Hydration of Oleic Acid by Enterococcus gallinarum, Pediococcus acidilactici andLactobacillus sp. Isolated from the Rumen

Three ruminal bacteria found to convert oleic acid to 10-hydroxystearic acid were identified asEnterococcus gallinarum , Pediococcus acidilactici and a lactobacillus physiologically similar toLactobacillus reuteri and L. fermentum. The oleic acid-hydrating properties of the three bacteria were determined and hydration was shown to be predominantly a feature of anaerobic culture with relatively little hydration occurring in aerobic culture. Hydration was highly pH dependent and not related to cell growth. At the optimal hydration pH for each bacterium, hydration yields were 97%, 93% and 76% for the E. gallinarum, P. acidilactici andLactobacillus strains, respectively. The enterococcus and the lactobacillus hydrated oleic acid only after growth ceased whereas the pediococcus hydrated oleic acid during the late logarithmic growth. Hydration was not specific for oleic acid with all three bacteria hydrating the oleic acid homologue palmitoleic acid. None of the bacteria hydrogenated oleic acid to stearic acid. Our results suggest that a capacity to hydrate oleic acid may be a property of many lactic acid bacteria.     

Conversion of oleic acid to 10-hydroxystearic acid by whole cells of <Emphasis Type="Italic">Stenotrophomonas nitritireducens</Emphasis>

The optimal reaction conditions for the conversion of oleic acid to 10-hydroxystearic acid by whole cells of Stenotrophomonas nitritireducens were: pH 7.5, 35°C, 0.05% (w/v) Tween 80, 20 g cells l⁻¹, and 30 g oleic acid l⁻¹ in an anaerobic atmosphere. Under these conditions, the cells produced 31.5 g 10-hydroxystearic acid l⁻¹ over 4 h with a conversion yield of 100% (mol/mol) and a productivity of 7.9 g l⁻¹ h⁻¹, indicating that oleic acid was converted completely to 10-hydroxystearic acid, with no detectable byproduct. This is the highest concentration, productivity, and yield of 10-hydroxystearic acid from oleic acid reported thus far.     

Fatty acid composition and preservation of the Tyrolean Iceman and other mummies

In anthropology, objective parameters to adequately describe storage conditions and the preservation of mummies have yet to be identified. Considering that fatty acids degrade to stable products, we analysed their profile in human mummies and in control samples by gas chromatography coupled to mass spectrometry (GC/MS). Originating from different epochs and civilizations, samples of the Tyrolean Iceman, other glacier corpses, a freeze dried mummy, corpses from a permafrost region, a corpse mummified immersed in water, and a desert mummy were evaluated. Chemometric analysis based on the concentrations of 16 fatty acids revealed the degree of similarity between anthropologic and fresh corpse samples, which was mainly influenced by the content of palmitic acid, oleic acid, and 10-hydroxystearic acid. The presence of 10-hydroxystearic acid was associated with immersion in water, whereas dry mummification was accompanied by high contents of oleic acid. Samples of the Tyrolean Iceman clustered between fresh tissue and those of other glacier corpses indicating the good preservation of this mummy. Thus, environmental post-mortem conditions were associated with characteristic fatty acid patterns suggesting that chemometric analysis of fatty acid contents may add to our knowledge about post-mortem storage conditions and the preservation of human corpses.     

Hydration of oleic acid by a Selenomonas strain from the ovine rumen

J.A. HUDSON, C.A.M. MACKENZIE AND K.N. JOBLIN. 1996. A Selenomonas sp., isolated from the ovine rumen, was characterized with regard to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydration occurred only in stationary phase in a medium containing 0.1%, 0.5% (w/v) galactose or 0.5% (w/v) glucose, but not in a medium containing 1% galactose. Growth under a hydrogen headspace did not result in the production of stearic acid, the biohydrogenated product of oleic acid. Linoleic and linolenic acids (0.1% v/v) were not hydrated. It is concluded that the growing bacterium is unlikely to contribute to oleic acid hydration in the rumen.     

