Oxidation of acylglycerols and phosphoglycerides by soybean lipoxygenase

Lipoxygenase (EC 1.13.11.12) catalyzes the incorporation of oxygen into polyunsaturated fatty acids, resulting in the formation of their corresponding hydroperoxides. The ability of a commercial preparation of soybean (Glycine max L. Merr.) lipoxygenase to catalyze the oxidation of acylglycerols and phosphoglycerides was investigated. The oxidation rate of trilinolein increased nearly 100% when 5 mM deoxycholate was added to the reaction medium. With further increases in the concentration of deoxycholate, the oxidation rate decreased slightly. The pH profile of trilinolein oxidation was bell-shaped. The rate of oxidation was maximal at pH 8, and it decreased to near zero at pH 5 and pH 11. Even under optimal conditions, the rate of trilinolein oxidation was only 3% of that of linoleic acid, and analysis of time course data showed that, at most, 15% of available linoleate was oxidized. In contrast to the slow rate of trilinolein oxidation, tested phosphoglycerides and diacylglycerols were oxidized at moderate rates. The rate of phosphoglyceride oxidation depended upon the structure of the polar head group and varied between 7–28% of the rate of linoleic acid oxidation. Diacylglycerols reacted at a rate that was 40% of that of linoleic acid. Analysis of the time course of 1,3-dilinolein oxidation showed that as much as 67% of the available linoleate could be converted to the corresponding hydroperoxide. Analyses by high-performance liquid chromatography revealed that more than 20% of the 1,3-dilinolein was converted to unidentified products that are not hydroperoxides.     

Oxidation of various lipid substrates with unfractionated soybean and wheat lipoxidase

Quantitative determinations of lipoxidase in wheat were made on five milling fractions from four wheat varieties, including two hard red winter and two hard red spring wheats. Lipoxidase content decreased with increasing refinement and was lowest in the flour fraction. In general, the mill fractions from spring wheats exhibited slightly higher lipoxidase content than the corresponding mill fractions from winter wheats. Aqueous extracts from soybeans (variety Hawkeye) and wheat (variety Selkirk, break shorts fraction) were adjusted in concentration so that the lipoxidase activity with respect to linoleic acid was approximately the same. The diluted extracts were then tested for their ability to oxidize other lipid substrates with the necessary 1,4 pentadiene system. Soybean lipoxidase was far more reactive toward methyl linoleate and trilinolein than wheat lipoxidase. Mono- and dilinolein were relatively unreactive in both systems although dilinolein was a better substrate in the soybean system than in the wheat. Neither system oxidized highly unsaturated digalactosyl diglycerides alone. However the soybean extract oxidized this substrate to a small extent in the presence of catalytic amounts of linoleic acid while, under identical conditions, the wheat extract remained inactive. Polyacrylamide gel electrophoresis of the aqueous extracts of soybeans and wheat-milling fractions coupled with a staining procedure specifically for lipoxidase indicated three to four isoenzyme bands in the soybeans and two to four isoenzyme bands in the wheat-milling fractions. Isoenzymes may be responsible for differences in enzymatic activity on various substrates.     

Formation of octadecadienoate dimers by soybean lipoxygenases

The aim of this investigation was to determine whether the regioselectivity found for lipoxygenases in the formation of fatty acid hydroperoxides from linoleic acid is reflected in the formation of dimeric products in secondary reactions involving linoleic acid, product hydroperoxide and lipoxygenase. A method was therefore developed for the separation and identification of dimers formed by fusion of two linoleic acid radicals or a linoleic acid radical and linoleate. The method includes solid-phase extraction, preparative separation of products by thin-layer chromatography, derivatization to the corresponding fully hydrogenated methyl esters and capillary gas chromatography (GC) coupled with electron impact mass spectrometry. We present evidence that the formation of octadecadienoate dimers, during the secondary reaction of soybean lipoxygenase-1 or lipoxygenase-3, is a nonenzymic process that can be envisaged by nonspecific association of intermediate fatty acid radicals (L^*) that have dissociated from the enzyme. We could show that the relative amounts of different octadecadienoate dimers formed remain unaltered, regard-less of pH and type of soybean isoenzyme used. Quantitative analysis by GC showed that under the reaction conditions used, the formation of dimers branching at the 13-position is preferred.     

