Extraction And Purification

Due to extremely low concentrations of PPO and laccase in plants and their instability, preparation and purification of PPO and laccase are not easy. PPO has been isolated from a variety of sources, but pigment contamination and the occurrence of multiple forms have frequently hampered its characterization. Oxidation reactions taking place during the isolation of the enzyme, due to natural substrates within the plant, result in changes in enzyme properties as well as in apparent multiplicity. Such reactions can be partially prevented by isolation under nitrogen gas, by using reducing agents such as ascorbic acid or cysteine, or by using phenol-adsorbing agents, such as poly(vinylpyrrolidone) or poly(ethylene glycol) (37,38). The binding of PPO to membranes in many tissues further complicates its isolation. Solubilization of acetone powder preparations or extracting with detergents also result in modification in structure and properties of the enzyme. To minimize these changes, all extraction steps should be carried out at temperature below 0°C, preferably at — 20 to — 30°C. Communition and homogenization are often carried out in liquid nitrogen or under a nitrogen atmosphere (39). Acetone precipitation followed by buffer extraction is one of the methods most often applied (16).

For the purification of the enzyme, several methods have been used that vary according to the enzyme source and the degree of purity to be attained. Most often, precipitation with ammonium sulfate of different saturation, gel chromatography on Sephadex columns, ion exchange chromatography, DEAE-cellulose or DEAE-Sephadex are applied. Recently, hydrophobic gel chromatography using Phenyl-Sepharose CL-4B has been used effectively (40,41). The separation of enzymes by electrophoresis is now a standard procedure. Sodium dodecylsulfate (SDS) techniques and electroblotting of catechol oxidase also have been successfully used (42,43).

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