Purification and Characterization of Polyphenol Oxidase Enzyme from Damson Plum (Prunus insititia) by Spectrophotometrically

Abstract

Polyphenol oxidase enzyme, performing browning reactions in fruits and vegetables, was purificated from damson plum (Prunus insititia) which has a high antioxidant activity. Firstly, partially purified polyphenol oxidase was treated by 0-80% ammonium sulfate precipitation and dialysis, respectively. Characterization studies were carried out by using catechol, 4-methyl catechol, pyrogallol and caffeic acid as 0.05M/ pH:7.2/ 25°C; 0.2M/ pH:4.5/ 10°C; 0.01M/ pH:6.8/ 5°C and 0.2M/ pH:8.5/ 10°C, respectively. The kinetic constants of Vmax and KM were calculated for the same substrates as 17219.97 U/(mL*min) and 11.67mM; 7309.72 U/(mL*min) and 5mM; 12580.12 U/(mL*min) and 3.74mM; 12100.41 U/(mL*min) and 6.25 mM, respectively. Catechol gave the highest Vmax value when compared to others. In the second step, purification was performed by using Sepharose 4B-L-Tyrosine-p-amino benzoic acid and Sepharose 6B-L-Tyrosine-p-amino benzoic acid affinity gels. A single band of approximately as 50-55 kDa was observed in SDS-PAGE and Native-PAGE. 90 and 10.2 purification folds were obtained for Prunus insititia PPO by the reference Sepharose-4B-L-Tyrosine-p-aminobenzoic acid and original Sepharose-6B-L-Tyrosine-p-aminobenzoic acid gels, respectively. PPO enzyme purification from Prunus insititia by affinity chromatography has not been investigated in literature yet.