Isolation of a pure isomer of linoleic acid hydroperoxide

A mixture of positional isomers of linoleic acid hydroperoxide was produced from the oxidation of linoleic acid by lipoxygenase from corn or soybean. Chromatography on a column of silicic acid separated 13-hydroperoxy-11,9-octadecadienoic acid in 99+% purity from the mixture obtained by soybean lipoxygenase oxidation of linoleic acid. Attempts at isolation of pure 9-hydroperoxy-10,12-octadecadienoic acid from hydroperoxides obtained by corn lipoxygenase oxygenation of linoleic acid were partially successful with isolation of the 9-hydroperoxide in 97% purity.     

Soybean lipoxygenase is active on nonaqueous media at low moisture: a constraint to xerophilic fungi and aflatoxins?

Previous workers have reported that certain products of the lipoxygenase pathway are detrimental either to the development and growth of Aspergillus species or to aflatoxin production by these organisms. Since Aspergillus often thrives on “dry” stored grains, depending on the level of the relative humidity, we sought to determine if lipoxygenase could catalyze the oxidation of linoleic acid on these “dry” substrates equilibrated at various relative humidities. A desiccated model system, previously adjusted to pH 7.5, was composed of soybean extract, linoleic acid, and cellulose carrier. The model system was incubated for up to 24 h at four relative humidities ranging between 52 and 95% to determine the extent of oxidation catalyzed by lipoxygenase, compared with heat-inactivated controls. Oxidation in the active samples was much greater than in the controls at all relative humidities, and oxidation was principally enzymatic as demonstrated by chiral analysis of the linoleate hydroperoxides formed. The main product was 13S-hydroperoxy-9Z,11E-octadecadienoic acid, accompanied by a significant percentage of 9S-hydroperoxy-10E,12Z-octadecadienoic acid. Since the products became more racemic with time of incubation, autoxidation appeared to be initiated by the lipoxygenase reaction in dry media. Additionally, the biological relevance of lipoxygenase activity was tested under these xerophilic conditions. Thus, enzyme-active and heat-inactivated defatted soy flour amended either with or without 3.5% by weight linoleic acid was inoculated with fungal spores and incubated at 95% relative humidity. Although fungal growth occurred on all treatments, samples inoculated with Aspergillus parasiticus showed significantly less aflatoxin in the enzyme-active samples, compared to inactivated flour. Addition of linoleic acid had little effect, possibly because the defatted soy flour was found to contain 1.7% residual linoleic acid as glyceride lipid.     

Lipoxygenase activity in olive (Olea europaea) fruit

The present work was designed to characterize lipoxygenase activity in olive fruit pulp, in order to determine its significance in the biosynthesis of virgin olive oil aroma. Lipoxygenase activity has been detected in particulate fractions of enzyme extracts from olive pulp subjected to differential centrifugation. The activity in different membrane fractions showed similar properties, with optimal pH in the range of 5.0–5.5 and a clear specificity for linolenic acid, which was oxidized at a rate double that of linoleic acid under the same reaction conditions. The enzyme preparations displayed very low activity with dilinoleoyl phosphatidylcholine, suggesting that olive lipoxygenase acts on nonesterified fatty acids. The enzyme showed regiospecificity for the Δ-13-position of both linoleic and linolenic acid, yielding 75–90% of Δ-13-fatty acid hydroperoxides. Olives showed the highest lipoxygenase activity about 15 wk after anthesis, with a steady decrease during the developmental and ripening periods. Olive lipoxygenase displayed properties that support its involvement in the biogenesis of six-carbon volatile aldehydes, which are major constituents of the aroma of virgin olive oil, during the process of oil extraction.     

