Terence Galliard

The enzymic breakdown of lipids in potato tuber by phospholipid- and galactolipid-acyl hydrolase activities and by lipoxygenase.

Abstract Homogenization of potato tubers at 0° results in rapid enzymic hydrolysis of the endogenous phospholipids and galactolipids to produce free fatty acids and fatty acid hydroperoxides. The enzymes responsible for these effects have been identified as acyl hydrolases and lipoxygenase. Monogalactosyl diglyceride is particularly susceptible to hydrolysis and monogalactosyl monoglyceride was detected as an intermediate product. Acyl transferase activity was also associated with the acyl hydrolase action. Properties of the various enzyme activities are described; all are present mainly in the particle-free supernatant fraction and have acidic pH optima. Evidence is presented which indicates that free lipids are hydrolysed more readily than the lipids of lipoprotein membranes. The results are discussed with particular reference to the lipid composition and enzyme activities of subcellular organelles prepared from tissue homogenates.

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.

The formation of cis-3-nonenal, trans-2-nonenal and hexanal from linoleic acid hydroperoxide isomers by a hydroperoxide cleavage enzyme system in cucumber (Cucumis sativus) fruits.

1. A particulate enzyme fraction and an acetone powder preparation from cucumber fruits cleaved 9- and 13-hydroperoxyoctadecadienoic acids to form volatile aldehydes and oxoacid fragments. 2. From the 9-hydroperoxide, the major volatile fragments were cis-3-nonenal and trans-2-nonenal using particulate enzyme and acetone powder preparations, respectively. 3. Hexanal was the only significant volatile fragment from the 13-hydroperoxide. 4. The particulate enzyme system was equally effective on both 9- and 13-hydroperoxide isomers and was fully active under anaerobic conditions and at pH 6.4. 5. An enzymic pathway for the biogenesis of hexanal, cis-3- and trans-2-nonenal (components of the characteristic flavour volatiles of cucumber) from linoleic acid is proposed. This involves the sequential activity of lipoxygenase, hydroperoxide cleavage and cis-3-: trans-2-enal isomerase enzymes.

The enzymic cleavage of linoleic acid to C9 carbonyl fragments in extracts of cucumber (Cucumis sativus) fruit and the possible role of lipoxygenase

1. Homogenates and acetone powders of cucumber fruits catalyse the enzymic conversion of linoleic acid to aldehyde and oxoacid fragments in high yield, up to 60% with acetone powder extracts. 2. The major products are trans2-nonenal--a major component of the characteristic odour of cucumber--and 9-oxononanoic acid. 3. The cleavage reaction is a heat-labile, aerobic process, optimal at pH 6 (approx.). 4. Substrate specificity studies indicate that a lipoxygenase-type of reaction is involved in the cleavage process. 5. The acetone powder extracts have lipoxygenase activity and the proportion of linoleic acid hydroperoxide to carbonyl fragments depends upon incubation conditions. 6. Linoleic acid hydroperoxide isomers are also converted to carbonyl fragments by acetone powder extracts; the 9-hydroperoxide is cleaved at the 9-10 position whereas 12-13 cleavage is predominant with the 13-hydroperoxide isomer.

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.

Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

Abstract A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.

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.

Phospholipase, galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Abstract Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.

Metabolic changes in excised fruit tissue—III. The development of ethylene biosynthesis during the ageing of disks of apple peel

Abstract The production of ethylene by disks of peel from pre-climacteric apples is induced by ageing the disks aerobically at 25°. The development of the ethylene-producing system is dependent upon protein synthesis. Ethylene evolution is stimulated by peroxidation products from linolenic acid, but not by methionine. The relationship between changes occurring during the ageing of pre-climacteric apple peel disks during the development of the climacteric in whole fruit is discussed. A rapid and sensitive method for the determination of ethylene is described.