| The enzymes ligninase and Mn-peroxidase have been implicated in the degradation of recalcitrant pollutants by the wood rotting fungus Phanerochaete chrysosporium. Objectives of this research were to elucidate the reaction chemistry for both of these peroxidases, and to evaluate their application potential in industrial wastewater treatment. Effects on enzyme activity and stability were evaluated for a number of different factors, including temperature, pH, enzyme concentration, and the presence of various chemical effectors. The roles of manganese and certain buffers in carrying out oxidation reactions by Mn-peroxidase were evaluated in detail. In the presence of stabilizing ligands (buffers), Mn(II) is enzymatically oxidized to a Mn(III)-buffer complex, which then oxidizes organic substrates. Stoichiometry and kinetics of enzymatic Mn(III)-tartrate formation were characterized. Mn(III)-tartrate was found to be decomposed by hydrogen peroxide in a manner that could be catalyzed by Cu(II). Most experimental observations on Mn-peroxidase in this study and by others therefore can be explained as a competition between enzymatic Mn(III) formation and decomposition by peroxide. Screening studies on Mn-peroxidase oxidation of phenolic pollutants indicated that o-cresol and 2-chlorophenol were oxidized via Mn(III)-tartrate. In preliminary tests 2-nitrophenol was removed in the absence of manganese, indicating that 2-nitrophenol interacted directly with enzyme intermediates. Screening tests with ligninase also resulted in at least partial removal of five phenolic pollutants. Using o-cresol as a model substrate, partial removal was demonstrated to result from enzyme inactivation during the course of the reaction. Inactivation involved both thermal denaturation and mechanism-based inactivation related to hydrogen peroxide. Mechanism-based inactivation was overcome by controlled addition of peroxide, so that essentially complete removal of o-cresol could be achieved. Further work to demonstrate the feasibility of ligninase application in wastewater treatment should be directed at characterizing reaction products. More fundamental research on the chemistry of Mn-peroxidase in the absence of manganese must be conducted before its application potential can be established. |