Preparation of fatty epoxy alcohols using oat seed peroxygenase in nonaqueous media

Peroxygenase is an enzyme of higher plants that is capable of using hydroperoxide and hydrogen peroxide for oxidation of a double bond to an epoxide. A microsomal fraction was prepared from dry oat (Avena sativa) seeds. The peroxygenase activity of this fraction was tested using fatty acid hydroperoxide 2a [13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid] and its methyl ester 2b as sources of peroxygen. These were prepared by the action of soybean lipoxygenase on linoleic acid. A high-performance liquid chromatographic assay was used to differentiate between peroxygen cleavage and peroxygen cleavage with accompanying double-bond oxidation Higher activity was obtained with 2b compared to 2a, and peroxygen cleavage activity was observed in both aqueous and organic solvent media. Double-bond oxidation activity was high only in aqueous media and nonpolar organic solvents. Structural elucidation of the epoxidized product showed it to be the oxylipid, methyl cis-9,10-epoxy-13(S)-hydroxy-11(E)-octade-cenoate 4b, demonstrating specificity for epoxidation of the cis double bond. Trihydroxy product was not detected, demonstrating that the epoxide was not hydrolyzed.     

Effect of triacylglycerol composition and structures on oxidative stability of oils from selected soybean germplasm

The oxidative stability of soybean oil triacylglycerols was studied with respect to composition and structure. Crude soybean oils of various fatty acid and triacylglycerol composition, hexane-extracted from ground beans, were chromatographed to remove non-triacylglycerol components. Purified triacylglycerols were oxidized at 60°C, in air, in the dark. The oxidative stability or resistance of the substrate to reaction with oxygen was measured by determination of peroxide value and headspace analysis of volatiles of the oxidized triacylglycerols (at less than 1% oxidation). The correlation coefficients (r) for rates of peroxide formation (r=0.85) and total headspace volatiles (r=0.87) were related positively to oxidizability. Rate of peroxide formation showed a positive correlation with average number of double bonds (r=0.81), linoleic acid (r=0.63), linolenic acid (r=0.85). Rate of peroxide formation also showed a positive correlation with linoleic acid (r=0.72) at the 2-position of the glycerol moiety. A negative correlation was observed between rate of peroxide formation and oleic acid (r=−0.82). Resistance of soybean triacylglycerols to reaction with oxygen was decreased by linolenic (r=0.87) and increased by oleic acid (r=−0.76)-containing triacylglycerols. Volatile formation was increased by increased concentration of linolenic acid at exterior glycerol carbons 1,3 and by linoleic acid at the interior carbon 2. Headspace analysis of voltiles and high-performance liquid chromatography of hydroperoxides indicated that as oxidation proceeded there was a slight decrease in the linolenic acid-derived hydroperoxides and an increase in the linoleic acid-derived hydroperoxides. The oxidative stability of soybean oil triacylclycerols with respect to composition and structure is of interest to the development of soybean varieties with oils of improved odor and flavor stability. Presented at the 81st Annual American Oil Chemists' Society Meeting, Baltimore, MD, April 18–21, 1990.     

大豆脂肪氧合酶酶促合成亚油酸氢过氧化物

研究了水相体系中大豆脂肪氧合酶酶促合成亚油酸(LA)氢过氧化物的反应,最佳反应条件为底物质量浓度4 11g/L,pH=9,5℃,反应时间120min,亚油酸氢过氧化物产率80%。当m(LA)∶m(surfactant)=1∶1时,Tween系列、CTAB、AOT、AEO4、OAPCG、S8390均能加速反应,其中AEO4使反应时间缩短至30min,产率提高12%。一些盐可以明显地加快反应进程,其中柠檬酸钠使反应时间缩短至15min。外源Fe3+在反应体系中的浓度为0 5μmol/L或外源Fe2+为5nmol/L时反应速度显著提高,消除了滞后期。     

