component enzymes of the pig heart trifunctional mitochondrial β-oxidation complex are suggested by analyzing the primary structure of the large subunit of this membrane-bound multienzyme complex [Yang S.-Y.et al. (1994) Biochem. biophys. Res. Commun. 198, 431–437] with those of the subunits of the E. coli fatty acid oxidation complex and the corresponding mitochondrial matrix β-oxidation enzymes. Long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase are located in the amino-terminal and the central regions of the 79 kDa polypeptide, respectively, whereas the long-chain 3-ketoacyl-CoA thiolase is associated with the 46 kDa subunit of this complex. The pig heart mitochondrial bifunctional β-oxidation enzyme is more homologous to the large subunit of the prokaryotic fatty acid oxidation complex than to the peroxisomal trifunctional β-oxidation enzyme. The evolutionary trees of 3-hydroxyacyl-CoA dehydrogenases and enoyl-CoA hydratases suggest that the mitochondrial inner membrane-bound bifunctional β-oxidation enzyme and the corresponding matrix monofunctional β-oxidation enzymes are more remotely related to each other than to their corresponding prokaryotic enzymes, and that the genes of E. coli multifunctional fatty acid oxidation protein and pig heart mitochondrial bifunctional β-oxidation enzyme diverged after the appearance of eukaryotic cells.     

Fat metabolism in higher plants. LXII. The pathway of ricinoleic acid catabolism in the germinating castor bean (Ricinus communis L.) and pea (Pisum sativum L.)

With cell-free extracts from both germinating peas and castor beans, O-¹⁴Cricinoleate (¹⁴C located at odd-numbered positions in the carbon chain) was β-oxidized at least to the C₁₀ level. With the pea system, formation of unsaturated hydroxy acid intermediates to the C₁₂ level occurred. Acetyl-CoA was the primary product of β-oxidation activity. Although the pathway beyond the C₁₂-intermediate level was not resolved conclusively, two alternative routes may exist in the castor bean system to convert 4-hydroxy-decanoic acid to 2-keto-octanoate, one involving 4-keto-decanoate, the other 2-hydroxy-octanoate. Subsequent degradation of the 2-keto-octanoate tentatively involves an α-oxidation step, releasing CO₂ and heptanoic acid. Further β-oxidation of the latter is envisaged to yield propionyl and acetyl CoA. All necessary enzymes for the catabolism of ricinoleic acid to propionate appear to be associated with the cytosomes.     

Schistosoma mansoni does not and cannot oxidise fatty acids,but these are used for biosynthetic purposes instead

Adult schistosomes, parasitic flatworms that cause the tropical disease schistosomiasis, have always been considered to be homolactic fermenters and, in their energy metabolism, strictly dependent on carbohydrates. However, more recent studies suggested that fatty acid β-oxidation is essential for egg production by adult female Schistosoma mansoni. To address this conundrum, we performed a comprehensive study on the lipid metabolism of S. mansoni. Incubations with [¹⁴C]-labelled fatty acids demonstrated that adults, eggs and miracidia of S. mansoni did not oxidise fatty acids, as no ¹⁴CO₂ production could be detected. We then re-examined the S. mansoni genome using the genes known to be involved in fatty acid oxidation in six eukaryotic model reference species. This showed that the earlier automatically annotated genes for fatty acid oxidation were in fact incorrectly annotated. In a further analysis we could not detect any genes encoding β-oxidation enzymes, which demonstrates that S. mansoni cannot use this pathway in any of its lifecycle stages. The same was true for Schistosoma japonicum and all other schistosome species that have been sequenced. Absence of β-oxidation, however, does not imply that fatty acids from the host are not metabolised by schistosomes. Adult schistosomes can use and modify fatty acids from their host for biosynthetic purposes and incorporate those in phospholipids and neutral lipids. Female worms deposit large amounts of these lipids in the eggs they produce, which explains why interference with the lipid metabolism in females will disturb egg formation, even though fatty acid β-oxidation does not occur in schistosomes. Our analyses of S. mansoni further revealed that during the development and maturation of the miracidium inside the egg, changes in lipid composition occur which indicate that fatty acids deposited in the egg by the female worm are used for phospholipid biosynthesis required for membrane formation in the developing miracidium.     

Fat metabolism in higher plants. Recent studies on plant alpha-oxidation systems

Both the seed and the leaf α-oxidation systems of higher plants require for activity a reductant and molecular oxygen. The reductant may be NADH or a coupled glucose + glucose oxidase system. d-2-Hydroxypalmitate is the only 2-hydroxy acid which is formed from palmitic acid under a variety of conditions; l-2-Hydroxypalmitate is not formed, but when added does inhibit the initial attack of the α-oxidative enzyme (s) on palmitate. Since the addition of glutathione + gluthathione peroxidase greatly increases the formation of d-2-hydroxypalmitate from palmitate, with a concomitant decrease in CO₂, it is strongly suggested that the intermediate in α-oxidation is d-2-hydroperoxypalmitate, which may then undergo decarboxylation to a pentadecanal and CO₂ or be reduced to d-2-hydroxypalmitate. Evidence in support of this hypothesis is presented.     

