Purification and Characterization of Polyphenol Oxidase in the Fruits of Opuntia ficus-indica.

Firstly, polyphenol oxidase (PPO) was purified from the fruits of Opuntia ficus-indica using Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity chromatography, and the enzyme was characterized. The PPO was purified 20.59-fold. Thereafter, PPO was performed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The kinetic parameters, optimum pHs, and optimum temperatures were investigated for three substrates. Opuntia ficus-indica PPO's optimum pH and optimum temperature were 9.0 and 20 °C; 7.5 and 20 °C; and 7.5 and 30 °C, respectively, when using pyrogallol, catechol, and 4-methyl catechol as substrates. For the pyrogallol, catechol, and 4-methyl catechol, the Km, Vmax, and Vmax/Km values were determined as 16.67 mM, 833.33 U/mLmin, and 50 U/mLminmM; 6.33 mM, 126.58 U/mLmin, and 20 U/mLminmM; and 5.38 mM, 107.53 U/mLmin, and 20 U/mLminmM, respectively. As a result, pyrogallol was a more appropriate substrate than catechol and 4-methyl catechol for the PPO from Opuntia ficus-indica.

Purification and Properties of Polyphenol Oxidase of Dried Volvariella bombycina.

Polyphenol oxidase (PPO) was purified and characterized from a dried wild edible and medicinal mushroom (V. bombycina). Using Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity chromatography, PPO was purified from the dried V. bombycina. The purification was completed with a 33.85-fold purification. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the purified enzyme migrated as a single band. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be about 25 kDa. Catechol, 4-methyl catechol, and pyrogallol were used as substrates to determine the enzyme activity and its kinetic parameters (Km and Vmax). At the optimum pH and temperature, dried V. bombycina PPO's Km and Vmax values for catechol, 4-methyl catechol, and pyrogallol were found to be 1.67 mM-833.33 U/mL, 3.17 mM-158.73 U/mL, and 2.67 mM-3333.33 U/mL, respectively. Also investigated were the effects of pH and temperature on the enzymatic properties of PPO in dried V. bombycina. The optimum pH and temperature values for dried V. bombycina PPO obtained by using catechol, 4-methyl catechol, and pyrogallol as substrates were 6.5, 15 °C; 9.0, 20 °C; and 8.0, 15°C, respectively. This is the first study on the purification and characterization of PPO from dried V. bombycina.