Development of an alternative method for purification and kinetic properties of polyphenol oxidase from some apple cultivars.

In this study, an alternative method to purify the polyphenol oxidase (PPO) was developed. For this purpose, a novel affinity gel was synthesized by first Sepharose-4B being activated by cyanogen bromide (CNBr), then L-tyrosine (as a spacer arm) was coupled to CNBr-activated Sepharose-4B, and then o-aminobenzoic acid, as a ligand for purification of the PPO, was coupled to L-tyrosine. PPO enzymes were purified from Starking Delicious, Granny Smith, and Golden Delicious apple cultivars grown in the Eğirdir district of Isparta/Türkiye by utilizing both the Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity gel and the novel affinity gel. 101.59, 22.69, and 12.81 purification folds were achieved from Starking Delicious, Granny Smith, and Golden Delicious cultivars, respectively, by using the novel gel. 128.95, 25.60, and 45.92 purification folds were achieved in the Starking Delicious, Granny Smith, and Golden Delicious cultivars, respectively, by utilizing Sepharose 4B-L-tyrosine-p-aminobenzoic acid. The Km, Vmax, and Vmax/Km values were calculated to be 20 mM, 25,000 U/mLmin, and 1,250 U/mLminmM; 40 mM, 20,000 U/mLmin, and 500 U/mLminmM; and 50 mM, 5,000 U/mLmin, and 100 U/mLminmM for the purified PPO enzymes of the Starking Delicious, Granny Smith, and Golden Delicious cultivars, respectively, by using catechol as substrate by the Lineweaver-Burk method. This study investigated the effect of para and ortho substituent positioning on ligand performance in affinity chromatography purification. The findings show that a novel affinity gel using an o-aminobenzoic acid ligand is effective for PPO purification, highlights the importance of ligand structure in affinity chromatography, and contributes significantly to enzyme purification.