Purification and characterization of olive (Olea europaea L.) peroxidase - Evidence for the occurrence of a pectin binding peroxidase

Peroxidase from olive fruit (Olea europaea L., cv Douro) in a black ripening stage was purified to electrophoretic homogeneity, resulting in four cationic and four anionic fractions. The anionic fractions accounted for 92% of recovered activity and showed molecular masses of 18-20 kDa. The anionic fraction PODa4, the predominant fraction that comprised about 70% of total recovered activity, showed an isoelectric point of 4.4 and optimum pH and temperature of, respectively, 7.0 and 34.7 degrees C, and apparent K(m) values of 41.0 and 0.53 mM, for phenol and H(2)O(2), respectively. From the activity-temperature profile, the denaturation temperature and the changes in enthalpy and heat capacity for unfolding of PODa4 were estimated as being, respectively, 36.5 degrees C, 411.2 and -13.6 kJ mol(-1) K(-1). The activation energy for phenol oxidation by PODa4 was 99.1 kJ mol(-1), corresponding to a calculated temperature coefficient (Q(10)) of 4. The arabinose (39 mol%) and galacturonic acid (38 mol%) content of the carbohydrate moiety indicated the existence of pectic material in the purified PODa4 fraction. Co-migration of the carbohydrate with the protein band in the isoelectric focusing electrophoresis, points to PODa4 fraction as being a pectin type binding peroxidase. (c) 2006 Elsevier Ltd. All rights reserved.