LACCASE ISOENZYMES OF PLEUROTUS-ERYNGII - CHARACTERIZATION, CATALYTICPROPERTIES, AND PARTICIPATION IN ACTIVATION OF MOLECULAR-OXYGEN AND MN2+ OXIDATION

Two laccase isoenzymes produced by Pleurotus eryngii were purified to electrophoretic homogeneity (42- and 43-fold) with an overall yield of 56.3%. Laccases I and II from this fungus are monomeric glycoproteins with 7 and 1% carbohydrate content, molecular masses (by sodium dodec yl sulfate-polyacrylamide gel electrophoresis) of 65 and 61 kDa, and p Is of 4.1 and 4.2, respectively. The highest rate of 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonate) oxidation for laccase I was reached at 65 degrees C and pH 4, and that for laccase II was reached at 55 de grees C and pH 3.5. Both isoenzymes are stable at high pH, retaining 6 0 to 70% activity after 24 h from pH 8 to 12. Their amino acid composi tions and N-terminal sequences were determined, the latter strongly di ffering from those of laccases of other basidiomycetes. Antibodies aga inst laccase I reacted,vith laccase Ii, as well as with laccases from Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus floridanus. Different hydroxy- and methoxy-substituted phenols and aromatic amines were oxidized by the two Laccase isoenzymes from P. eryngii, and the influence of the nature, number, and disposition of aromatic-ring subs tituents on kinetic constants is discussed. Although both isoenzymes p resented similar substrate affinities, the maximum rates of reactions catalyzed by laccase I were higher than those of laccase II. In reacti ons with hydroquinones, semiquinones produced by laccase isoenzymes we re in part converted into quinones via autoxidation, The superoxide an ion radical produced in the latter reaction dismutated, producing hydr ogen peroxide. In the presence of manganous ion, the superoxide anion was reduced to hydrogen peroxide with the concomitant production of ma nganic ion. These results confirmed that laccase in the presence of hy droquinones can participate in the production of both reduced oxygen s pecies and manganic ions.