Purification and characterization of a novel laccase from the edible mushroom Clitocybe maxima

Abstract A novel laccase from the edible mushroom Clitocybe maxima was purified and characterized. The purification protocol involved ammonium sulfate saturation, ion-exchange chromatography on DEAE-cellulose, SP-Sepharose, and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. It was a monomeric protein with a molecular mass of 62 kDa as estimated by SDS-PAGE. Its N-terminal amino acid sequence was DIGPVTPLAI, which exhibited partial sequence homology to those of published mushroom laccases. Its optimum pH was 3.0 and its optimum temperature was 60 °C. It manifested degrading activity towards a variety of phenolic compounds. The most sensitive substrate for the enzyme was 2,7′-azinobis[3-ethylbenzothiazolone-6-sulfonic acid]diammonium salt (ABTS), with a K m of 61.7 μM at pH 3.0 and 37 °C. The ranking of degradation activity toward phenolic substrates was ABTS > hydroquinone > N,N-dimethyl-1,4-phenylenediamine > pyrogallol > catechol > 2-methylcatechol. The laccase showed antiproliferative activity against Hep G2 and MCF-7 tumor cells with IC 50 of 12.3 μM and 3.0 μM, respectively. It also lowered the activity of human immunodeficiency virus-1 (HIV-1) reverse transcriptase with an IC 50 of 14.4 μM.