Figure 2.

Structural alignment between the protein designed by iGEM TecCEM (red, magenta, blue and green) and the crystallized structure of the Human Estrogen Receptor Alpha 2IOG (white). The alignment shows a preserved structure in the Ligand Binding Domain of the modified ESR1. Protein visualized with Chimera [3].

The same was done for part BBa_K3809011, which was a reporter we designed to test experimentally in the lab whether the protein was folding correctly and if it could be immobilized in the QCM. The results we got from the alignment of the modified mRFP of BBa_K3809011 and mRFP were the same as for ESR1 of part BBa_K3809010; both retained a similar structure to their reference structures. 

To further prove the functionality of our protein, we conducted a Docking Analysis in which we tested the ability of the receptor to bind and capture a group of Endocrine Disruptive Chemicals which includes Estradiol (the natural ligand), Benzene, Bisphenol A, Dimethyl Phthalate, Ethylene Glycol, Phenol, Chloroprene and Perfluorooctanoic Acid. To prepare the model for the analysis, we added hydrogens to it, improving the quality of the assay. To this end, we used the tool MolProbity [4]. The ligands were obtained from PubChem [5] and the Docking was conducted using the Software tool SwissDock [6]. The results we obtained were then visualized in Chimera in search of the type of interaction the ligands had with the receptor. We found that in most cases, the ligands bound to the protein formed a canal instead of a pocket. Besides, from the best result of the docking, we obtained its Free Gibbs Energy and used it to calculate the Association Constant. Furthermore, we evaluated the formation of hydrogen bonds between the receptor and the small molecule. The results are seen below. 

Figure 3.

Chloroprene-ESR1 Complex. The amino acids Thr71, Cys103 and Lys118 act as a spatial reference and are the ones closest to the molecule. 

The conclusion we got from these results was that our protein retained its functionality while keeping the characteristics we added and without compromising the affinity towards the ligands. As shown in Table I, we observed that EDCs had a similar affinity towards the receptor as Estradiol (natural ligand); and although the site of the interaction varied, the binding of the ligands with ESR1 was acceptable. What we concluded from this part of the experiments we performed is that we had reasons to believe that ESR1 would be able to work as the capturing molecule of EDCs. 

What did we accomplish in the Lab?

After we did the first analysis of the protein, we knew that we had what’s necessary to begin working in the Laboratory. We had gathered enough information and had the material in the Lab just to begin our experiments. So, after we got our permission to enter our Campus, we did just that. 

We planned our experiments as follows:

• Obtain our Biobricks (either from the Synthesis of gBlocks through IDT or retrieving them from past iGEM plates).

• Insert our gBlocks in pSB1C3 vectors.

• Transform our DNA in a Cloning System to get more copies.

• Extract the plasmids out of the Cloning System.

• Make digestions of the plasmids containing the Biobricks.

• Ligate our Biobricks with the ones we obtained from iGEM plates.

• Transform our constructs in a Cloning System to get more copies.

• Extract the constructs out of the Cloning System.

• Transform our constructs in an Expression System.

• Retrieve our protein

• Immobilize the protein onto the Quartz Crystal Microbalance.

The parts we synthesized from IDT were BBa_K3809010 and BBa_K3809011 while the part we retrieved from iGEM Plates was BBa_K081005. We assembled parts BBa_K3809012 (ESR1)  and BBa_K3809013 (mRFP) by merging  BBa_K3809010 with BBa_K081005 and BBa_K3809011 with BBa_K081005 respectively. For more information about our Parts, read here.
 

However, due to the pandemic we were unable to achieve the goal of the protein purification and the subsequent immobilization onto the QCM. However, we did plan the immobilization protocol. It consists of the immobilization using the cysteine residue we added to the protein and a functionalized QCM. The functionalization would be done using L-cysteine, genipine and chitosan and the formation of a crosslinked structure that would give the protein stability. 

pSB1C3, mRFP and ESR1

We started out by observing the plasmids pSB1C3 we had on an agarose gel electrophoresis to determine with which one was going to work.

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We chose plasmid pSB1C3 labelled as B (lane 8), as it presented the clearest band between 2 and 3 Kb on the gel.

Next, we transformed 3 DH5a cultures with chosen pSB1C3 plasmid, made an extraction and ran an agarose gel electrophoresis to verify integrity and desired size.

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We noted clear bands of approximately 2 kb. pSB1C3 desired plasmid is 2070 kb, so we were able to verify its size.

Next, a digestion with restriction enzymes EcoRI and PstI was carried out to make sure those enzymes cut the plasmid at the desired sites, producing expected fragment sizes.

