Purification and characterization of polyphenol oxidase enzyme and investigation of immobilization with different clays and their inhibition effects using local apple variety Arap Kizi

The Polyphenol oxidase enzyme was purified from Arap kızı apple (Malus domestica), a local apple cultivar, grown in Isparta province. The PPO enzyme was extracted in phosphate buffer and purified by the sequential use of ammonium sulfate precipitation, dialysis, and Sepharose 4-B-L-tyrosine-p-aminobenzoic acid affinity gel. The enzyme molecular weight was estimated by SDS-PAGE. To determine the optimum pH and temperature values, the activities were measured using three different substrates at different pH (4.5-9.0) and temperature ranges (10-45˚C). KM and Vmax values of PPO enzyme against different substrates were determined by the Linewear-Burk method as 50 mM and 2500 U/mLmin for catechol substrate; 2 mM and 2000 U/mLmin for 4-methyl catechol substrate; 6 mM and 10000 U/mLmin for pyrogallol substrate. Pyrogallol substrate was found to be the most suitable substrate for the PPO enzyme. Optimum pH was 8.5 and optimum temperature was 10˚C for the pyrogallol substrate. Purified PPO enzyme was immobilized on to three different clays (red, green and white). Inhibition types, IC50 and Ki values for immobilized PPO and free enzyme, which was added during the measurements, were determined. The most effective inhibitor for both the immobilized and the free enzyme was white clay.