A novel laccase (Tolacc-T) from white rot fungus Trametes orientalis was enriched to apparent homogeneity with a specific activity of 20.667U/mg protein and recovery yield of 47.33%. The SDS-PAGE gave a single band indicating that Tolacc-T appears as a monomeric protein with a molecular mass of 44.0kDa. Domain structure analysis revealed that Tolacc-T contained a typical copper II binding domain and shared three potential N-glycosylation sites, but had no copper I binding domain, demonstrating that the enzyme is really a laccase, but a novel laccase. Optimal pH and temperature of Tolacc-T was 4.0 and 80°C, respectively, and it retained more than 80% of its original activity after 2h incubation at 10°C to 50°C. The enzyme exhibited strict substrate specificity towards ABTS but showed no or trace activities towards other substrates. Among the metals tested, Mn2+ was proved to be the best activator for enhancing the laccase activity. A strongly inhibiting effect was found when NaN3, L-cysteine, and DTT were added to the enzyme. However, Tolacc-T activity was little bit inhibited in the presence of chelator EDTA. Furthermore, the enzyme was capable of degrading structurally different synthetic dyes in the absence of a redox mediator.
Keywords: Characterization; Laccase; Purification.
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