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All fragments were the expected size, as well as the uncut plasmid in lane 8.

At the same time, we amplified by PCR our mRFP and ESR1 genes in order to obtain multiple copies and be able to clone each one with pSB1C3.

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Both amplified products were the desired size. mRFP amplification was expected to be 997 bases and ESR1 amplification was expected to be 1971 bases. Both amplifications are observed in the agarose gel near the desired sizes.

In parallel, DH5a chemocompetent cells were transformed with BBa_K081005 and inoculated in a selective culture medium.

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​Transformation was successful because the LB+agar+CAM culture medium presented growth, which means the bacteria acquired the CAM resistance gene inside BBa_K081005.

Next, a digestion with restriction enzymes SpeI and PstI was carried out to make sure those enzymes cut the plasmid at the desired sites, producing expected fragment sizes.
All fragments were the expected size.

After that, DH5a transformed cells with both constructions (pSB1C3+mRFP and pSB1C3+ESR1) were propagated in culture media (solid and liquid). 30 colonies were chosen from the pSB1C3+ESR1 cultures and a colony PCR was performed to them in order to verify the presence of ESR1. The colonies that present DNA fragments of approximately 2 Kb, since ESR1 is 1971 bases.

A few colonies of the 30 that were chosen presented the expected size (colonies 8, 13, 18, 21, 23 and 26, as illustrated above), so those DNA fragments were extracted and BL21 cells were transformed with them.

Protein Expression:
Our Final Result

We were able to express the protein only for cells from colonies 18 and 23. The results were verified through an SDS-PAGE. However, the best band was seen for colony 23. The results are shown below. 
We retrieved samples during the incubation period so that we could see the differential expression of ESR1.

In conclusion:
The results shown are promising and demonstrate that we can produce this protein in order to immobilize it in our QCM. Due to the pandemic, we weren’t able to do that just yet; but, we want to continue with this project and develop our biosensor so that we can fulfill the implementation we proposed.
 

This figure shows the band corresponding to the protein ESR1 (69.6 kDa). 
 

The other side of our project: Degradation through a Laccase.

As we stated in our Project Description, one of our goals was to find a Laccase that could degrade EDCs in water. Here we describe how our screening process was done and how we characterized the Laccase and establish a purification protocol.

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In silico analysis

For this section of our project, we wanted to compare the Laccase we were using (BBa_K863010) with a Commercial Laccase (if you want to see how we selected this Laccase, please read the information below). We did some experiments in which we predicted the structure of the enzyme to test the affinity of the Laccase towards any given substrate through Molecular Docking. We uploaded the amino acids sequence of the enzyme to I-TASSER, a server which runs a platform that predicts the structure of a protein and its function through a model [7] and compared the model we got with a Laccase from Trametes versicolor obtained from PDB under the code 1KYA [8] using a structural alignment (seen in figure 9). 

Figure 9.

Structural alignment of the model of the Laccase from BBa_K863010 predicted using I-TASSER (Light Brown) and the Laccase 1KYA from Trametes versicolor obtained from PDB (Light Blue). Image visualized using Chimera [3].

The results of these experiments showed a similar folding to the Laccase 1KYA from Trametes versicolor, which makes us think that the affinity for their substrates may be also similar. To test whether this last statement was true, we performed a Docking using SwissDock [6] and obtained the Free Gibbs Energy of the interaction, as well as the Affinity Constant for a group of EDCs (the same group we used for the Docking of ESR1). The Docking was done for the Modeled Protein and the Laccase 1KYA. 

Table II

Results obtained from the Docking Analysis for Laccase from BBa_K863010

Table III

Results obtained from the Docking Analysis for Laccase from Trametes versicolor.

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In this case, the results were similar between both Laccases, with a similar affinity towards the same substrates.

Color Assay:

To demonstrate activity of the lacasse purified, we conducted a dye degradation assay. Owing to their broad substrate specificity, laccases have attracted considerable interest in terms of applications to many fields, such as environmental detoxification. Some fungal laccases have been reported to perform dye decolorization of a variety of dyes, such as azo, anthraquinone, and aromatic methane. Synthetic dyes are widely used in several industries, including textiles, food processing, paper printing, cosmetics, and pharmaceuticals. Three dyes were selected according to previous reports of laccase degradation, malachite green, methylene blue and rose Bengal (Cheriaa J. & Bakhrouf A., 2009 [10]; Forootanfar, H., 2012; Pramanik, S., & Chaudhuri, S. 2018 [11]). Based on the chemical structure of chromogenic groups, dyes are classified as azo, heterocyclic/polymeric or triphenylmethanes. About 60% of produced dyes belong to the azo group which are categorized as monoazo, diazo, and triazo dyes. Malachite green is a triphenylmethane dye belonging to a basic dyes class, used extensively for dyeing silk, wool and cotton. Rose Bengal, an Azo dye is used in apoptosis assays, biological staining photography, recording industry, etc., this dye is genotoxic and microbial toxic. Methylene blue is a heterocyclic aromatic compound that has a wide use in biological and medicine applications in addition to textile-processing industries.

