Single electron transfer by an extracellular laccase from the white-rot fungus Pleurotus ostreatus

Abstract

Two different bands with laccase activity were obtained after nondenaturing PAGE of the culture filtrate of Pleurotus ostreatus. Immunoblot analysis revealed that antisera raised against laccase I were not reactive to laccase II. Laccase I, which exhibited faster mobility on nondenaturing polyacrylamide gel, was purified 42.9-fold with an overall yield of 10.8%. Gel filtration and SDS-PAGE revealed that laccase I is a single polypeptide with a molecular mass of approximately 64 kDa. Laccase I contained 12.5% carbohydrate by weight and 3.9 mol copper (mol protein)-1. The absorption spectrum of laccase I showed a type 1 signal at 605 nm and EPR spectra showed that the parameters of the type 1 and type 2 Cu signals were g parallel = 2.197 and A parallel = 0.009 cm-1, and g parallel = 2.263 and A parallel = 0.0176 cm-1, respectively. The data obtained from the pH profiles suggested that two ionization groups, whose pKa values were 5.60-5.70 and 6.70-6.85, may play an important role in the active site of laccase I as the ligand of copper metal. The optimal pH and temperature for the activity of laccase I were 6.0-6.5 and 30-35 degrees C, respectively. The enzyme had affinity for various lignin-related phenolic compounds: the Km values for ferulic acid and syringic acid were 48 and 89 microM, respectively. EPR spectroscopic study of the action of laccase I on 3,5-dimethoxy-5-hydroxyacetophenone indicated that this enzyme catalyses single electron transfer with the formation of the phenoxy radical as an intermediate.