Carboxylic acids used in common buffer systems inhibit the activity of fungal laccases

The inhibition of fungal laccases by carboxylic acids has been studied. Steady-state kinetics performed with recombinant laccase from Trametes villosa and ABTS as a substrate revealed an s-linear, i-parabolic mixed inhibition type for acetate, while formate exhibited a linear, non-competitive inhibition type. Although Ki values were several orders of magnitude higher than those for azide, inhibition levels for acetate were substantial (10-60% of initial activity) at concentrations commonly used in routine laccase assays (10-100 mM). The first order inactivation rate constant for acetate was low (0.39 min(-1)) and similar to that of propionate and butyrate. However, inhibition by di- and tricarboxylic acids was considerably less pronounced (up to 20% at 100 mM) and instantaneous. Therefore. citrate and particularly succinate appear much more suitable for laccase assays and applications than acetate which should be avoided. Wild-type laccases from several Trametes species were found to be inhibited to a similar extent, while laccase from Pleurotus eryngii and some other species were not affected or even stimulated by carboxylic acids. These results collectively suggest that fungal laccases do not share a common structural feature responsible for their inhibition by carboxylic acids. (C) 2009 Elsevier B.V. All rights reserved.