Jennifer A. Matthew

A simple method for the preparation of pure 9-D-hydroperoxide of linoleic acid and methyl linoleate based on the positional specificity of lipoxygenase in tomato fruit.

Incubation of linoleic acid with crude homogenate of tomato fruit gave a high yield (69%) of linoleic acid hydroperoxides with a ratio of 9- to 13-hydroperoxide isomers of 96∶4. After chromatography of the products, as free acids or methyl esters, hydroperoxides with 9- to 13-isomeric ratios of >99∶1 were obtained. The major product was characterized as 9-d-hydroperoxy-octadeca-trans-10,cis-12-dienoic acid. The results demonstrate the positional specificity of lipoxygenase from tomato fruit.

The mechanism of the rearrangement of linoleate hydroperoxides

Linoleate hydroperoxides undergo rearrangement leading to their isomerisation in which the OOH group is relocated or the stereochemistry of a double bond changed, or both. The reaction was studied mainly with pure isomers of methyl hydroperoxylinoleates since conditions could be found in which rearrangement occurred with little accompanying decomposition. The rearrangement was found to be non-stereoselective and took place by a free-radical chain mechanism. Using 18O-labelled hydroperoxide on 18O2, it was shown that the oxygen atoms of the OOH group of the hydroperoxides exchanged with surrounding molecular oxygen during the rearrangement. A mechanism for the rearrangement is proposed.

Lipoxygenase-mediated cleavage of fatty acids to carbonyl fragments in tomato fruits

Abstract Homogenates of tomato fruits catalysed the enzymic conversion of linoleic and linolenic acids (but not oleic acid) to C 6 aldehydes in low (3–5%) molar yield. Hexanal was formed from linoleic acid; cis -3-hexenal and smaller amounts of trans -2-hexenal were formed from linolenic acid. With the fatty acids as substrates, the major products were fatty acid hydroperoxides (50–80% yield) and the ratio of 9- to 13-hydroperoxides as isolated from an incubation with linoleic acid was at least 95:5 in favour of the 9-hydroperoxide isomer. When the 9- and 13-hydroperoxides of linoleic acid were used as substrates with tomato homogenates, the 13-hydroperoxide was readily cleaved to hexanal in high molar yield (60%) but the 9-hydroperoxide isomer was not converted to cleavage products. Properties of the hydroperoxide cleavage system are described. The results indicate that the C 6 aldehydes are formed from C 18 polyunsaturated fatty acids in a sequential enzyme system involving lipoxygenase (which preferentially oxygenates at the 9-position) followed by a hydroperoxide cleavage system which is, however, specific for the 13-hydroperoxy isomers.

Improvement of the gelation properties of sugarbeet pectin following treatment with an enzyme preparation derived from Aspergillus niger - comparison with a chemical modification

Abstract Pectin derived from sugarbeet pulp is extracted and then subjected to either enzymic or chemical modification. The enzyme is derived from the fungus Aspergillus niger . A partial purification of the enzyme yields a preparation which is devoid of polygalacturonase but which effects a number of changes in the pectin molecule; some deacetylation, some demethoxylation and a large reduction in the arabinose content. The resulting treated pectin possesses considerably enhanced gelling properties. Chemical modification of the pectin results in a greater degree of deacetylation and demethoxylation, but a much lower percentage removal of arabinosyl residues. Gels formed from the chemically modified pectin are firmer than those formed from the untreated material. However, they are more brittle and less like a conventional gel than those formed from the enzymatically treated pectin.

The enzymic formation of long chain aldehydes and alcohols by α-oxidation of fatty acids in extracts of cucumber fruit (Cucumis sativus)

1. An enzyme system that catalyses the alpha-oxidation of fatty acids to shorter chain products is present in acetone powders of cucumber fruits. 2. In the absence of NAD+, the predominant product from palmitic acid is pentadecanal. Addition of NAD+ gives rise to a homologous series of n-alkanals, the concentrations of which are in the same order as that reported in the volatile products formed on homogenization of cucumbers, i.e. C15 greater than C14 greater than C13 greater than C12. 3. Pentadecan-1-ol is also formed from palmitic acid in the absence of added NAD+; C15, C14 and C13 n-alkanols are produced in the presence of NAD+. 4. The substrate specificity for saturated fatty acids is in the order C12 less than C14 greater than C16 greater than C18. Unsaturated C18 acids are oxidized more readily than stearic acid. 5. The alpha-oxidation system is inhibited by dithiothreitol, cysteine, imidazole and certain metal ligands (CN-, N3-, diphenylthiocarbazone) but not by EDTA. 6. Differences between the alpha-oxidation system in cucumber and those previously reported in other plants are discussed.