Stereospecificity of Microbial Hydrations of Oleic Acid to 10-Hydroxystearic Acid

We recently described a simple method for ascertaining the stereochemical purities of hydroxy fatty acids (S. H. El-Sharkawy, W. Yang, L. Dostal, and J. P. N. Rosazza, Appl. Environ. Microbiol. 58:2116-2122, 1992) based on the ¹H-nuclear magnetic resonance spectral analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates. This report describes the stereochemistries of microbial hydrations of oleic acid to 10-hydroxystearic acid by Nocardia aurantia (also known as Rhodococcus rhodochrous) ATCC 12674, Nocardia restrictus ATCC 14887, Mycobacterium fortuitum UI-53387, Pseudomonas species strain NRRL-2994, Pseudomonas species strain NRRL B-3266, and baker's yeast. 10(R)-hydroxystearic acid isolated from Pseudomonas species strain NRRL-2994 was the standard for use in the ¹H-nuclear magnetic resonance spectral technique to permit simple assignments of the absolute configurations of 10-hydroxystearic acid produced by different microorganisms. While the R. rhodochrous ATCC 12674-mediated hydration of oleic acid gave mixtures of enantiomers 10(R)-hydroxystearic acid and 10(S)-hydroxystearic acid, Pseudomonas species strain NRRL-B-3266 produced optically pure 10(R)-hydroxystearic acid. The remaining microorganisms stereoselectively hydrated oleic acid to 10(R)-hydroxystearic acid containing between 2 and 18% of the contaminating 10(S)-hydroxystearic acid.     

Oxidation of oleic and elaidic acids in rat and human heart homogenates

Parallel incubations with uniformly 14C-labeled oleic and elaidic acids were conducted to compare oxidation rates in tissue homogenates prepared from rat and human hearts. Radioactivity in 14CO2 and 14C-labeled chain-shortened acid-soluble products was used to measure the extent of oxidation. Oxidation rates (pmol/min per mg heart protein) determined on 14C-labeled acid-soluble products suggest that oleic acid was oxidized 35-40% faster than elaidic acid by both male and female rat heart homogenates, whereas human heart homogenates oxidized these fatty acids at equal rates. Rates for female heart homogenates were somewhat higher than those for males in rats and humans. Rates of formation of 14CO2 were the same for each acid in rat and human heart tissue. Comparative rates of formation of oxidation products expressed as oleic/elaidic ratios from parallel incubations confirm that preferential oxidation of oleic acid occurred with rat heart homogenates, but not with the human heart homogenates. These data suggest that the presence of the trans double bond in elaidic acid does not impair its utilization for energy by human heart muscle.     

Factors affecting the formation of 10-hydroxystearic acid from oleic acid by a ruminal strain of Enterococcus faecalis

 A ruminal strain of Enterococcus faecalis was characterised with respect to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydroxy fatty acid was produced after growth had ceased and the carbon source was almost exhausted. Hydroxy fatty acid production was equally rapid whether the inoculum had been grown in the presence of oleic acid or not, and almost complete conversion was achieved when oleic acid was present at a concentration of up to 0.5% (v/v). Incubation under a hydrogen headspace did not result in biohydrogenation of oleic acid. In pH-controlled batch culture the proportion of oleic acid hydrated varied with the pH of incubation, with more hydration at lower pH. Growth was retarded in the presence of 0.1% (v/v) linoleic acid, inhibited by the same concentration of linolenic acid and did not result in the formation of hydrated products from these substrates. If this organism is able to transform oleic acid in the rumen then the only product likely to be formed is 10hydroxystearic acid. Received: 17 July 1995/Received last revision: 24 October 1995/Accepted: 30 October 1995     

Production of 10-hydroxystearic acid from oleic acid by whole cells of recombinant Escherichia coli containing oleate hydratase from Stenotrophomonas maltophilia

