Production , Purification and Properties of Extracellular Laccase of Agaricus bisporus

Extracellular laccase (EC 1 .14.18.1) of the cultivated mushroom Agaricus bisporus was produced constitutively in defined or complex media. No enzyme induction was found after treatment with cycloheximide or with other potential inducers such as toluidine or xylidine. The enzyme was purified to homogeneity by ammonium sulphate precipitation, ion-exchange chromatography, gel filtration and affinity chromatography. It eluted as a single peak from ion-exchange, gel filtration and affinity columns and sedimented as a single band on centrifugation. It showed four enzymically active bands on electrophoresis and a diffuse band on isoelectric focusing. Its molecular weight was estimated to be about 100000 and the enzyme contained 15% carbohydrate and two atoms of copper per molecule. The substrate specificity was similar to that of other fungal laccases. Antiserum prepared against the purified enzyme gave one major precipitin band. Production of extracellular laccase (EC 1 .14.18.1) is a common feature of many higher basidiomycete fungi, particularly those associated with wood decay or the terminal stages of decomposition of leaf litter The production, regulation and properties of fungal laccases have been examined in a number of species. In cultures of Polyporus versicolor extracellular laccase can be induced above basal levels by treatment of cultures with the aromatic amines toluidine or xylidine (Fahraeus et al., 1958). Similar inducing effects are found with the extracellular laccase of Neurospora crassa and in this organism increased laccase production is also induced by treatment with cycloheximide or actinomycin D (Froehner & Eriksson, 1974 b). The laccase of Aspergiltus nidulans appears during conidiation and is responsible for a step in the synthesis of conidium pigment (Clutterbuck, 1972). In the basidiomycete Schizo-phyllum commune laccase activity is positively correlated with the ability of monokaryons to fruit (Leonard, 1971) and with the stage of fruiting development in dikaryotic cultures (Leonard & Philips, 1973). The purified fungal laccases are normally blue, copper-containing, proteins capable of oxidizing 0-and p-phenols and aromatic amines and differ from tyrosinases on the basis of their substrate specificity (Hewitt & Smith, 1975). Differences have been reported in many of the properties of the various fungal laccases