Produção, imobilização por Cross-Linked Enzyme Aggregates (CLEAS) e aplicação de lacases fúngicas na biodegradação de ciprofloxacino

Abstract

The occurrence and adverse impacts on health and the environment caused by emerging contaminants (EC) have been of considerable concern in recent years. Especially for antibiotics, their widespread presence in the environmental compartments, due to inadequate disposal or high percentage of excretion after administration, can trigger the development and dissemination of bacterial resistance, a serious public health problem. Until now, several approaches have been investigated for the removal of drugs present in effluents through alternative to conventional treatment strategies. In this context, enzymatic biodegradation, mainly with the use of laccases (EC 1.10.3.2), has received considerable attention. To improve enzymatic stability and enable the reuse of laccases in biocatalytic cycles, different immobilization techniques are employed, including the Cross-Linked Enzyme Aggregates (CLEAs) methodology. In this work, laccases were produced under two conditions using white rot fungi: (i) by monoculture of Trametes villosa and (ii) by co-culture of T. villosa with Pycnoporus sp., reported for the first time in the literature. The crude extract of T. villosa and Pycnoporus sp., expressed as main proteins laccases with apparent molecular masses between 55 and 45 kDa and both were identified in the co-culture. The crude extracts from the two cultures were immobilized by CLEAs, using 75% ammonium sulfate as a precipitant and glutaraldehyde as a crosslinking agent at concentrations of 200 mM for T. villosa laccases (Lac-TvL) and 162.5 mM for co-culture laccases (Lac-CO). After optimization, the enzymatic recoveries corresponded to 27.77± 1.07% for T. villosa CLEAs (CLEAs-TvL) and 35.38 ± 0.95% for co-culture CLEAs (CLEAs-CO), after 3h of reaction at 22 °C, statically. Lac-CO in its free form showed greater thermal and pH stability than Lac-TvL. Free and immobilized laccases showed optimal temperature for catalytic activity between 50 to 60 °C and optimal acidic pH (between 2.4 and 3.0). The immobilization of extracts increased the thermostability at 40 °C, retaining 100% of the residual activity after 180 min of reaction, while the free forms Lac-TvL and Lac-CO maintained 82.08 ± 1.14% and 89.98± 1.90% activity, respectively. At 55 °C, CLEAs-TvL maintained 79.37 ± 0.40%, a value 1.5 times greater than that obtained for the free form after 180 min; while CLEAs-CO presented 88.59 ± 0.52% of residual activity, 11% more than free Lac-CO. Free laccases and CLEAs removed with the same efficiency the drug ciprofloxacin (2.5 mg L-1), reaching 53 to 62% of biodegradation after 24h of reaction (40 °C and 150 rpm) with the use of 4-hydroxybenzoic acid (HBA) as a mediator, with reduced antimicrobial action on strains of Escherichia coli.