Characterization of polyphenol oxidase in coffee

Polyphenol oxidase (PPO) was characterized in partially purified extracts o f leaves (PPO-L) and fruit endosperm (PPO-E) of coffee (Coffea arabica L.). PPO activity was higher in early developmental stages of both leaves and e ndosperm of fruits. Wounding or exposure of coffee leaves to methyl jasmona te increased PPO activity 1.5-4-fold. PPO was not latent and was not activa ted by protease treatment. PPO activity was stimulated 10-15% with sodium d odecyl sulphate (SDS) at 0.35-1.75 mM, but at higher concentrations activit ies were similar to the control samples, without detergent. Prolonged incub ation of extracts with trypsin or proteinase K inhibited PPO activity but p epsin had no effect. Inhibition of PPO with proteinase K was increased in t he presence of SDS. PPO activity from both tissues was optimal at pH 6-7 an d at an assay temperature of 30 degreesC. Activity was highest with chlorog enic acid as substrate with a K-m of 0.882 mM (PPO-L) and 2.27 mM (PPO-E). Hexadecyl trimethyl-ammonium bromide, polyvinylpyrrolidone 40, cinnamic aci d and salicylhydroxamic acid inhibited PPO from both tissues. Both enzymes were inactivated by heat but the activity in endosperm extracts was more he at labile than that from leaves. The apparent M-r, determined by gel filtra tion was 46 (PPO-L) and 50 kDa(PPO-E). Activity-stained SDS-polyacrylamide gel electrophoresis (PAGE) gels and western blots probed with PPO antibodie s suggested the existence of a 67 kDa PPO which is susceptible to proteolyt ic cleavage that generates a 45 kDa active form. (C) 2000 Elsevier Science Ltd. All-rights reserved.