Bioremediation and Biodegradation Effect of Various Pollutants and Soil-Like Constituents on Laccase from Cerrena unicolor M.T. Filazzola, F. Sannino, M. A. Rao, and L. Gianfreda* ABSTRACT Laccase from Cerrena unicolor catalyses the oxidation of a wide range of aromatic compounds, either xenobiofic or naturally occurring phenols, leading to the formation of polymeric products. These are characterized by their low solubility and often may formprecipitates or aggregates. The oxidizing efficiency of the enzyme is strictly depen- dent on the number of hydroxyl groups and the position of substituents on the phenolic molecules. During the reaction with some substrates, the enzyme is inactivated, because of possible adsorption of laccase molecules on newly formedpolyphenols. By contrast, the oxidation of humic precursors (i.e., resorcinol, gallic acid, andpyrogallol) does not influence greatly the residual laccase activity. The triazinic herbi- cides, triazine and prometryn (2,4-bis(isopropylamino)-6-methylthio- s-triazine), are not substrates oflaccase. They, however, inhibit laccase activity assayed with 2,4-dichlorophenol (2,4-DCP) or catechol substrates. The reductionofsubstrate oxidation rates is usually accom- paniedby the retention of higher levels of residual enzymatic activity. These results, together with the slight recovery in laccase activity following dialysis of the assay mixture, provide further evidence that the enzymemay be incorporated into or adsorbed onto polyphenolic products, with a consequentreduction in the concentration of active forms of laccase. E CCASES (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) are copper-dependent phenol oxidases, which are widespread in plants and fungi (Nerud and Misurcova, 1996), and whose physiological function is mainly connected with lignin transformation (Gianfreda et al., 1998; Youn et al., 1995; Rodriguezet al., 1996). The catalytic mechanism of laccase action has been thor- oughly reviewed by Yarapolov et al. (1994). A charac- teristic of laccase molecules is their prosthetic group, which contains four copper atoms that differ in their redox potential, accessibility to solvents, spectra, and in the features of their surroundings. Usually, the reaction is characterized by a two-step mechanism,initially in- volving the formation of a free radical from an organic substrate and the subsequential reduction of O:. The organic substrate (which may be represented by various compounds, including o,p-diphenols, aminophenols, polyphenols, polyamines, lignin, some inorganic ions and aryl diamines), is oxidized (Bollag et al., 1988; Faure et al., 1996), and molecular oxygen is reduced directly to water by a four-electron without involvement of any mediator (Yarapolov et al., 1994). Organic pollutants are transformed by laccase into highly reactive radicals, Dipartimento di Scienze Chimico-Agrarie, Universith di Napoli "Fed- erico II," Via Universit/a 100, 80055Portici (Napoli), Italy. Received 31 July 1998. *Corresponding author (gianfred@unina.it). Published in J. Environ. Qual. 28:1929-1938 (1999). which may spontaneously polymerize, giving rise to less soluble, high molecular weight compounds (Roy- Arcand and Archibald, 1991), or which may interact with organic soil colloids, thus being incorporated into these latter and removed from the ecosystem (Rutti- mann-Johnson and Lamar, 1996). The enzymatic laccase oxidation process may provide a suitable alternative technology for the treatment of polluted aquatic environments and industrial wastewa- ters. The insoluble precipitates produced by laccase- mediated polymerization of xenobiotic substances may be easily removed from water by sedimentation or filtra- tion (Dec and Bollag, 1990). Furthermore, the incorpo- ration of xenobiotics in humic-like materials mayrender laccase a potential detoxifying agent for the remediation of polluted soil (Bollag et al., 1980). The development such bioremediation methodologies, however, requires that attention should be devoted to factors, which may influence laccase activity in heterogeneous environ- ments, for example, the inhibitory effect of organic and inorganic soil colloids on laccase activity (Gianfreda and Bollag, 1994), and the possible enhancementof the transformation of less-reactive pollutants through the use of phenolic and nonphenolic cosubstrates (Roper et al., 1995; Bourbonnais et al., 1995). The direct effect of phenolic compounds on laccase activity should also be considered. Indeed, several experimental findings have demonstrated that aromatic compounds such as phenols, phenol derivatives, and humic precursors, may behave as inhibitors of various enzymes. Gianfreda et al. (1993) and Rao et al. (1998) demonstrated that activity of invertase, acid phosphatase, and urease were differently inhibited by tannic acid. The purpose of the present study was to evaluate and quantify the performance of a laccase from the fungus Cerrena unicolor, in the presence of phenolic com- pounds from different origin. In particular, we consid- ered precursors of humic acids--such as pyrogallol, gallic acid, and resorcinol--and various phenolic pollut- ants, which are easily detected in waste waters from herbicide polluted sites or from olive oil mill waste waters. Furthermore, we have examined the possible en- hancement and/or counteracting effects of compounds used in combination (such as herbicides from combined formulation or their intermediate products). Particular attention has been devoted to the residual activity levels of the enzyme, after its initial catalytic action on added substrates, to evaluate the effectiveness of such a cata- lytic process for a cost-effective realistic application. 1929 Published November, 1999