Izmir grape polyphenol oxidase (Vitis vinifera L.):: Partial purification and some kinetic properties

Abstract

Polyphenol oxidase (PPO) was extracted from the Izmir grape fruit (Vitis vinifera L.) and its characteristics were studied. The PPO enzyme was purified 2.2-fold by (NH4)(2)SO4 precipitation and 26.1-fold by gel filtration chromatography. This partially purified enzyme obtained from dialysis migrated as one band with catechol substrate, four bands with Coomassie brilliant R-250 (NATIVE-PAGE) native system and as six bands of 34, 30, 22, 18, 16 and 15 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The sample obtained from dialysis after ammonium sulfate precipitation was used for the characterization of partially purified enzyme. Substrate specificity experiments were carried out with catechol, 4-methyl catechol, L-tyrosine, pyrogallol, p-cresol, gallic acid and caffeic acid. Michael-Menten constant and maximum velocity values were 3.65 mM and 1,971.6 Delta A/dak, respectively, with catechol substrate. Optimum pH for the PPO enzyme were determined at 7.2, 7.0, 7.4, 7.2, 6,4, 6.7 and 5.7 using catechol, 4-methyl catechol, L-tyrosine, pyrogallol, p-cresol, gallic acid and caffeic acid substrates, respectively. Optimum temperature for maximum PPO activity was 25C for catechol. Heat inactivation studies showed a significiant decrease in enzymatic activity at temperatures above 45C. Activation energy was calculated to be 12.4 kcal/mol. The most potent inhibitors for the grape PPO were thiourea, sodium diethyldithiocarbamate and sodium azide. Inhibition constants were calculated for L-ascorbic acid, L-cysteine and beta-mercaptoethanol at the optimum pH of PPO activity.