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

SIMPLE SUMMARY: Opuntia ficus-indica (L.) Mill. (OFI) is a member of the Cactaceae family. Its fruits are available in a wide range of colors, including orange, lime green, purple, and red. In many countries, the cacti are cultivated and used as foods and medicinal plants. They can grow in dry and semi-arid environments and have substantial economic potential. In the current study, for the first time, polyphenol oxidase (PPO) was purified from the fruits of O. ficus-indica, whose color was orange, through utilizing the Sepharose 4B–L-tyrosine–p-aminobenzoic acid column, and the purified enzyme was characterized. ABSTRACT: 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.