SUMMARY: Two different bands with laccase activity were obtained after nondenaturing PAGE of the culture filtrate of 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). 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 | = 2-197 and | = 0-009 cm, and | = 2-263 and | = 0-0176 cm, respectively. The data obtained from the pH profiles suggested that two ionization groups, whose pvalues 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 °C, respectively. The enzyme had affinity for various lignin-related phenolic compounds: the values for ferulic acid and syringic acid were 48 and 89 μM, 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.


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