Purification of polyphenol oxidase enzym from tortum apple (Malus sylvestris.) by affinity chromatography, characterization and investigate the effect of some chemicals on the enzym activity

Polyphenol oxidase enzyme causes the enzymatic browning in some fruits and vegetables. In this study, the PFO enzyme was purified for the first time using the Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column from Tortum apple. The purity of the enzyme was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimum pH, optimum temperature, optimum ionic strength, stable pH, KM and Vmax values of PPO enzyme were determined using catechol and 4-metil catechol substrates. The optimum pH and temperature of PPO were determined as 5,0-5.5, 10°C. Optimum ionic strength of this enzyme was found as 0,05 M for both substrates. KM constants and Vmax values of enzyme for the used two substrates were found as 6,17-4,16 mM and 3,38-7,75 µmol/dk, respectively. Ascorbic acid, sodium azide, thiourea, GSH and DTT were studied in inhibitor studies. IC50 values were determined in five different inhibitor concentrations for these inhibitors. Ki constant and the types of inhibition of these inhibitors were determined by using Lineweaver-Burk graphics. Ascorbic acid was appointed to be the most effective inhibitor according to the Ki constants and values of IC50.