EXTRACTION, PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM WALNUT (Tetracapidium conophorum)

Abstract

Polyphenol oxidase (PPO) is an enzyme that uses molecular oxygen to oxidize wide range of phenols to yield quinone products capable of auto-polymerization to form melanin pigments. In this study, PPO was isolated and purified from walnut (Tetracarpidium conophorum) by combination of ammonium sulphate precipitation, DEAE Sephacel and Sephacryl S-200 column chromatography. Biochemical properties such as molecular weight, temperature and pH effect on enzyme activity and stability were determined. Effects of metal ions, activators and inhibitors were also determined on the PPO activity. Three PPO isoforms were purified from walnut (Tetracarpidium conophorum). The three isoenzymes have the same optimum pH of 8.0 and optimum temperature of 60 oC. All the PPO isoenzymes retained more than 60% of their initial activity after 6 hours incubation with buffers 4.0 and 8.0. The molecular weights obtained for Walnut PPO are 59.05 and 63.82 kDa respectively. The PPO isoenzymes activity were effectively activated in presence of SDS, Cu2+, Zn2+, Fe2+, Mg2+ and Mn2+ ion at all concentrations investigated while urea, EDTA, Na+, Ca+, K+ ion and ascorbic acid inhibit the PPO isoenzymes activity. The Km values obtained in this study for the three PPO isoenzyme purified from Walnut are 13.58 mM, 7.10 mM and 8.12 mM whereas their Vmax values are 25.25 U/ml, 13.35 U/ml and 4.58 U/ml respectively. The substrate specificity studies revealed that walnut PPO has equal monophenolase and diphenolase activities.