| Popis: |
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. |
