Purification of polyphenoloxidase from coffee fruits

Polyphenoloxidase (PPO) was purified from coffee beans at the pinhead stage. Purification was carried out by precipitation of contaminating proteins with 30% (NH4)2SO4, dialysis of the supernatant and chromatography on phenyl sepharose and DEAE columns. A purification factor of 364 was obtained. Two bands (29 and 64 kDa) of the purified PPO were detected after SDS-PAGE which presented activity in partially denaturing SDS-PAGE and gave a positive response when probed in Western blots with an apple 27 kDa PPO antibody. It is suggested that the 29 kDa band was a cleavage product of the 64 kDa PPO. The N-terminal sequencing of the 29 kDa protein did not show similarity with others PPOs. Other reports have shown that the 64 kDa is a pre-protein which is converted to a 45 kDa mature form by action of a chloroplastid protease. The 45 kDa was not detected in pinhead fruits but usually detected in the leaves and endosperm of coffee. It is suggested that the appearence of the 45 kDa form might be controlled by the developmental stage of the fruit. Chlorogenic acid was the preferred substrate for the purified PPO.