Summary auto-generated
This study describes the production, purification, and characterization of extracellular laccase from the cultivated mushroom Agaricus bisporus. The enzyme was produced constitutively in both defined and complex media, with no induction observed following treatment with cycloheximide, toluidine, or xylidine—compounds that induce laccase in other fungi. Laccase was purified to homogeneity through a five-step purification scheme involving ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and affinity chromatography. The purified enzyme had a molecular weight of approximately 100,000 Da, contained 15% carbohydrate and two copper atoms per molecule, and showed four enzymatically active bands on electrophoresis. The enzyme exhibited substrate specificity typical of fungal laccases, oxidizing both ortho- and para-phenols and aromatic amines. A single major precipitin band formed when the enzyme was tested against antiserum, indicating immunological homogeneity. The research provided biochemical characterization data for comparison with other fungal laccases and generated purified enzyme and antibodies for further studies on laccase activity during mushroom fruiting.
Key findings
- Laccase from A. bisporus is produced constitutively and cannot be induced by compounds (toluidine, xylidine, cycloheximide) that effectively induce the enzyme in other fungi.
- The purified enzyme is a copper-containing glycoprotein with molecular weight ~100,000 Da, containing 2 copper atoms and 15% carbohydrate.
- Electrophoresis revealed four enzymatically active bands, yet the enzyme eluted as a single peak from multiple chromatography columns and sedimented as a single species, suggesting post-translational modifications.
- The enzyme exhibits typical laccase substrate specificity, oxidizing ortho- and para-phenols and aromatic amines but not diphenols, and is inhibited by copper-binding agents (azide, cyanide) and detergents.
- Immunological analysis showed a single major precipitin band, confirming the homogeneity of the purified enzyme preparation.
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Abstract
SUMMARY: 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.