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Selecting our Laccase:

The following table describes the four laccases with which we started our experiments. At first we carried out the assays described next to characterize each laccase and deduce which one works best.

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We used three colorants (methylene blue, malachite green and bengal rose) at the beginning of our experimentation to verify laccase functionality. We observed the color changes in each colorant when interacting with the pellet (soluble fraction) and with the supernatant (culture medium) of the BL21 LB+CAM culture with which we expressed the laccases.

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Citrate Buffer With Colorants - August 18

No apparent difference between the RIPA buffer control and laccase B pellet laccase is found. The rest of the pellet tubes and all the supernatant tubes do present color differences compared to their controls. This suggests that the laccases A, C and D present functionality when they are and they are not secreted by the cells, and laccase B presents functionality only when it is not secreted by the cells.

All tubes do present color differences compared to their controls. This suggests that the four laccases present functionality when they are and they are not secreted by the cells.

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Laccase B was discarded and we decided to keep working with laccase C (15 A, BBa_K863010).

Citrate Buffer With Colorants - August 18

Once laccase functionality was verified and one laccase was chosen, the experiments that followed were carried out to quantify enzymatic activity.

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The same three colorants were used to verify laccase enzymatic activity. We observed the color changes in each colorant when interacting with the pellet (soluble fraction) and with the supernatant (culture medium) of the BL21 DE3 LB+CAM culture with which we expressed laccase 15A. We confirmed our observations by measuring the absorbances (at the respective wavelength of each colorant) of our control, soluble fractions and culture medium samples. 
 

A constant behaviour of the interaction between laccase 15 A and the three colorants is that the absorbances of soluble fraction samples are smaller than their control (citrate buffer). In contrast, absorbances of culture medium samples are greater than their control (nicker affinity buffer). This demonstrates that protein in the soluble fraction presents greater enzymatic activity than protein in the culture medium.

To rule out that the loss of color was due to a change in pH in the medium, pH strips were used to determine the pH of the tests carried out.

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The samples for the 3 dyes and the two protein concentrates analyzed (soluble fraction or culture medium) were observed values between pH 8 and 7. pH to which no color change is attributed. This verifies / validates the activity observed in soluble fraction.

Citrate Buffer With Colorants - October 02

Then again, the same three colorants were used to verify laccase enzymatic activity with the same procedure of the assay described above.

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Interaction with methylene blue exhibits a different behaviour, compared to the interaction with the other two colorants. Soluble fraction and culture medium samples present smaller absorbance values than their respective colorants. So, in terms of absorbance, in methylene blue it is not very clear if the activity is greater in the soluble fraction or culture medium. However, we can tell from pure observation that it is more evident in the soluble fraction samples.

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For malachite green assays, the absorbances of soluble fraction samples are smaller than their control (citrate buffer). In contrast, absorbances of culture medium samples are greater than their control (nicker affinity buffer). This demonstrates that protein in the soluble fraction presents greater enzymatic activity than protein in the culture medium.

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For bengal rose assays, the absorbances of neither the soluble fraction samples nor the culture medium samples are smaller than their respective control (citrate buffer). This shows no enzymatic activity on any samples.

Again, to rule out that the loss of color was due to a change in pH in the medium, pH strips were used to determine the pH of the tests carried out.

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The samples for the 3 dyes and the two protein concentrates analyzed (soluble fraction or culture medium) were observed values between pH 8 and 7. pH to which no color change is attributed. This verifies / validates the activity observed in soluble fraction.

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The typical curves were made for each type of dye. Thus, we obtained a percentage of degradation of the dye by the laccases obtained in soluble fraction or culture medium. The dye was prepared as indicated in the lab notebook and that color was analyzed at 0% degradation. The corresponding dilutions were made to dilute in 80%, 55% and 25% and only buffer without dye as 0%. Samples and readings were made in triplicate. The obtained curves and the corresponding average absorbance value are presented. The correlation factor R was> 0.98 for the three cases. The samples of culture medium concentrated by precipitation with ammonium sulfate always showed an intense brown color. So the absorbance was read directly from the sample for each of the wavelengths at which each dye absorbed.

