Copper dissociation as a mechanism of fungal laccase denaturation by humic acid

Effects of humic acid on removal of hydroxy polychlorobiphenyls (PCBs) with laccase from Trametes versicolor were studied. In the absence of humic acid, hydroxy PCBs were rapidly degraded by laccase. However, the rate constants decreased with increasing humic acid concentration, the reactions being completely inhibited at 150 mg l(-1) of humic acid. Peroxidase from Arthromyces ramosus was not inhibited by the same treatment. The activity of humic acid-deactivated laccase was completely restored by copper ions (500 muM of Cu2+ in 150 mg l(-1) of humic acid), but not by other metal ions (Zn2+, Fe2+ and Hg2+). Humic acid-deactivated laccase purified by gel permeation chromatography (GPC) showed no activity against 2,2'-azino-bis(3-ethylbenzthiazoline sulfonic acid) diammonium salt and 3,5-dichloro-4-hydroxybiphenyl, but its activity was restored by copper ion treatment. Humic acid-deactivated laccase showed similar properties, such as GPC retention time and copper ion requirements for activity, to those of laccase deactivated by nitrilotriacetic acid. The extent of humic acid inhibition, expressed as activity non-recoverable by copper ion treatment, increased over time more rapidly than that of the humic acid-free control. These results suggest that short-term inactivation of laccase, i.e., less than 1 day, is attributable to a depletion of copper ion.