Purification, characterization of polyphenol oxidase enzyme from posof badele apple (Malus domestica L.) and investigation of effects of some chemicals and metal ions on enzyme activity

In the current study, performed with Posof Badele apple (Malus domestica L.), polyphenol oxidase (PBPPO) enzyme was partially purified by acetone precipitation method, then purified by affinity chromatography and kinetic properties of enzyme were investigated. When SDS-PAGE was done with pure enzyme samples, PPO was seen as a single band and purity of enzyme was proved. Spesific substrat of PPO was determined by using L-Tyrosine, 4-methylcatechol (4-MK) and catechol substrates. The optimum pH of the polyphenol oxidase with 4-MK substrate was found as 5.5. KM and Vmax values for catechol were determined as 33.3 mM and 5.86 EU/mLmin, for 4-MK substrate these values were determined as 8.53 mM and 3.54 EU/mLmin respectively. For optimum temperature, pH stability, thermal stability, and inhibition studies enzyme activities were examined with 4-methyl catechol substrate. Stable pH of the enzyme and optimum temperature were found as 6.0 and 10°C respectively. When thermal stability of PPO was examined, it was found that after incubation for 1 hour at 15 minute intervals, it was stable at 0-10-20-30°C. Besides, effects of some metal ions (Mg2+, Al3+, K+, Cu2+ and Na+) and chemicals on pure enzyme activity were investigated. Al3+ and Cu2+ metal ions were found to activate the enzyme. Ki and I50 values were calculated and inhibition types were determined. It was found that the strongest inhibitory effect on the PPO from Posof Badele apple (Malus domestica L.) was made by ascorbic acid.