A putative fatty acid hydratase from Stenotrophomonas maltophilia was cloned and expressed in Escherichia coli. The recombinant enzyme showed the highest hydration activity for oleic acid among the fatty acids tested, indicating that the enzyme is an oleate hydratase. The optimal conditions for the production of 10-hydroxystearic acid from oleic acid using whole cells of recombinant E. coli containing the oleate hydratase were pH 6.5, 35°C, 0.05% (w/v) Tween 40, 10 g l(-1) cells, and 50 g l(-1) oleic acid. Under these conditions, whole recombinant cells produced 49 g l(-1) 10-hydroxystearic acid for 4 h, with a conversion yield of 98% (w/w), a volumetric productivity of 12.3 g l(-1) h(-1), and a specific productivity of 1.23 g g-cells(-1) h(-1), which were 18%, 2.5-, and 2.5-fold higher than those of whole wild-type S. maltophilia cells, respectively. This is the first report of 10-hydroxystearic acid production using recombinant cells and the concentration and productivity are the highest reported thus far among cells.     

Conversion of oleic acid to 10-hydroxystearic acid by Nocardia paraffinae

Resting cells of Nocardia paraffinaeCBS 255 58 anaerobically converted oleic acid to 10-hydroxystearic acid (optimum activity at 35°C and pH 7). Addition of oleic acid (0.22 mg/ml of medium), as an inducer, improved the hydroxylating activity. The biocatalyst was inactivated by sonication, lyophilization and heating, but kept 65% of its initial activity after five freeze-thawing treatments. This activity appeared to be located either in the cells or bound to the plasmic membrane.     

Production of 10-hydroxystearic acid from oleic acid and olive oil hydrolyzate by an oleate hydratase from Lysinibacillus fusiformis

A recombinant enzyme from Lysinibacillus fusiformis was expressed, purified, and identified as an oleate hydratase because the hydration activity of the enzyme was the highest for oleic acid (with a k (cat) of 850?min(-1) and a K (m) of 540?μM), followed by palmitoleic acid, γ-linolenic acid, linoleic acid, myristoleic acid, and α-linolenic acid. The optimal reaction conditions for the enzymatic production of 10-hydroxystearic acid were pH 6.5, 35?°C, 4% (v/v) ethanol, 2,500?U ml(-1) (8.3?mg?ml(-1)) of enzyme, and 40?g l(-1) oleic acid. Under these conditions, 40?g l(-1) (142?mM) oleic acid was converted into 40?g l(-1) (133?mM) 10-hydroxystearic acid for 150?min, with a molar yield of 94% and a productivity of 16?g l(-1)?h(-1), and olive oil hydrolyzate containing 40?g l(-1) oleic acid was converted into 40?g l(-1) 10-hydroxystearic acid for 300?min, with a productivity of 8?g l(-1)?h(-1).     

Conversion of fatty acids by Bacillus sphaericus-like organisms

Bacillus sphaericus species are mesophilic round-spored organisms that readily utilize fatty acid-based surfactants during growth, but their ability to modify fatty acids is unknown. Among 57 B. sphaericus-like strains tested for fatty acid transformation activity in Wallen fermentation (WF) medium, ten converted oleic acid to a new product determined by gas chromatography – mass spectrometry (GC-MS) to be 10-ketostearic acid (10-KSA). Additionally, a few other strains converted ricinoleic acid and linoleic acid to new products that remain to be characterized. Unlike most microbial hydrations of oleic acid, which produce a mixture of 10-KSA and 10-hydroxystearic acid, the conversion of oleic acid by B. sphaericus strains was unique in that 10-KSA was the sole reaction product. By replacing dextrose with sodium pyruvate in WF and adjusting to pH 6.5, conversion of oleic acid to 10-KSA by strain NRRL NRS-732 was improved from about 11% to more than 60%. Using the defined optimal conditions, the conversion reaction was scaled up in a stirred-batch reactor by using technical-grade oleic acid as substrate. This is the first report on the characterization of fatty acid conversions by B. sphaericus species. Received: 17 December 2001 / Accepted: 17 January 2002     

