Purification of polyphenol oxidase enzyme from Iğdır city's grape kırmızı kişmiş (Vitis Vinifera L.) by affinity chromatography, characterization and inhibition

In this study, the PPO enzyme from the Kirmizi Kismis (Vitis vinifera L.), a historical grape variety, was purified by affinity chromatography using the cold acetone precipitation followed by Sepharose-4B-L-tyrosine-p-aminobenzoic acid affinity column and the kinetic properties of the enzyme were investigated.The purified enzyme was appeared as a single band on native- and SDS- polyacrylamide gel electrophoresis. Optimum PPO activity was determined using 4-methyl catechol, catechol, L-Tyrosine substrates.The optimum pH of the enzyme was found to be 5,0 in the presence of 4-methyl catechol substrate.The Michaelis-Menten constant (Km) and the maximum reaction velocity (Vmax) were calculated for 4-methylcatechol, catechol substrates.Menten constant (Km) and maximum velocity (Vmax) values were determined as 7,25 mM and 2500 U/mL.dak protein for 4-methyl catechol and 24,21 mM and 2000 U/mL.dak protein for catechol.PFO enzyme pH stability, optimum temperature and thermal stability, enzyme activity on the properties of some metals and inhibitors such as the effect of 4-methylcatechol substrate was examined in the presence.The optimum temperature was found as 30 °C and the stable pH value was found as 7,0.When the thermal stability profile of the enzyme was analyzed, it was very stable in the range of 10-40 °C, it was observed that after 1 hour incubation. I50 and Ki values and inhibition types were determined for four different food additive and ascorbic acid showed the strongest inhibitory effect on the Vitis vinifera L. PPO activity. The effect of Na+ , K+ , Mg2+, Cu2+ and Al3+ ions on PFO activity at final concentrations of 1 and 10 mM was investigated.