PURIFICATION AND CHARACTERIZATION OF AN ENZYME WITH BOTH BILIRUBIN OXIDASE AND LACCASE ACTIVITIES FROM MYCELIUM OF THE BASIDIOMYCETE <i>Pleurotus ostreatus</i>
An enzyme from mycelium of the white-rot Basidiomycete <i>Pleurotus ostreatus </i>with both bilirubin oxidase and laccase activity was isolated and characterized by biochemical methods. The yield of the purified enzyme was 127 ug/g dry biomass. The enzyme had specific activities of 21 U/mg and 261 U/mg with bilirubin and a laccase substrate ABTC, respectively. LC-MS/MS analysis identified the intracellular oxidoreductase from mycelium of <i>P. ostreatus </i>as bilirubin oxidase (BOX) with very high degree of homology with the <i>pox2 </i>gene product. The enzyme displayed maximal laccase activity at 50-55°C with all substrates examined. The pH optimum, however, was substrate-dependent. Whereas a pH optimum of 3.0 was observed for ABTC, a pH optimum of 7.0 was found for syringaldazine and guaiacol. Although the enzyme maintained activity over a broad pH range, its long-term stability at pH below 4.0 was low. Bilirubin oxidase from mycelium of <i>P. ostreatus </i>possesses high thermal stability, but it is sensitive to metal chelators. Complete bleaching of trypan blue at concentration of 5 mg/ml in water was achieved after 180 min of incubation with 20 mE/ml of the purified enzyme.