Production of 10-Ketostearic Acid from Oleic Acid by Flavobacterium sp. Strain DS5 (NRRL B-14859)

A microbial isolate, Flavobacterium sp. strain DS5, produces 10-ketostearic acid (10-KSA) from oleic acid in 85% yield. This is the first report on this type of reaction catalyzed by a Flavobacterium strain. The product was purified to give white, plate-like crystals melting at 79.2°C. The optimum time, pH, and temperature for the production of 10-KSA are 36 h, 7.5, and 30°C, respectively. A small amount of 10-hydroxystearic acid (10-HSA) (about 10% of the amount of the main product, 10-KSA) is also produced during the bioconversion. 10-KSA is not further metabolized by strain DS5 and accumulates in the medium. In contrast to growing cells, a resting-cell suspension of strain DS5 produces 10-HSA and 10-KSA in a ratio of 1:3. The crude cell extract obtained from ultrasonic disruption of the cells converts oleic acid mainly to 10-HSA (10-HSA/10-KSA ratio, 97:3). This result strongly suggested that oleic acid is converted to 10-KSA via 10-HSA. Enzymes catalyzing the hydration and secondary alcohol dehydrogenation are cell associated. Product 10-HSA from strain DS5 is 66% enantiomeric excess in the 10(R) form. The oleic acid conversion enzyme(s) reacts with unsaturated fatty acids in the order oleic acid > palmitoleic acid > linoleic acid > linolenic acid > γ-linolenic acid > myristoleic acid.     

Production of 10-Ketostearic Acid and 10-Hydroxystearic Acid by Strains of Sphingobacterium thalpophilum Isolated from Composted Manure

Six strains of Sphingobacterium thalpophilum were isolated from a compost mixture enriched with oleic acid. These strains converted oleic acid to 10-ketostearic acid (10-KSA; 87–94% of the total conversion product) and to 10-hydroxystearic acid (10-HSA; 6–13%) exhibiting three levels of total product yields. The predominant production of 10-KSA by these new S. thalpophilum isolates is in contrast to strain 142b (NRRL B-14797) previously isolated from a commercial compost, which produces exclusively 10-HSA. The production yield of greater than 75% 10-KSA was achieved in 36 h, acting on 0.26 g of oleic acid in 30-ml fermentation broth incubated with agitation at 28°C. For easy maintenance, fast-growth, and high bioreactivity, these S. thalpophilum strains are suited for developing a large-scale production of 10-KSA and 10-HSA. Received: 20 July 1999 / Accepted: 20 August 1999     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

Biochemistry of development in insects. Incorporation of fatty acids into different lipid classes.

1. To study the different metabolic behaviour of various stages of development of the insect Ceratitis capitata, the incorporation of labelled decanoic, lauric, myristic, palmitic, stearic, oleic, and linoleic acids into triacylglycerols by insect homogenates was investigated. The time-course of incorporation of labelled fatty acids was firstly studied by using oleic acid; it showed that after 10 min of incubation the levels of radioactivity incorporated into triacylglycerols and those remaining in the free fatty acids were practically unchanged. 2. All labelled fatty acids were efficiently incorporated by larval homogenates; however, most of the radioactivity remained as free fatty acids in the presence of pharate adult homogenates, palmitic, and stearic acids being the most scarcely incorporated by this stage of development of the insect. 3. Plots of triacylglycerol and free fatty acid radioactivites versus the stage of development defined a crossing-zone in coincidence with the larval-pupal apolysis. This metabolic difference between larval and pharate adult homogenates could not be explained through differences in the acyl-CoA synthetase activity of the insect; this enzyme activity was notably higher in pharate adult homogenates than in the larval homogenates whatever would be the nature of the fatty acid. 4. [14C]Triolein was scarcely hydrolyzed by both larval and pharate adult homogenates. 5. Double-label experiments were carried out by incorporating either [3H]oleic acid or [3H]-palmitic acid and [14C]glycerol 3-phosphate by larval and pharate adult homogenates at different incubation intervals. Diacylglycerols, triacylglycerols, and phosphoglycerides were isolated and the 14C/3H molar ratio calculated. Results suggest the existence of a different acyltransferase activity in the different stages of development of the insect.     

