Separation, partial purification and characterization of a fatty acid hydroperoxide cleaving enzyme from apple and tomato fruits

Abstract

A membrane-bound enzyme catalysing the cleavage of 13-hydroperoxy-(Z)-9, (£)-l 1-octadecadienoic acid (13-LHPO) and 13-hydroperoxy-(Z)-9, (£)-ll, (Z)-15-octadecadienoic acid (13-LnHPO) to C6-aldehydes was isolated and partially purified from apples and tomatoes. Attempts to employ Ultrogel AcA 34 and AcA 22 in a gel chromatographic purification step were partially frustrated by reaggregation phenomena. However, by using Sepharose CL-4 B an enzyme fraction (MW 200 000 Da) with lipoxygenase and fatty acid hydroperoxide cleaving activity could be separated from a high molecular-weight active eluate. By applying preparative isoelectric focussing tothe tomato protein we succeeded in separating the fatty acid cleaving activity from the lipoxygenase, because of their different isoelectric points of pH 5.8 -6.1 and pH 5.0, respectively, An 8.4-fold purification of the fatty acid cleaving activity was achieved. A pH-optimum of 5.5 and a Km-value of 2.6 x 10-5 m/l for the 13-hydroperoxide of linoleic acid were measured. p-Chloromercuribenzoic acid (1 mM) showed significant inhibitory effect on the fatty acid hydroperoxide cleaving enzyme, but no evidence of inhibition was found with 1mM H2O2, KCN, DABCOand EDTA or superoxide dismutase (270 U). The maximum amount of fatty acid hydroperoxide decomposition (C6-aldehyde formation) was determined to be 59%. © 1982, Walter de Gruyter. All rights reserved.