Characterization of polyphenoloxidase from wild pear (Pyrus elaegrifolia)

Abstract

Wild pear polyphenoloxidase (PePPO) was extracted and purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. Optimum conditions for pH, temperature and heat inactivation were determined. At the optimum pH and temperature, K-M and V-max values for PePPO with catechol and pyrogallol were determined. The V-max/K-M showed that PePPO has the greatest activity toward catechol. Optimum pH for PePPO was pH 6.0 using catechol as substrate. Optimum temperatures of PePPO for pyragallol and catechol were 65 and 35C, respectively. Enzyme activity decreased because of heat denaturation with increasing temperature. Inhibition of PePPO was investigated using p-aminobenzoic acid, ethyleneglycol, L-cysteine, L-tyrosine, sodium azide, p-aminobenzenesulfonamide, beta-mercaptoethanol and dithiothreitol and catechol as substrate. Competitive-type inhibition was obtained with ethyleneglycol, L-cysteine, L-tyrosine, p-aminobenzenesulfonamide and dithiothreitol. Uncompetitive inhibition was obtained with beta-mercaptoethanol, sodium azide and p-aminobenzoic acid. These results show that the most effective inhibitor for PePPO was dithiothreitol and that the type of inhibition depended on the origin of PPO.