Chlorobenzoic acid degradation by Lentinus (Panus) tigrinus: In vivo and in vitro mechanistic study-evidence for P-450 involvement in the transformation

Abstract Aim of this work was to investigate the ability of Lentinus (Panus) tigrinus to degrade and detoxify a chlorobenzoate (CBA) mixture composed of mono-, di- and tri-chlorinated isomers. The degradation process was investigated as a function of both the growing medium ( i.e. low N Kirk's and malt extract-glucose medium) and cultivation conditions ( i.e. stationary and shaken cultures). The majority of CBAs were quantitatively degraded within the early 15 d from spiking with the notable exception of the double ortho -chlorinated compounds, 2,6-di-, 2,3,6-tri- and 2,4,6-tri-CBA. Analysis of the degradation intermediates indicated the occurrence of side chain reduction, hydroxylation and methylation reactions. Although CBAs stimulated laccase production, in vitro experiments with a purified L. tigrinus laccase isoenzyme demonstrated its inability to participate in the initial attack on CBAs even in the presence of redox mediators; similar results were found with a Mn-peroxidase isoenzyme. Conversely, prompt degradation was observed upon 1 h incubation of CBAs with a purified microsomal fraction containing cytochrome P-450 monooxygenase. The nature of some reaction products ( i.e. hydroxylated derivatives), the dependency of the reaction on NADPH and its susceptibility to either CO or piperonyl butoxide inhibition confirmed the involvement of L. tigrinus cytochrome P-450 in the early steps of CBA degradation.