Team:TU Darmstadt/Project/Pharmaceutical Degradation
Outlook: What would we have done with more time?
Unfortunately, it was not possible for us to come to the lab and conduct actual experiments this year. Nevertheless, we came up with a lab plan with all the experiments and assays we would have done to get our project up and running.
At the beginning of our lab time we would have transformed our two laccases, CueO and CotA, into E. coli. After a successful expression of the enzymes, we would have used a 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay to check the laccase activity in vitro.[1] The kinetic activity or the determination of an effective laccase concentration would have been detected by HPLC. We were also planning a toxicity assay. Since our plan is to render diclofenac and other toxic substances less harmful to the environment by oxidation via our laccases, the detection of the substrate concentration with the lowest toxicity is a crucial step. Hence, we were planning an assay with zebrafish embryos, considering they are not classified as animal testing and commonly used in this respect.[2] (see Tox-Assay, Hyperlink).
After the detection of the activity of laccase in vitro, the affinity of the substrate conversion would be increased using a quick-change PCR.
We planned to perform the analytical assays with our own expressed laccases as well as with laccase from Trametes versicolor in order to generate comparable values.
As we try to degrade a wide range of substances in wastewater with our laccase (see modular biofilm, hyperlink), we also wanted to measure the kinetic degradation values for different substances. Since it was very difficult to find comparable degradation values for different substances during our research, we would have also done this with the laccase from Trametes versicolor, so our work would have been useful for future projects.
In addition to our laccases, we were also focusing on the degradation of azithromycin via the enzyme EreB. Here too, we would have transformed and expressed the enzyme into E. coli at the beginning of our laboratory time. To detect the degradation of azithromycin, we were planning a Kirby-Bauer assay.[3] Just as with the laccases, we would have performed HPLC to measure kinetic activity, as well as a LC-MS. To increase the substrate affinity of the enzyme, we would have performed site saturation mutagenesis. Our modeling team is also working on a rational design of the enzyme. (see modeling, hyperlink)
All assays would have been performed with both the naturally occurring enzyme variant and the optimized variant to generate comparability.
The next step would have been an implementation of our selected enzymes in a B. subtilis biofilm. We planned to realize this via a tasA fusion protein. To prove our concept, our biofilm sub-group has worked on an assay used to immobilize a fusion protein from tasA and sfGFP in the biofilm matrix (see Filmies, Hyperlink). The assay has already been performed with larger proteins, so we are positively encouraged to have it performed successfully with our laccases or EreB. A laccase-tasA and EreB-tasA fusion protein has already been designed for this purpose and would have been transformed and expressed in B. subtilis if it had been possible to go into the laboratory. This way, we would have managed to immobilize the active enzyme in the outer biofilm matrix, which harmful drugs and pharmaceuticals in wastewater reach without complications.
Sources:
[1]: Ivana Eichlerová, Jaroslav Šnajdr and Petr Baldrian, Laccase activity in soils: consideration for the measurement of enzyme activity, Chemosphere, 2012, 88: 1154-1160, https://doi.org/10.1016/j.chemosphere.2012.03.019
[2]: A. V. Hallare, H.-R. Köhler, R. Triebskorn, Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO, Chemosphere, 2004, 56: 659-666, https://doi.org/10.1016/j.chemosphere.2004.04.007
[3]: Jan Hudzicki, Kirby-Bauer Disk Diffusion Susceptibility Test Protocol, American Society for Microbiology, 2009