A novel white laccase from Pleurotus ostreatus

Two laccase isoenzymes (POXA1 and POXA2) producedby Pleurotus ostreatus were purified and fullycharacterized. POXA1 and POXA2 are monomeric glycoproteinswith 3 and 9% carbohydrate content, molecularmasses of about 61 and 67 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, of about 54 and 59kDa by gel filtration in native conditions, and of 61 kDaby matrix-assisted laser desorption ionization massspectrometry (only for POXA1) and pI values of 6.7 and4.0, respectively. The N terminus and three tryptic peptidesof POXA1 have been sequenced, revealing clearhomology with laccases from other microorganisms,whereas POXA2 showed a blocked N terminus. The stabilityof POXA2 as a function of temperature was particularlylow, whereas POXA1 showed remarkable highstability with respect to both pH and temperature.Both enzymes oxidize syringaldazine and ABTS (2,2*-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) togetherwith a variety of different substituted phenolsand aromatic amines with the concomitant reduction ofoxygen, but POXA1 is unable to oxidize guaiacol. Bothenzymes were strongly inhibited by sodium azide andthioglycolic acid but not by EDTA.UV/visible absorption spectra, atomic adsorption, andpolarographic data indicated the presence of 4 copperatoms/mol of POXA2 but only one copper, two zinc, andone iron atoms were found/mol of POXA1.The neutral pI and the anomalous metal content ofPOXA1 laccase render this enzyme unique in its structuralcharacteristics. The lack of typical absorbance at600 nm allows its classification as a "white" laccase.