PURIFICATION AND PROPERTIES OF POLYPHENOL OXIDASE IN HEAD LETTUCE (LACTUCA-SATIVA)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (-)-epicatechin. The K(m) values for the enzyme, using chlorogenic acid (pH 4.5, 30-degrees-C) and (-)-epicatechin (pH 7.0, 30-degrees-C) as substrate, were 0.67 mM and 0.91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (-)-epicatechin oxidase activities were 4.5 and 7.8, respectively, and both activities were stable in the pH range 6-8 at 5-degrees-C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca2+, Mn2+, Co2+ and Ni2+ for chlorogenic acid oxidase activity was stronger than that for (-)-epicatechin oxidase activity.