MEASUREMENT OF CHLOROPHYLLASE ACTIVITY IN OLIVE FRUIT (OLEA-EUROPAEA)

Chlorophyllase activity in olive fruit (Olea europaea) was measured us ing the enzyme solubilized from the protein precipitate. The reaction was stopped by freezing the mixture at -20 degrees C, to avoid dilutio n of the sample and consequent reduction of the substrate levels to be low the detection limits of the analytical system. Separation of the s ubstrates and products of the enzymatic reaction was performed by reve rse-phase HPLC using a gradient solvent system of water and ion suppre ssor/methanol/acetone. These conditions allowed direct resolution of t he reaction mixture prior to centrifugation, without the need for the transfer of any of the components to other solvents. Olive chlorophyll ase in the crude enzymatic extract showed maximum activity at 50 degre es C and the optimum pH was 8.5 in acetate-phosphate-borate buffer for all substrates used, chlorophylls (a and b) and pheophytins (a and b) . The K-m and V-max values obtained for hydrolysis of these substrates showed that chlorophyllase had a greater a affinity for chlorophyll b , while the highest maximum rate of reaction occurred with pheophytin a. Substrate inhibition was observed with pheophytin b.