Enzymic formation of carbonyls from linoleic acid in leaves of Phaseolus vulgaris

Homogenization of Phaseolus vulgaris leaves at acid pH results in the evolution of hexanal, cis-3- and trans-2-hexenal. With cell-free extracts of leaves, linoleic and linolenic acids are enzymically converted to their hydroperoxides (predominantly the 13-hydroperoxide isomers) and to hexanal or hexenal respectively. Activity was highest in young, dark-green leaves and was stimulated by Triton X-100. Oleic acid is not a substrate for these reactions. Both 9- and 13-hydroperoxides were cleaved to carbonyl fragments and are proposed as intermediates in the formation of volatile aldehydes and non-volatile ω-oxoacids in P. vulgaris leaves. Properties of the enzyme systems are described.

Enzymic reactions of fatty acid hydroperoxides in extracts of potato tuber II. Conversion of 9- and 13-hydroperoxy-octadecadienoic acids to monohydroxydienoic acid, epoxyhydroxy- and trihydroxymonoenoic acid derivatives

1. Crude extracts and partially purified enzyme preparations from potato tubers catalyse, at pH 5-7, the conversion of linoleic acid hydroperoxides to a range of oxygenated fatty acid derivatives. 2. 9-D- and 13-L-hydroperoxide isomers are converted at similar rates to equivalent (isomeric) products. 3. The major products from the 13-hydroperoxide isomer were identified as the corresponding monohydroxydienoic acid derivative, threo-11-hydroxy-trans12,13-epoxy-octadec-cis9-enoic acid and 9,12,13-trihydroxy-octadec-trans10-enoic acid. The corresponding products from the 9-hydroperoxide were the monohydroxydienoic acid, 9,10-epoxy-11-hydroxy-octadec-12-enoic acid and 9,10,13-trihydroxy-octadec-11-enoic acid. 4. No separation of activities forming the different products was achieved by partial purification of enzyme extracts. 5. Product formation was unaffected by EDTA, CN-, sulphydryl reagents or glutathione but was reduced by boiling the extracts. 6. This system is compared with the 9-hydroperoxide-specific enzymic formation of divinyl ether derivatives by potato extracts.

Partial purification and properties of a cis-3: trans-2-enal isomerase from cucumber fruit

Abstract An enzyme, which catalyses the isomerisation of cis-3-enals to trans-2-enals, has been partially purified from cucumber fruit. The isomerase activity has been resolved from significant contamination by the related activities, lipoxygenase and hydroperoxide cleavage enzymes. An examination of the substrate specificity of the isomerase enzyme showed it to be specific for the cis-3-enals. The most efficient isomerisation was achieved with cis-3-hexenal and cis-3-nonenal which are, physiologically, the two most significant substrates. The trans-3-enal and cis-3-enol were not suitable substrates for the enzyme.

Gelation of sugarbeet and citrus pectins using enzymes extracted from orange peel.

Abstract Sugarbeet pectin is shown to form gels in the presence of calcium using an enzyme preparation extracted from orange peel. The gels were transparent and exhibited no syneresis. The mechanism of gelation is chain association arising from both lowered pectin solubility and from formation of a limited network of calcium-linked junction zones. The gelation reaction involves limited pectin demethoxylation, the release of acetate presumably from C-2 or C-3 of galacturonyl residues, and a decrease in pH. The enzymes responsible are pectinesterase (EC 3.1.1.11) and pectin acetylesterase. We suggest that the latter is a novel activity associated with triacetin acetylesterase (EC 3.1.1.6). The gels are compared to citrus pectin gels made in the same way.

The enzymic degradation of lipids resulting from physical disruption of cucumber (Cucumis sativus) fruit

Abstract Homogenization of fresh tissue from cucumber fruits results in a loss of endogenous lipid catalysed by acyl hydrolase enzymes. Deacylation of lipids is not accompanied by accumulation of free fatty acids. The levels of both saturated (mainly palmitic) and polyunsaturated (linoleic and linolenic) fatty acids in the lipids are reduced. Losses of the major acyl lipid constituents of cucumber (triacylglycerols and phospholipids) are mainly responsible for the observed hydrolysis. Triacylglycerol acyl hydrolase (lipase), phospholipase D and polar lipid acyl hydrolase enzyme activities were demonstrated. It is suggested that hydrolytic attack on endogenous lipids is the initial event on disruption of cucumber tissue, in the formation of lipid degradation products, amongst which are the volatile carbonyl compounds responsible for the characteristic flavour of cucumber.