Synthesis and characterization of combined cross-linked laccase and tyrosinase aggregates transforming acetaminophen as a model phenolic compound in wastewaters

Laccase (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1) are ubiquitous enzymes present in nature as they are known to originate from bacteria, fungi, plants, etc. Both laccase and tyrosinase are copper-containing phenoloxidases requiring readily available O2 without auxiliary cofactor for their catalytic transformation of numerous phenolic substrates. In the present study, laccase and tyrosinase have been insolubilized as combined crosslinked enzyme aggregates (combi-CLEA) using chitosan, a renewable and biodegradable polymer, as crosslinker. The combi-CLEA, with specific activity of 12.3U/g for laccase and 167.4U/g for tyrosinase, exhibited high enzymatic activity at pH5–8 and temperature at 5–30°C, significant resistance to denaturation and no diffusional restriction to its active site based upon the Michaelis–Menten kinetic parameters. Subsequently, the combi-CLEA was applied to the transformation of acetaminophen as a model phenolic compound in samples of real wastewaters in order to evaluate the potential efficiency of the biocatalyst. In batch mode the combi-CLEA transformed more than 80% to nearly 100% of acetaminophen from the municipal wastewater and more than 90% from the hospital wastewater. UPLC–MS analysis of acetaminophen metabolites showed the formation of its oligomers as dimers, trimers and tetramers due to the laccase and 3-hydroxyacetaminophen due to the tyrosinase. [Display omitted] •Laccase and tyrosinase were successfully insolubilized as combi-CLEA.•The pH profiles of the combi-CLEA were broader than those of the individual free enzymes.•The catalytic efficiencies Kcat/KM were higher for the combi-CLEA than the free enzymes.•The combi-CLEA displayed considerable denaturation-resistance to the various inhibitors compared to the free enzymes.•The treatment of acetaminophen in real wastewaters showed high transformation of the drug.