Insolubilization of Tramates versicolor laccase as cross-linked enzyme aggregates for the remediation of trace organic contaminants from municipal wastewater

Abstract

The novelty of this study deals with the biocatalytic treatment of trace organic contaminants (TrOCs) from municipal wastewater by insolubilized laccase. Laccase from Trametes versicolor was aggregated by three-phase partitioning technique followed by cross-linking with glutaraldehyde to produce insolubilized laccase as cross-linked enzyme aggregates (CLEAs). The optimal conditions for CLEAs preparation include ammonium sulphate concentration of 83% (w/v), crude to t-butanol ratio of 1.00: 1.05 (v/v), pH 5.3, and glutaraldehyde concentration of 20 mM obtained via statistical design. The efficiency of insolubilization of the CLEAs laccase based on the kcat/km ratio was approximately 4.8-fold greater than that of free laccase. The developed CLEAs showed greater resistance to product inhibition mediated by ABTS than the free enzyme and exhibited excellent catalytic activity even after the tenth successive cycle. Further, free laccase and the synthesized CLEAs laccase were utilized to treat five analgesics, two NSAIDS, three antibiotics, two antilipemics, and three pesticides in the municipal wastewater. Under the batch process with operating conditions of pH 7.0 and 20 °C, 1000 U/L of CLEAs, laccase removed 11 TrOCs in the range of about 20–99%. However, the inactivated CLEAs only adsorbed 2–25% of TrOCs. It was observed that acetaminophen, mefenamic acid, trimethoprim, and metolachlor depicted almost complete removal with CLEAs laccase. The performance of CLEAs laccase in a perfusion basket reactor was tested for the removal of TrOCs from municipal wastewater.