Purification of a novel low-molecular-mass laccase with HIV-1 reverse transcriptase inhibitory activity from the mushroom Tricholoma giganteum

Abstract

A laccase with a novel N-terminal sequence, a low molecular mass of 43 kDa smaller than those of previously reported laccases, a pH optimum of 4, and a temperature optimum at 70 °C was isolated from fresh fruiting bodies of the mushroom Tricholoma giganteum. The activity of the enzyme rose steadily from 20 to 50 °C, increased very slowly from 50 to 70 °C, and fell slightly when the temperature was further increased to 80 °C. The activity of the laccase underwent little changes over the pH range 3.0–5.0. However, the enzyme activity dwindled to nothing after exposure to 100 °C for 10 min and when the ambient pH was 7 or above. The procedure used for purifying the enzyme included ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and FPLC-gel filtration on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and CM-cellulose. It inhibited HIV-1 reverse transcriptase with an IC 50 of 2.2 μM.