Team Aboa 2021

The Lac Case - Utilization of laccases for pharmaceutical waste detoxification

Pharmaceutical waste is one of the most deleterious pollutants in the Baltic Sea. Especially the non-steroidal anti-inflammatory drug diclofenac is causing severe harm to this delicate ecosystem. The current removal efficiency of diclofenac is only 27% at our local wastewater treatment plant. The project objective was to contribute to the development of a microbial wastewater treatment system for the detoxification of this compound. The approach was to overexpress and extract three heterologous laccases, specific enzymes which are capable of catalyzing the conversion of diclofenac into less harmful derivatives, in engineered E. coli. We were able to successfully produce and purify CotA (from B. subtilis) and CueO (from E. coli), of which CotA was shown to have catalytic activity in vitro. Implementation of this work would include the expression of this laccase in photosynthetic cyanobacteria in a closed bioreactor system, integrated as a part of the wastewater purification process.

A graphical illustration of our project. In the first step, there are three plasmids of which each contains one laccase gene name. From plasmids arrows point to the bacteria, under which there is a text “E. coli BL21(DE3)”. Arrows point from the bacteria to structures of laccases and further to one 96 well-plate. From this well-plate one arrow is pointing to a graph and further to a petri dish containing cyanobacteria. Finally, an arrow is pointing to a picture in which there are green tubes indicating a photobioreactor as well as gray pipelines and a tank indicating a wastewater treatment plant.
Figure 1. Overview of our project. First, we designed plasmids for the overexpression of the laccase enzymes CotA from Bacillus subtilis, CueO from Escherichia coli and Yak from Yersinia enterocolitica (1). Then we transformed the plasmids into E. coli BL21(DE3) cells (2) and purified the produced laccases (3). We measured laccase activities with ABTS and syringaldazine assays (4). Our aim was to compare the laccase activities at conditions simulating our local wastewater treatment plant (5), and express the most promising laccase in the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 (6). These laccase-producing cyanobacteria could then finally be implemented as a part of a wastewater treatment plant in the form of a photobioreactor (7).