Interactive effects of protein and carbohydrates on production of microbial metabolites in the large intestine of growing pigs

The study aimed at determining the effect of protein type and indigestible carbohydrates on the concentration of microbial metabolites in the large intestine of pigs. The experiment involved 36 pigs (15 kg initial body weight) divided into six groups, fed cereal-based diets with highly digestible casein (CAS) or potato protein concentrate (PPC) of lower ileal digestibility. Each diet was supplemented with cellulose, raw potato starch or pectin. After 2 weeks of feeding, pigs were sacrificed and samples of caecal and ascending, transverse and descending colon digesta were collected for analyses of microbial metabolites. PPC increased the concentration of ammonia, p-cresol, indole, n-butyrate, isovalerate and most of the amines in comparison with CAS. Pectin reduced the production of p-cresol, indole, phenylethylamine and isovalerate in the large intestine compared with potato starch. Starch and pectin increased mainly the concentration of n-butyrate and n-valerate in the colon compared to cellulose. Interaction affected mainly amines. Feeding PPC diet with potato starch considerably increased putrescine, cadaverine, tyramine and total amines concentrations compared with PPC diets with pectin and cellulose, whereas feeding CAS diet with starch reduced their concentrations. There was also a significant effect of interaction between diet and intestinal segment on microbial metabolites. In conclusion, PPC intensifies proteolysis in the large intestine and also n-butyrate production. Raw starch and pectin similarly increase n-butyrate concentration but pectin inhibits proteolysis more efficiently than starch. The interactive effects of both factors indicate that pectin and cellulose may beneficially affect fermentative processes in case of greater protein flow to the large intestine.     

Volatile Cucumis melo components: Identification of additional compounds and effects of storage conditions

Additional volatile compounds were isolated from muskmelon fruit by means of a water recycling apparatus, separated by GLC, and identified principally by MS and GLC retention data. Compounds reported for the first time as melon components are: n-hexanol, 1-octen-3-ol, cis-3-nonen-1-ol, n-butyl acetate, isobutyl acetate, 2-methylbutyl acetate, n-hexyl acetate, ethyl n-butyrate, ethyl 2-methylbutyrate, benzyl acetate, β-phenethyl acetate, and γ-phenylpropyl acetate. Muskmelon fruit stored frozen prior to steam distillation-extraction yielded an essence which, when compared with that obtained from freshly harvested fruit, contained considerably larger amounts of trans-2-nonenal, n-nonanol, cis-3-nonen-1-ol, cis-6-nonen-1-ol, and the methyl and ethyl esters of linoleic and linolenic acids. Marked decreases in the relative amounts of benzyl acetate, β-phenethyl acetate, and γ-phenylpropyl acetate resulted from freezing. All 21 compounds examined were present in the essences prepared from fresh, refrigerated, and frozen fruit.     

Isomerization ofn- andi-butyrate in anaerobic methanogenic systems

Studies of the degradation of the two isomeric forms of butyrate in different anaerobic environments showed isomerization betweenn- andi-butyrate. Degradation rates were similar for the different examined systems and degradation rates forn-butyrate degradation were generally higher than fori-butyrate. Degradation rates forn-butyrate ranged from 0.52 to 1.39 day^–1, while the rates fori-butyrate were from 0.46 to 1.15 day^–1. Production of isomers was not observed when the volatile fatty acid degradation was inhibited by addition of bromoethane sulfonic acid, indicating that isomerization was coupled to the methanogenic degradation of the acid. The degree of isomerization observed duringn-butyrate degradation was similar to the degree duringi-butyrate degradation. Experiments indicated that the isomerization degree was higher for the thermophilic than for the mesophilic inocula.     

Anaerobic fermentation for n-caproic acid production: A review

Anaerobic fermentation-based technologies are used for treating organic residues, and producing high value-added products, such as solvents, gases, and organic acids. Among several organic acids, n-caproic acid can be used as antimicrobial agent, additive in animal feed, flavor additive, and feedstock for chemical and biofuel industries. n-Caproic acid formation occurs through a carboxylic acid chain elongation process, which uses reverse β-oxidation of acetic and/or n-butyric acid, and ethanol or lactic acid as an electron donor. This review addresses important issues in commercial n-caproic acid production: metabolic pathways, kinetics and thermodynamics, substrates, reactors, inhibition of competing biological activities, pH, and acid extraction. Additionally, a mathematical model to describe the reverse β-oxidation kinetics was evaluated from existing literature. Current investigations show a wide range of n-caproic acid production rates (3.0–55.8 g/(L·d)), using different open cultures, fermentation conditions, and methods for inhibiting the methanogenesis. Clostridium kluyveri presence and a dominance of the Clostridium spp. were identified as determinant when ethanol was provided as electron donor. Continuous n-caproic acid extraction through pertraction is a promising technology, which combines selective extraction and enhanced production rates. However, confirming the industrial feasibility of this process requires further investigation.