Team:UChicago/Description

Project Description

Background

Lake Michigan serves as the water supply for 48 million people and supports a vast and interconnected ecosystem [1]. But recently, pharmaceutical pollutants have found their way into our water. From farms, hospitals, and unused medications, chemicals are seeping into the lake with harmful consequences. Excess antibiotics overturn the ecosystem’s careful microbial balance, in addition to exacerbating antibiotic resistance and rendering medical treatments ineffective. These chemicals are toxic to keystone species such as algae and are ingested by wildlife where it undergoes biomagnification - potentially finding its way back to us. [2]

One such pollutant is triclosan, an antibacterial agent present in 93% of gels, foams, and soaps marketed as “antibacterial” or “antimicrobial” [3]. Triclosan works by inhibiting fatty acid synthesis, and bacterial resistance to triclosan is also cross-linked to antibiotic resistance, which makes this chemical all the more troubling [4]. It has been found as one of the top 4 pollutants in Lake Michigan [5], making this chemical a pressing local threat.



Currently, water treatment to remove triclosan remains a challenge for wastewater plants. Although its removal efficiency from water is quite high, the chemical is simply relocated into sludge - the physical waste produced by treatment plants - which is then reintroduced back into the environment through programs that convert sludge into compost [6]. So, although triclosan may physically change locations, until it is chemically deactivated its toxic effects are still felt in the environment.

One common tool to deactivate these antibiotics and antifungals is laccase, an enzyme that can oxidize a range of aromatic compounds. Laccases function by oxidizing a substrate, which is then coupled with the reduction of oxygen gas to water. The electron transfer occurs along a highly conserved pathway within the core of the enzyme, facilitated by four Cu2+ ions embedded within the protein [7]. We chose the laccase from the fungus Trametes Versicolor for its high redox potential and because it is well-documented, having been used widely in industrial purposes [8].

But due to its promiscuity, laccases are not optimized to remove triclosan in a water treatment plant’s environment. Instead, the optimal activity found for the degradation of triclosan by laccase is at a pH of 5 and a temperature of 25 degrees Celsius [9]. This specific pH and temperature does not translate well to wastewater treatment plant conditions, which is where we would want to implement our solutions. So, the goal of our project is to engineer a tailored enzyme that most effectively deactivates triclosan at wastewater conditions. This would make it easier for wastewater treatment plants to purify water and prevent environmental threats to the community.

Sources
[1] - “Facts and Figures about the Great Lakes”. United States EPA. https://www.epa.gov/greatlakes/facts-and-figures-about-great-lakes
[2] - Olaniyan LW, Mkwetshana N, Okoh AI. Triclosan in water, implications for human and environmental health. Springerplus. 2016;5(1):1639. Published 2016 Sep 21. doi:10.1186/s40064-016-3287-x
[3] - Weatherly LM, Gosse JA. Triclosan exposure, transformation, and human health effects. J Toxicol Environ Health B Crit Rev. 2017;20(8):447-469. doi:10.1080/10937404.2017.1399306
[4] - Herbert P Schweizer, Triclosan: a widely used biocide and its link to antibiotics, FEMS Microbiology Letters, Volume 202, Issue 1, August 2001, Pages 1–7, https://doi.org/10.1111/j.1574-6968.2001.tb10772.x
[5] - Benjamin D. Blair, Jordan P. Crago, Curtis J. Hedman, Rebecca D. Klaper. Pharmaceuticals and personal care products found in the Great Lakes above concentrations of environmental concern, Chemosphere, Volume 93, Issue 9, 2013, Pages 2116-2123, ISSN 0045-6535, https://doi.org/10.1016/j.chemosphere.2013.07.057.
[6] - Lozano N, Rice CP, Ramirez M, Torrents A. Fate of Triclocarban, Triclosan and Methyltriclosan during wastewater and biosolids treatment processes. Water Res. 2013 Sep 1;47(13):4519-27. doi: 10.1016/j.watres.2013.05.015. Epub 2013 May 20. PMID: 23764601.
[7] - Mot AC, Silaghi-Dumitrescu R. Laccases: complex architectures for one-electron oxidations. Biochemistry (Mosc). 2012 Dec;77(12):1395-407. doi: 10.1134/S0006297912120085. PMID: 23244736.
[8] - Kurniawati S, Nicell JA. Characterization of Trametes versicolor laccase for the transformation of aqueous phenol. Bioresour Technol. 2008 Nov;99(16):7825-34. doi: 10.1016/j.biortech.2008.01.084. Epub 2008 Apr 11. PMID: 18406607.
[9] - Kim YJ, Nicell JA. Laccase-catalyzed oxidation of aqueous triclosan. J Chem Technol Biotechnol 81:1344–1352 (2006). https://doi.org/10.1002/jctb.1507

Design

Because Chicago has a climate that varies dramatically in temperature between summer and winter, we wanted to ensure that our enzyme would be functional at both extremes. So, we reached out to our local wastewater treatment plant to get a sense of the temperature fluctuations, and based our simulation conditions on the data they gave us.

Stickeney Wastewater Treatment Plant Monthly Average Temperature and pH


We cross-checked these conditions with Team Aboa and found that their wastewater temperatures and pH were consistent with ours. This gave us the following simulation conditions:

Control Conditions
Laccase complexed with triclosan at pH 5 and 25 degrees Celsius. Found to be the optimal triclosan degradation conditions by Kim and Nicell, 2006


Wastewater Treatment Plant Conditions
We took the average temperature of the three warmest months (July, August, and September) and the average temperatures of the three coldest months (January, February, March). Since there was no variation in the pH, we just took its yearly average.

Method

End goal: Mutated laccase that is optimized to bind to triclosan at wastewater pH and temperature. Find structural basis for differences in pH and temperatures to guide future rational design and characterization

Workflow: We first run MD simulations through Gromacs of our control conditions - wildtype/unedited laccases with their respective ligands, at a known pH and temperature which we can compare to literature values. Once the MD simulations are run, we can analyze the data using methods that asses the stability of various residues on the active site. This gives us baseline information about the enzyme that we can then compare to later on. Additionally, this will give us information about which residues we can potentially mutate. Then, through literature review and information obtained from MD, we will determine which specific mutations we want to induce in our laccase. We run new simulations with the above mutations to determine if our changes were effective or not at increasing the enzyme’s activity at a desired pH and temperature.

Procedure
1. Obtain coordinate files of laccase and triclosan
2. Create a new coordinate file of triclosan docked with laccase
3. Create topology files of docked laccase and triclosan
4. Use coordinate and topology files as inputs for MD simulation via GROMACS
5. Analyze results
6. Generate hypothesis for potential mutations to make, create new coordinate files with those mutations
7. Repeat steps 4-6