PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF POLYPHENOL OXIDASE FROM SEEDS OF MELON (Citrullus colocynthis)

Abstract

Polyphenol oxidases (PPOs) are enzymes that catalyze the oxidation of a wide range of phenolic compounds by utilizing molecular oxygen. Purification and biochemical characterization of polyphenol oxidase from Citrullus colocynthis, a typical variety of melon was achieved. Polyphenol oxidase was extracted and purified using a procedure that consisted of ammonium sulphate precipitation, ion exchange chromatography on DEAE Sephadex A-50 and gel filtration chromatography on Sephadex G-200. The effects of pH and temperature on the enzyme activity and stability were investigated, effect of metal ions and other chemical agent, substrate specificity, kinetic parameters and molecular weight were determined. The existence of polyphenol oxidase was supported with the appearance of a single dark band on SDS PAGE which indicates that the enzyme was homogenous with the molecular weight of about 42kDa. The yield of enzyme was about 26.3% while the purification fold was 126. The optimum pH and temperature activity were found to be 7.0 and 60ºC respectively and more than 80% of activity was retained at pH 7.0 after six hours incubation period while about 79% and 36% of its initial activity were retained after 10 minutes of incubation time at 70oC and 80oC temperature conditions respectively. The enzyme activity was inhibited by low concentration of Sodium dodecyl sulphate and enhanced in the presence of Cu2+. The Michealis- Menten constant ( Km) of the enzyme using catechol, gallic acid, L- DOPA and tyrosine as substrates were 5.04 mM, 6.38 mM, 7.48 mM and 7.89 mM respectively. The activity of polyphenol oxidase may be the underline causes of the often observed changes in the organoleptic properties of melon (egusi) soup prepared using mechanically dehulled melon seeds.