Characterization and heterologous production of a novel laccase from Melanocarpus albomyces

Laccases (EC 1.10.3.2) are multicopper oxidases that catalyze oxidation of various substituted phenolic compounds, aromatic amines and even certain inorganic compounds by using molecular oxygen as the electron acceptor. Their substrate versatility makes laccases highly interesting for various applications, including textile dye bleaching, pulp bleaching and bioremediation, where enzymatic catalysis could serve as a more environmentally benign alternative than the currently used chemical processes. However, most laccases studied thus far are not well-suited for the applications because of their low stability at high temperatures or pH values. This work focused on identifying and characterizing novel fungal laccases having potential for the applications as well as on development of efficient production methods for laccases. Laccase-producing fungi were screened from various environmental samples by plate tests using the indicator compounds guaiacol, tannic acid and the polymeric dyes Remazol brilliant blue R and Poly R-478. A total of 26 positive fungal strains were isolated, and their ability to produce laccase was studied in liquid media. Four fungal strains produced significant amounts of laccase, and these enzymes were preliminarily characterized. The novel laccases were found to be rather typical basidiomycete laccases, although they had notably high thermostabilities as compared to other fungal laccases. A novel laccase from the ascomycete Melanocarpus albomyces was purified and biochemically characterized. The substrate specificity and susceptibility towards inhibitors were shown to be typical for laccases. Spectral data measured for the purified laccase indicated that the characteristic three types of copper were present. Interestingly, M. albomyces laccase showed good thermostability and it had a pH optimum at neutral pH with phenolic substrates. Both of these are unusual properties for fungal laccases. The crystal structure of M. albomyces