Characterization of a Coprinus cinereus laccase

A wild-type Coprinus cinereus laccase and its recombinant form expressed in an Aspergillus oryzae host have been purified and characterized. The mature laccase had a molecular mass of 58 kDa by mass spectrometry, an isoelectric point near 4, and two absorption maxima at 278 and 614 nm. Photometric titration with 2,2'-biquinoline showed a Cu/protein(subunit) stoichiometry of approximate to 4. The electron paramagnetic resonance spectrum showed typical type 1 and type 2 Cu signals, and the circular dichroism showed a typical coordination geometry of the type 1 Cu(II). At pH 5.5, the enzyme had a redox potential of 0.55 V vs. normal hydrogen electrode at its type 1 site. The laccase could oxidize 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) and syringaldazine with optimum pH of 4 and 6.5, respectively. Halides inhibited the laccase. At pH 8.5, the laccase had an optimum temperature between 60 degrees C and 70 degrees C. At the same pH, the laccase had a half-life of >200 or 21.8 min in the presence of 0 or 2 mM H2O2, respectively, at 40 degrees C. Mediated by several phenols and phenothiazines, the laccase was able to oxidatively bleach Direct Blue 1 dye at alkaline pH, making it a promising industrial enzyme candidate. (C) 1999 Elsevier Science Inc. All rights reserved.