The isolation and specificity of alfalfa lipoxygenase

Partial purification of the lipoxygenase from alfalfa seed was accomplished by fractionation of the protein with (NH₄)₂SO₄, phosphate, heavy metal salts and ultracentrifugation. About 24% of the original activity was recovered. The partially-purified alfalfa lipoxygenase enzyme was free of hydroperoxide-decomposing activity and was used to determine the positional specificity of linoleic acid oxidation by alfalfa lipoxygenase. Combined gas liquid chromatography-mass spectrometry was used to analyze known mixtures of 10- and 12-hydroxystearic acid derivatives and was satisfactory for the quantitative determination of the ratio of each component. This combination was used to analyze mixtures containing position isomers of hydroxy fatty acids without separation of each individual compound by other methods. Hydroperoxides produced from linoleic acid oxidation catalyzed by alfalfa lipoxygenase were converted by sodium borohydride reduction, catalytic hydrogenation and bis(trimethylsilyl)acetamide silylation to their corresponding trimethylsilyl either esters and the positional distribution was studied. The 9- and 13-linoleate hydroperoxides produced by alfalfa lipoxygenase were in equal concentrations (50∶50) whereas the distribution for soybean lipoxygenase was 70% 13- and 30% 9-hydroperoxides. Paper No. 3780 of the journal series of the Pennsylvania Experiment Station.     

Effects of soybean lipoxygenase-1 on phosphatidylcholines containing furan fatty acids

Naturally occurring tetraalkylsubstituted furan fatty acids (F-acids) were tested as potential substrates for soybean lipoxygenase-1. For this purpose, F-acid methyl ester and phosphatidylcholines containing F-acids at thesn-2 position of the glycerol residue wer incubated with the enzyme. Oxidation of F-acids only occurs in the presence of linoleic acid as co-substrate. Linoleic acid is converted by lipoxygenase to the corresponding hydroperoxide that oxidizes the F-acid, probably in a radical reaction, to form an unstable dioxoene compound. This intermediate the forms, dependent on pH, unsaturated furanoid acids or isomers with cyclopentenolone structure that can be detected by gas chromatography/mass spectrometry (GC/MS). F-acids located at thesn-2 position of a synthetic phosphadidylcholine (PC), containing linoleic acid in thesn-1 position, are co-oxidized to a greater extent by incubation with soybean lipoxygenase-1 than are F-acids bound to PC with myristic acid in thesn-1 position when subjected to the enzyme in the presence of a great excess of linoleic acid. The results suggest that F-acids may play a strategic role in antioxidative processes in plant cells.     

Sulfite-induced lipid peroxidation

Sulfite initiated the peroxidation of linoleic acid and linolenic acid emulsions via a free radical mechanism. Peroxidation of these fatty acids required oxygen and sulfite and occurred with concomitant oxidation of sulfite to sulfate. In reaction mixtures containing linoleic acid, the formation of conjugated diene equaled the formation of hydroperoxide. In reaction mixtures containing linolenic acid emulsions, thiobarbituric acid reactive materials were also formed. Peroxidation was pH-dependent; peroxidation of linoleic acid proceeded between pH 4 and 7, but linolenic acid peroxidation was significant only if pH was below pH 6. The linoleic acid hydroperoxides thus formed were reduced and methylated to methyl hydroxystearate. Analysis of methyl hydroxystearate by gas chromatographymass spectrometry indicated that sulfite-induced peroxidation gave rise to the 9- and 13-hydroperoxy isomers. In addition to the hydroperoxides, sulfite adducts were detected. Hydroquinone, butylated hydroxytoluene and α-tocopherol effectively inhibited both sulfite oxidation and hydroperoxide formation. Conjugated diene formation also was inhibited by 4-thiouridine, suggesting that the reaction is mediated by the sulfite radical. No significant inhibition was observed with the addition of superoxide dismutase, catalase, or the hydroxyl radical scavengers, mannitol ort-butanol. A possible mechanism is presented to account for sulfite-induced peroxidation of linoleic acid.     

