Partial purification and characterisation of polyphenol oxidase from faba bean (Vicia faba) coat

Introduction.The aim of this study is to isolate polyphenol oxidase (PPO) enzyme from faba bean coat, and to determine its optimum pH and temperature, Km and Vmax parameters, to investigate biochemical properties such as pH and temperature stability, substrate selectivity, storage stability of enzyme, and also effects of some metals and inhibitors on its activity. Materials and methods.The protein precipitation was done by using ammonium sulfate salt and then, dialysis was performed to remove salt ions after precipitation.Protein content of the samples at each step was determined according to Bradford method, and enzymatic activity was determined spectrophotometrically.Results and discussion.The properties of PPO enzyme, which was purified 3.1-fold, were determined using catechol as substrate.Km and Vmax values of the enzyme were determined to be 5.53 mM and 4424,58 U.ml -1 min -1 , respectively.The enzyme showed the highest activity at pH 5.0 and 10 ºC.It saved about 60% of its activity after a 30 min incubation period at its optimum pH.Substrate specificity of PPO was studied using eight substrates including pyrogallol and 4-methylcatechol.When the thermal stability profile was examined in the 10-60 ºC temperature range, it exhibited more than 50% of its activity in the range of 10-40 ºC after 1 hour of incubation.While L-ascorbic acid, sodium sulfite and L-cysteine showed strong inhibitory effects, the studied metals showed variable effects on the enzyme activity.Conclusions.The polyphenol oxidase enzyme activity causes undesirable changes and nutritional value loss in some fruits and vegetables.The results obtained provide information about faba bean PPO and give clues to the effective methods for controlling browning during storage.