Conversion of oleic acid to 10-hydroxystearic acid by two species of ruminal bacteria

Bacteria able to convert oleic acid to 10-hydroxystearic acid were isolated from the ovine rumen. The solid hydroxy fatty acid produced from bacterial fermentations containing oleic acid was recovered by filtration, extraction into ether and crystallisation. The identity of the product was confirmed by HPLC and gas chromatography/mass spectrometry. One 10-hydroxystearic-acid-producing bacterial group was represented by two strains of an anaerobic gram-negative curved rod with tufts of flagella on the concave surface of the cell. The morphology and other characteristics enabled the strains to be tentatively identified as Selenomonas ruminantium. Another bacterium capable of the same transformation, represented by two strains of a facultatively anaerobic gram positive chain-forming coccus, was identified as Enterococcus faecalis. Since unsaturated fatty acids entering the rumen are normally hydrogenated, hydration of oleic acid represents an alternative fate of unknown significance in vivo.     

Identification of the peroxidation product hydroxystearic acid in Lewis lung carcinoma cells

Whole cell lipids were extracted from the Lewis lung carcinoma in vitro line C108. The fatty acids were derivatized to methylesters in order to identify endogenous oxidized derivatives by gasmass spectroscopy. The presence of 9-hydroxystearic acid and 10-hydroxystearic acid was thus evidenced for the first time in cultured mammalian cells. Moreover a linear correlation was found between the concentration of these products expressed as percentage of total fatty acid methylesters and the cell density in tissue culture flasks. This finding suggests an involvement of hydroxystearic acid in cellular functions related to the cell density in monolayer cultures.     

alpha-Chitinase activity among lactic acid bacteria

Chitin is a polysaccharide widely distributed in nature. Among 115 strains from 29 species of lactic acid bacteria only strains belonging to Carnobacterium divergens and Carnobacterium maltaromaticum hydrolyzed alpha-chitin. This activity was not affected by temperature (10 degrees C versus 30 degrees C) and in most cases not subject to glucose catabolite repression.     

Determination of endogenous levels of cyclic ADP-ribose in rat tissues

Cyclic ADP-ribose (cADPR) is a potent mediator of calcium mobilization in sea urchin eggs. The cADPR synthesizing enzyme is present not only in the eggs but also in various mammalian tissue extracts. The purpose of this study was to ascertain whether cADPR is a naturally occurring nucleotide in mammalian tissues. Rat tissues were frozen and powdered in liquid N2, followed by extraction with perchloric acid at -10 degrees C. [32P]cADPR was prepared and used as a tracer. The acid extracts were chromatographed on a Mono-Q column and cADPR in the fractions were determined by its ability to release Ca2+ from egg homogenates. That the release was mediated by cADPR and not inositol trisphosphate (IP3) in the extracts was shown by the fact that the homogenates, subsequent to Ca2+ release induced by active fractions, were desensitized to authentic cADPR but not to IP3. Furthermore, the Ca2+ release activity was shown to co-elute with [32P]cADPR. The endogenous level of cADPR determined in rat liver is 3.37 +/- 0.64 pmol/mg, in heart is 1.04 +/- 0.08 pmol/mg and in brain is 2.75 +/- 0.35 pmol/mg. These results indicate cADPR is a naturally occurring nucleotide and suggest that it may be a general second messenger for mobilizing intracellular Ca2+.