Purification of tea leaf (Camellia sinensis) polyphenol oxidase by using affinity chromatography and investigation of its kinetic properties

Polyphenol oxidase (PPO) was purified from tea leaves (Camellia sinensis) via affinity chromatography for the first time and the purified enzyme was characterized. The purity of enzyme and molecular weight was determined by SDS-PAGE and non-denaturing PAGE (native PAGE). Single bands were observed with both electrophoretically methods. The PAGE results indicated that the molecular weight of PPO from tea leaf was approximately 50 kDa. The pH, temperature, and kinetic parameters were studied. K-m values were 3.782 and 3.881 mM for catechol and 4-methylcatechol respectively. V-max values for catechol and 4-methylcatechol were also determined as 1.676 and 1.912 mu mol/L min respectively. Additionally, the inhibition effects of sodium metabisulfite, sodium sulfite, ascorbic acid, glutathione, dithioerythritol were investigated on the enzyme activity and IC50, K-i values were calculated for these substances.