Purification and characterization of laccase from wood-degrading fungus Trichophyton rubrum LKY-7

A new wood-degrading fungus Trichophyton rubrum LKY-7 secretes a high level of laccase in a glucose-peptone liquid medium. The production of laccase by the fungus was barely induced by 2,5-xylidine. The laccase has been purified to homogeneity through three chromatography steps in an overall yield of 40%. The molecular mass of the purified laccase was about 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE). The purified laccase had the distinct blue color and the basic spectroscopic features of a typical blue laccase: two absorption maxima at 278 and 610 nm and a shoulder at 338 nm. The N-terminus of the laccase has been sequenced, revealing high homology to laccases from wood-degrading white-rot fungi such as Ceriporiopsis subvermispora, but little similarity to laccases from non-wood-degrading fungi such as Agaricus bisporus and Cryptococcus neoformans. The enzyme had a low redox potential of ∼0.5 V, yet it was one of the most active laccases in oxidizing a series of representative substrates/mediators. Compared with other fungal laccases, the laccase has a very low Km value with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as a substrate and a very high Km value with violuric acid as a substrate. The laccase had the isoelectric point of 4.0. The laccase had very acidic optimal pH values (3–4), although it was more stable at neutral pH than at acidic pH. The laccase oxidized hydroquinone faster than catechol and pyrogallol. The oxidation of tyrosine by the laccase was not detectable under the reaction conditions. The laccase was strongly inhibited by sodium azide and sodium fluoride.