Antioxidant activity of mung bean hulls

The antioxidant activity of methanolic extracts of mung bean hulls was investigated. Extracts at a concentration of 100 ppm exhibited stronger antioxidant activity than 100 ppm dl-α-tocopherol (Toc) or 100 ppm butylated hydroxyanisole on the peroxidation of linoleic acid. Moreover, a synergistic effect was observed when 100 ppm of the extract was mixed with 100 ppm Toc. Also, the extracts showed good inhibitory activity in soybean oil oxidation, which was examined by peroxide value, thiobarbituric acid, and gas chromatography of oxidized fatty acid methyl esters. The extracts can reduce the formation of both primary and secondary oxidation products of soybean oil. The extracts had reducing power and scavenged 1,1-diphenyl-2-picrylhydrazyl radical, which may in part be responsible for their antioxidant activity. Based on the results obtained, mung bean hulls are a potential source of natural antioxidants owing to their marked antioxidant activity.     

Distribution of Triacylglycerols and Fatty Acids in Soybean Oil with Thermal Oxidation and Methylene Blue Photosensitization

Profiles of triacylglycerols (TAG) and fatty acids were compared in soybean oil thermally oxidized at 180 °C for 60 min or methylene blue photosensitized for 10 h. Headspace oxygen in thermally oxidized and photosensitized soybean oil decreased significantly (p < 0.05) as oxidation time increased. Relative contents of linoleic and linolenic acids decreased and those of oleic acid increased during oxidation. In both thermal and photosensitized oxidation, TAG with lower than 44 equivalent carbon number including dilinoleoyllinolenoylglycerol (LLLn, 40), trilinolein (LLL, 42), oleoyllinoleoyllinolenoylglycerol (OLLn, 42), dilinoleoyloleoylglycerol (LLO, 44), and dilinoleoylpalmitoylglycerol (PLL, 44) significantly decreased, while those with dioleoyllinoleoylglycerol (OOL, 46) increased significantly in relative peak areas (p < 0.05). Photosensitized oxidation decreased TAG containing linoleic and linolenic acids significantly faster than thermal oxidation in soybean oil (p < 0.05), which may be due to the singlet oxygen reaction. Photosensitized soybean oils can be differentiated from thermally oxidized samples using the distributions of TAG by principal component analysis.     

Commercial and potential utilization of lipoxygenase

The high specificity and activity of lipoxygenase (EC 1.13.1.13) have not been widely exploited in commercial applications. An analytical application of substrate specificity is exemplified by the Canadian Food and Drug Official Method FA 59 for determining unisomerized linoleic acid in hydrogenated fats. An attractive potential application of lipoxygenase is the lipoxygenase oxidation of linoleic acid for conversion of a renewable resource into a valuable chemical intermediate. Hydroxy-conjugated octadecadienoic acids (HCD) have been prepared by oxidation of a 10% soybean soapstock solution with an aqueous soy flour extract followed by reduction of the hydroperoxide. High yields and a 20-min reaction time are features of this procedure. These laboratory-scale experiments indicate that the processing cost to produce hydroxy-conjugated octadecadienoic acids can be estimated at 21 cents per lb. This cost does not include the cost of the soapstock. The combined hydroxy, conjugated diene, and fatty acid groups in HCD give it the potential of being a versatile chemical intermediate. HCD is readily converted to hydroxystearate or conjugated triene and can compete directly with tung oil acids or hydrogenated castor oil acids. Other reactions can be visualized based on functional group modification to yield products with potential application in the formulation of coatings, lubricants, emulsifiers, and plasticizers.     

A soy extract catalyzes formation of 9-oxo-trans-12,13-epoxy-trans-10-octadecenoic acid from 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid

In the presence of oxygen, a crude soy extract converted 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid into numerous products, from which 9-oxo-trans-12,13-epoxy-trans-10-octadecenoic acid was isolated. Additionally, the soy extract oxidized linoleic acid to the oxo-epoxyoctadecenoic acid, presumably via a sequential reaction involving lipoxygenase oxidation of linoleic acid followed by degradation of the resultant linoleic acid hydroperoxide. However, the linoleic acid substrate yielded two isomeric linoleic acid hydroperoxides and because of this, two isomeric oxoepoxyoctadecenoic acids. Presented in part at the 13th Congress, International Society for Fat Research, Marseilles, France, August 30–September 4, 1976.     

