Purification and characterization of polyphenol oxidase from Henry chestnuts (Castanea henryi)

Abstract

Polyphenol oxidase from Castanea henryi nuts was partially purified. Some characteristics of the enzyme were then determined to help devise measures for the prevention of undesirable enzymatic browning during storage and processing of the nuts. Preparation of acetone powder extracts of the nuts, ammonium sulfate precipitation, dialysis, and gel filtration resulted in 37-fold purification of the enzyme with a yield of 13%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the excitation spectrum confirmed the enzyme preparation to be homogeneous. The approximate molecular weight of the enzyme was determined by gel filtration to be 69 kD. The enzyme catalyzed the oxidation of catechol and pyrogallic acid as substrates but did not affect cresol or tyrosine. Using catechol as substrate, p-nitrophenol, thiourea, orcinol, and naphthol showed strong inhibition. The optimal pH and temperature for the enzyme were pH 5.0 and 40°C, respectively. The enzyme proved heat labile. When the enzyme was incubated at 70°C for 30 min, the remaining activity of the enzyme was only 8%. Possible approaches to applying the results of this study to the prevention of enzymatic browning in the production of C. henryi nuts is also discussed.