Purification, characterization of polyphenol oxidase from Lactarius piperatus and investigation of its catalytic efficiency on synthetic chemistry

In this study, a polyphenol oxidase (PPO) was purified from a wild edible mushroom, Lactarius piperatus, by using a Sepharose-4-B-L-tyrosine-p-aminobenzoic acid affinity column and characterized. The purified enzyme migrated as a single band on native- and SDS- polyacrylamide gel electrophoresis. The optimum pH and optimum temperature of the enzyme were found to be 7.0 and 20 °C, respectively, in the presence of catechol as a substrate. After 24 h of incubation at 4 °C, the L. piperatus PPO was extremely stable in the range of pH 3.0-9.0. The enzyme retained over 60% of its original activity in the range of pH 3.0-9.0 after 72 h incubation at 4 °C. When the thermal stability profile of the purified enzyme was analyzed, it was determined that the enzyme was highly stable in the range of 0-50 °C after 4 h incubation. The Km and Vmax values of L. piperatus PPO were calculated as 1 mM and 25000 U/mg protein, respectively, from the Lineweaver-Burk plot. According to I50 data, in the presence of some PPO inhibitors like sodium metabisulfite, ascorbic acid, sodium azide and benzoic acid, L. piperatus PPO was all inhibited, especially by sodium metabisulfite and ascorbic acid. In the experiments done with metal ions, it is established that the enzyme activity was very sensitive to metal ions.It was investigated that L. piperatus PPO is an effectively biocatalysis using catechin as substrate in the organic solvents of heptan, toluene and dichloromethane.All the verities support the presence of an active diphenolase in L. piperatus having similar properties to plant polyphenoloxidases.