Analysis of lipoxygenase kinetics by high-performance liquid chromatography with a polymer column

Soybean lipoxygenase (LOX; EC 1.12.11.12) catalyzes the oxygenation of polyunsaturated fatty acids, acylglycerols and phosphoglycerols, producing a regio-and enantiospecific hydroperoxide product. The goal of this work was to measure the relative rate of LOX-catalyzed oxidation of mixtures of lipids containing linoleate, using high-performance liquid chromatography (HPLC) and a light-scattering detector (LSD). Previous literature sugested that reversed-phase HPLC with silicabased columns could be used for the separation of individual fatty acids, acylglycerols, phosphoglycerides and their oxidation products. However, these columns produced ineffective separations of phosphoglycerides unless choline chloride and a strong base, such as KOH, are present in the mobile phase. Such modifiers precluded the use of the LSD. It was found that a reversed-phase column based upon an organic polymer support, rather than on silica, was able to separate these mixtures with a ternary solvent gradient of methanol/water/acetonitrile without the need for the addition of modifiers. The oxidation time course of a mixture of linoleic acid, trilinolein and 1-linoleoyl-2-stearoyl-sn-glycero-3-phosphocholine was followed using the developed HPLC method. The results showed that trilinolein and phosphatidylcholine reacted at one-tenth the rate of linoleic acid. The diacylglycerol, 1,3-dilinolein, was oxidized at a rate that was approximately 40% that of linoleic acid, with the formation of mono-and dihydroperoxides as well as other unidentified products.     

Comparison of methods for determining fatty acid oxidation produced by ultraviolet irradiation

Summary The thiobarbituric acid (TBA) reaction for fatty acid oxidation has been compared with Lundberg and Chipault's method for peroxides, the Kreis test for aldehydes, and with the degree of conjugation, using fatty acid esters exposed to ultraviolet light for various periods. The TBA test paralleled the other methods for methyl linolenate and methyl linoleate but was essentially negative for methyl oleate oxidation. The sensitivity of the TBA test for linolenate was 30–80 times that for linoleate at the same peroxide values. The TBA test appears to be a reliable method of estimating the oxidation products of linolenic and linoleic acids in tissues and other biological material. Supported by a grant from the Atomic Energy Commission.     

Oxidative stability of conjugated linoleic acids relative to other polyunsaturated fatty acids

Contrary to current opinion, conjugated linoleic acids (CLA) as a mixture of several isomers have been previously shown to function as prooxidants in the form of free fatty acids and methyl esters in heated canola oil. Furthermore, CLA oxidizes considerably faster than linoleic acid. However, stability of CLA relative to other polyunsaturated fatty acids remains undetermined. The present study was therefore undertaken to examine the relative oxidation rate of CLA compared with that of linolenic acid (LNA), arachidonic acid (AA), and docosahexaenoic acid (DHA) in air at 90°C. CLA, both in the form of free fatty acids and triacylglycerols, were extremely unstable to the same extent as DHA, but they oxidized considerably faster than LNA and AA. The mechanism by which CLA were readily decomposed was probably due to formation of the unstable free-radical intermediate.     

Lipoxygenase inhibition by naturally occurring monoenoic fatty acids

Lipoxygenase activity was not detected in preparations from several varieties of rapessed. Erucic acid, one of the main components of rapessed oil, competitively inhibited both soybean and peanut lipoxygenases. Various other long-chain monoenoic fatty acids were assayed for their effects on lipoxygenase activity with linoleic acid as substrate. Fatty acid chain length, from C₁₆, to C₂₄, was not a significant factor, but position of the point of unsaturation did affect enzyme activity. The position of the double bond from the carboxyl group seemed to be more significant than the distance between the point of unsaturation and the methyl terminal group in terms of inhibition.