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In this case, it was observed that the background noise due to the color of the sample was significant and contributed to the reading in the spectrophotometer. The background value of the samples and the typical curve obtained by diluting each dye with a citrate buffer were considered.

Citrate Buffer With Colorants - October 16

For our next experiment, we now had standardized conditions and took absorbance measurements at the respective wavelength of each colorant (Bengal Rose: 562 nm, Malachite Green: 617 nm and Methylene Blue: 664 nm) to document absorbance through time. The first measurement was taken after 16 hours of incubation at 37°C, and then every 24 hours for 3 days. We had 3 repetitions of 5 different samples of each colorant: culture medium, soluble fraction, purified culture medium, purified soluble fraction and Trametes versicolor commercial laccase. Curves of absorbance vs time were built, as well as typical curves for each colorant. As we focused on the activity measurement as a percentage of degradation through time, the 3 controls from blue, green and rose were considered as the 100%.

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We didn’t have the exact concentration of colorants in our samples, so we measured the activity as a percentage of degradation of each colorant, where a 100% of substrate was considered as the absorbance of the control. Thus, the enzymatic degradation would be expressed as the loss of color. The samples we worked with were laccase in culture medium,  culture soluble fraction, purified culture medium, purified soluble fraction and commercial laccase from T. versicolor. 

The results are presented below as graphs for each colorant.

The samples from the soluble fraction were the ones that presented the greater colorant degradation at the end of the assay, followed by culture medium and T. versicolor samples. Purified soluble fraction samples were the ones that presented the least degradation.

The samples from T. versicolor were the ones that presented the greater colorant degradation at the end of the assay, followed by purified culture medium samples. Purified soluble fraction samples were the ones that presented the least degradation.

The samples from T. versicolor were the ones that presented the greater colorant degradation, followed by soluble fraction samples. Purified soluble fraction samples were the ones that presented the least degradation.

From the previous graphs, we noted a similar behaviour in Methylene Blue and Bengal Rose, where soluble fraction and T. versicolor samples presented the greatest degradation. However, in Malachite Green, the Laccase from Trametes versicolor had a higher degradation rate.

The percentage of degradation of each sample for each colorant at the end of the assay are presented below:

Overall, the best results were obtained by the T. versicolor commercial laccase, followed by the soluble fraction and the purified soluble fraction. However, it is important to remember that T. versicolor has a higher concentration since it was prepared at 1 mg/mL (purified laccases had concentrations of 0.334 µg/mL in the culture medium and 0.1298 µg/mL in the soluble fraction).

These concentrations were determined through BCA quantification protocol, by creating a BCA curve, which is shown below.

Expression
Laccase Solubility Analysis - September 18

We made polyacrylamide gels to verify if there is a difference in laccase expression when induced with IPTG in soluble fractions, insoluble fractions and culture medium samples. The first gel was not run with standardized conditions, so the second gel was made.
 

The appearance of a differentially expressed band observed in the total protein after induction with IPTG, with standardized electrophoretic profiles (same total protein load) proves that the expression of a higher quantity of laccase C (15 A) is potentially enhanced with IPTG.

BPA degradation and detection

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Bisphenol A (BPA), an alkylphenolic compound, is a main monomer in the production of polycarbonates, epoxy resins, and other plastics. Detection and removal of bisphenol A from industrial waste/soil/drinking water are critical to minimize human consumption.

Elimination of hazardous phenolic compounds using laccases has gained attention due to its efficiency. Purified laccase from Paraconiothyrium variabile (PvL) has been reported with high removal percentage of BPA [9].  Also, it was found that BPA degradation was along with the occurrence of laccase production in solid state fermentation with Trametes versicolor [10]. In vitro, the optimum pH range of BPA degradation with laccase was in the acidic region with the optimal performance around pH 5.0 [1]. As we have observed great enzymatic activity at pH of 5.7, we decided to perform our BPA degradation assay with this condition.

The main method for BPA determination is High-performance liquid chromatography (HPLC). However, it is not a very accessible method, and it is very expensive. Thus, we make a review in literature to know alternatives to monitor BPA degradation. Determination of BPA was reported also by spectrophotometer by Ali H. in 2019 [11] , where a molecularly imprinted nanocomposite composed of a covalently connected 3D network of reduced graphene oxide, b-cyclodextrin and polyacrylate was used for selective electrochemical detection of bisphenol A. The nanocomposite selectively captures bisphenol A, it was demonstrated by the removal of bisphenol A (figure 10) It was examined by UV-absorbance study along with HPLC as shown in figure 10, it was observed that the absorbance peak of bisphenol A at 276 nm was diminished, confirming the successful capture of bisphenol A by the nanocomposite.

