Purification and Characterization of Laccase from Basidiomycete Fomitella fraxinea

A laccase was isolated from the culture filtrate of the basidiomycete Fomitella fraxinea. The enzyme was purified to electrophoretical homogeneity using ammonium sulfate precipitation, anion-exchange chromatography, and gel-filtration chromatography. The enzyme was identified as a monomeric protein with a molecular mass of 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography, and had an isoelectric point of 3.8. The N-terminal amino acid sequence for the enzyme was ATXSNXKTLAAD, which had a very low similarity to the sequences previously reported for laccases from other basidiomycetes. The optimum pH and temperature for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) were 3.0 and $70^{\circ}C$, respectively. The enzyme also showed a much higher level of specific activity for ABTS and 2,6-dimethoxyphenol (DMP), where the $K_m$ values of the enzyme for ABTS and 2,6-DMP were 270 and $426{\mu}M$, respectively, and the $V_{max}$ values were 876 and $433.3{\mu}M/min$, respectively. The laccase activity was completely inhibited by L-cysteine, dithiothreitol (DTT), and sodium azide, significantly inhibited by $Ni^+,\;Mn^{2+}$, and $Ba^{2+}$, and slightly stimulated by $K^+$ and $Ca^{2+}$.