Determination of the activity of laccase, and mediated oxidation of a lignin model compound, in aqueous-organic mixed solvents

The activity and stability of laccase from Poliporus pinsitus in ten mixed solvents, consisting of 1:1 buffered-water/organic-cosolvent, is studied by resorting to the new spectrophotometric assay based on 3-hydroxyantranilic acid (HAA). The enzyme is found to initially retain at least 70% of its activity, for content as high as 60% (w/w) of cosolvents such as dioxane, isopropanol, ethylene glycol, and acetonitrile, and the results obtained by use of the HAA assay are compared with those obtained with other chromogens and laccases of different origin. The enzyme activity does not remain stable in the mixed solvents, but decreases as a function of time; the decrease becomes substantial over a 24-hours period in the case of dioxane, isopropanol, or acetonitrile, whereas ethylene glycol does not affect the enzymatic activity. The major result of the present study is the evaluation of the effect caused by the cosolvents on the efficiency of oxidation of an hydrophobic non-phenolic substrate when using four laccase-mediator (ABTS, HPI, VLA, TEMPO) systems. Some mediator-specific solvent effects emerge. (C) 2004 Elsevier B.V All rights reserved.