Purification of polyphenol oxidase from cancur plum (Prunus domestica L.) and investigation of plum' s antoxidant properties

Polyphenol oxidases (PPO) are copper-containing metalloenzymes that catalyze the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to o-quinones in the presence of oxygen. In this study, PFO was purified from Cancur plums (Prunus domestica L.), collected from Türkgözü village in Posof, Ardahan, Turkey, using Sepharose-4B-L-tyrosine-p-aminobenzoic acid affinity gel. The PPO was characterized with natural and SDS-polyacrylamide gel electrophoresis, and in terms of biochemical properties. It was determined to have two isoenzymes by electrophoresis chromatogram on Cancur plum PPO. It was detected that the enzyme showed the highest activity at between 30-40 °C and pH 5.0-7.0 in the presence of L-tyrosine, 3,4-dihydroxyhydrocinnamic acid, 3,4-propanoic acid, p-coumaric acid, catechol and 4-methyl catechol as substrates. Each substrate of cancur plum PPO was found to comply with Michaelis-Menten kinetics. Kinetic data as a Michaelis-Menten constant (Km) and a maximum velocity (Vmax) calculated via the drawn-Lineaweaver Burk plot. Cancur PPO was found to have both monophenolase and diphenolase activity. It was determined that catechol is most interested in as substrate and followed by 4-methylcatechol, DHHSA, L-tyrosine, DHPPA and p-coumaric acid as substrates, respectively. The PPO was inhibited by ascorbic acid, sodium metabisulfite, benzoic acid and sodium azide in the presence of 4-methyl catechol as a substrate. The Ki values for these inhibitors were found as 0.02, 0.12, 6.0 and 18.3 mM, respectively. The total amount of phenol in aqueous extracts of Cancur plum was determined as 114.6 ± 5.0 mg GAE / 100 g. The antioxidant activity of the extract was found as 30.19 ± 0.96 (mg TEAC / g) with CUPRAC method, as1.98 ± 0.02 (mg / mL) wih DPPH method and as 2.17 ± 0.01 (mg / mL) with ABTS method.