Purification of hydroperoxide lyase from cucumbers

Hydroperoxide lyase (HPL) was extracted from cucumber fruit (Cucumis sativus) and purified by centrifugation, solubilization with detergent, ion-exchange chromatography and hydroxyapatite chromatography. 9-Hydroperoxy-linoleic acid and 13-hydroperoxy-linoleic acid lysing activities were purified 88-fold and 82-fold, respectively. The purified HPL preparation consisted of a single major band following SDS-electrophoresis with a molecular weight of about 55 000 Da; pH 6 was optimum for the lysis of both 9-hydroperoxy-linoleic acid and 13-hydroperoxy-linoleic acid substrates. The enzyme was relatively stable and retained more than two thirds of original activity after 3 weeks at 4 degrees C, but lost half of its activity after 2 min at 50 degrees C. Apparent K-m values for 9-hydroperoxy-linoleic acid, 9-hydroperoxy-linolenic acid, 13-hydroperoxy-linoleic acid and 13-hydroperoxy-linolenic acid were 6.76, 6.02, 5.46 and 12.4 mu M respectively. Corresponding V-max values were 19.3, 12.0, 7.58 and 11.4 mu mol min. The V-max (app)/K-m(app) values for 9-hydroperoxy-linoleic acid, 9-hydroperoxy-linolenic acid, 13-hydroperoxy-linoleic acid and 13-hydroperoxy-linolenic acid were 2.86, 1.99, 1.39 and 0.92, respectively. It is suggested that cucumber mesocarp contains only one type of HPL which is able to more efficiently catalyse the lysis of the 9-acyl hydroperoxides and especially 9-hydroperoxy-linoleic acid. (C) 1999 Elsevier Science Ltd. All rights reserved.