Chlorophyll catabolism in olive fruits (var. Arbequina and Hojiblanca) during maturation.

The central reaction of chlorophyll (chl) breakdown pathway occurring during olive fruits maturation is the cleavage of the macrocycle pheophorbide a to a primary fluorescent chl catabolite (pFCC) and it is catalyzed by two enzymes: pheophorbide a oxygenase (PaO) and red chl catabolite reductase (RCCR). In subsequent steps, pFCC is converted to different fluorescent chlorophyll catabolites (FCCs) and nonfluorescent chlorophyll catabolites (NCCs). This work demonstrated that RCCR activity of olive fruits is type II. During the study of evolution of PaO and RCCR activities through the olive fruits maturation in two varieties: Hojiblanca and Arbequina, a significant increase in PaO and RCCR activity was found in ripening stage. In addition, the profile and structure of NCCs present in epicarp of this fruit was studied using HPLC/ESI-TOF-MS. Five different NCCs were defined and for the first time the enzymatic reactions implied in chlorophyll degradations in olive fruits elucidated.

Characterisation of chlorophyll oxidation mediated by peroxidative activity in olives (Olea europaea L.) cv. Hojiblanca.

The oxidation of chlorophyll a (chl a) catalysed by peroxidase (POD) from mesocarp of the olive fruit (Olea europaea L., cv Hojiblanca) in the presence of H2O2 and 2,4-dichlorophenol (2,4-DCP), is characterised via the individualised quantification of the products of the enzymatic reaction using a new methodology of HPLC-UV spectrometry. This innovation has allowed the discovery that, in addition to 13(2) OH chl a and 15(1) OH lactone chl a, which are the first products of POD on chl a, the reaction process sequentially creates another series of oxidised chlorophyll derivatives which have not been previously described. Their origins have been linked to POD activity in the presence of 2,4-DCP. Likewise, a study of the effect of the concentration of the various cosubstrates on the POD reaction rate demonstrated that the correct establishment of the relative concentrations of the same ([H2O2]/[2,4-DCP]/[Chl]=1:3:0.02) is crucial to explaining inhibition effects by substrates and carrying out optimum measurements. Therefore, new essential parameters for the determination of POD activity on a chlorophyll substrate are established.