Purification and characterization of a laccase from the edible wild mushroom Tricholoma mongolicum.

A novel laccase from Tricholoma mongolicum was purified by using a procedure that entailed ion-exchange chromatographies on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and FPLC-gel filtration on Superdex 75. The purified enzyme was obtained with a specific activity of 1,480 U/mg-protein and a final yield of 15%. It was found to be a monomeric protein with a molecular mass of 66 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was GIGPVADLYVGNRIL, similar to some but also different to other mushroom laccases. The optimum pH and temperature for the purified enzyme were pH 2 to pH 3 and 30°C, respectively. It displayed a low K m toward 2,7-azinobis (3-ethylbenzothiazolone-6-sulfonic acid) diammonium salt (ABTS) and high k cat /K m values. The purified laccase oxidized a wide range of lignin-related phenols, but exerted maximal activity on ABTS. It was significantly inhibited by Hg 2+ ions, and remarkably stimulated by Cu 2+ ions. It inhibited HIV-1 reverse transcriptase and proliferation of hepatoma HepG2 cells and breast cancer MCF7 cells with an IC 50 of 0.65 μM, 1.4 μM, and 4.2 μM, respectively, indicating that it is also an antipathogenic protein.