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 Pleurotus ostreatus 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 P. ostreatus as bilirubin oxidase (BOX) with very high degree of homology with the pox2 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 P. ostreatus 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.