Isolation and Partial Purification of Polyphenol Oxidase from Seed of Melon (Cucumeropsis edulis)

Abstract

Polyphenol oxidase (PPO) from Cucumeropsis edulis was extracted and partially purified through (NH4)2SO4 precipitation, dialysis, and ion-exchange chromatography on DEAE-Sephadex-A50. The spectrophotometric method was used to assay the enzyme activity in C. edulis using L-DOPA as substrate, the physicochemical properties such as the effect of pH and temperature, substrate specificity, kinetic constants - maximum enzyme velocity (Vmax), and Michaelis - Menten constant (Km) for three substrates namely, L-Dopa catechol and tyrosine were determined. The effects of inhibitors and metal ions on PPO activity were also investigated. The optimum pH and temperature values were found to be pH 6.5 and 50 °C, and the inhibitory effects of inhibitors such as ascorbic acid, EDTA, SDS, and metal ions were enhanced positively with increased concentration except with divalent metals such as Cu2+, Fe2+, and Zn2+ reflecting an activating effect on C. edulis PPO. Moreover, the enzyme solution showed both monophenolase and diphenolase activity with L-DOPA having the highest Vmax/Km value. However, the data obtained in this research provided a theoretical basis for the prevention of enzymatic browning of C. edulis during processing.