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Figure 10. BPA absorbance spectra reported by different authors, the value of λmax is indicated by the pink arrow. A) Absorbance spectra showing the complete removal of bisphenol A (BPA) measured by spectrophotometer (a) and HPLC (b) [11]. B) Bisphenol A UV–VIS absorbance spectrum in water [12]. C) UV-VIS spectra of BPA in a reaction system with CC450 [13].

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To determine the concentration of BPA that can be detected by spectrophotometer, we found two reports where BPA was measured by this method, the first one reports a solution of 0.1 mM (25ppm) [12 and the second at 8.3 mM [11]. As BPA degradation by a laccase was reported to be initiated at 4 mM [1], we obtained the UV spectrum of this concentration in aqueous solution in the range of 200-600 nm. The results showed significant UV absorption with λmax at 290 nm (figure 11). Although we observe a very different wavelength from other references (274.6, 265 and 276 nm) as is shown in the figure 10, differences could be explained by the combination with other substances in those reports [11][12][13].
 

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Figure 11. UV spectrum of 4 mM BPA in aqueous solution in the range of 200-600 nm. Spectrophotometer Thermo Scientific Genesys 10S UV-VIS

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Then we developed a standard curve with BPA (0.5-4 mM) and observed a linear behavior between 0.5-2.5 mM (figure 12).

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Image description. The BPA degradation assay calibration curve. Following the concentrations of the TABLE IV. 

The standard calibration curve was obtained from the following samples of different concentrations, starting from 0.5 mM to 4 mM, following the amounts in microliters of the following image. These further UV-spectrophotometer each sample at a wavelength of 290 nm. And obtaining the results shown in Table IV. for the graphing of the standard curve.

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​Table V.  Preparation of each sample used in the obtention of the standard curve with the absorbance given by the UV- spectrophotometer. Using the values of H2O and citrate column for the BPA - absorbance graph in.

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BPA degradation assay by laccase

The laccase-catalyzed degradation of BPA was conducted. BPA stock solution was prepared at 25 mg/ml dissolved in methanol (according to the supplier's instructions, Aldrich 239658), then BPA working solution at 4mM was prepared in water. The reaction mixture (final volume of 1.1 mL) was prepared as follow: 0.5 ml of 4 mM BPA was added to 0.5 mL citrate buffer (100 mM Sodium citrate and 100 mM citric acid, pH 5.7), followed by introducing of the purified laccase from Trametes versicolor at 5 U/mL to the reaction mixture and incubation at 35°C and 250 rpm for different times. The negative control was designed by inserting a citrate buffer to the reaction mixture instead of laccase. To determine BPA degradation, the absorbance (at 290 nm) was measured.

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An average of the absorbances obtained was collected at the next image in order to graph further.

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Table VI. Results of averaging results obtained from graphing the absorbance averages measured at an initial time and after 16 hours have passed.

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Conclusion.

In this interpretation, It is noted that under the conditions carried out, the concentration of BPA decreases from time zero to the second dose 16 hours later. Since the control remains more or less constant with an indifferent variable. This being a support for mentioned literature where in the presence of laccase, BPA is degraded.
Further testing with different conditions, concentrations, temperatures, and different laccases is necessary to obtain more accurate information, as an assay with our purified laccase would be the next step in this assay. As well as performing this experiment with other EDCs other than BPA.
 

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References:

[1] Y. Song et al., “High-resolution comparative modeling with RosettaCM”, Structure, vol 21, no 10, bll 1735–1742, Sep 2013.

[2] K. D. Dykstra et al., “Estrogen receptor ligands. Part 16: 2-Aryl indoles as highly subtype selective ligands for ERα”, Bioorganic & Medicinal Chemistry Letters, vol 17, no 8, bll 2322–2328, 2007.

[3] E. F. Pettersen et al., “UCSF Chimera--a visualization system for exploratory research and analysis”, J Comput Chem, vol 25, no 13, bll 1605–1612, Oct 2004.

[4]  C. J. Williams et al., “MolProbity: More and better reference data for improved all-atom structure validation”, Protein Sci, vol 27, no 1, bll 293–315, Nov 2017.

[5] S. Kim et al., “PubChem in 2021: new data content and improved web interfaces”, Nucleic Acids Res, vol 49, no D1, bll D1388–D1395, Jan 2021.

[6] A. Grosdidier, V. Zoete, en O. Michielin, “SwissDock, a protein-small molecule docking web service based on EADock DSS”, Nucleic Acids Res, vol 39, no Web Server issue, bll W270-7, May 2011.

[7]  A. Roy, A. Kucukural, en Y. Zhang, “I-TASSER: a unified platform for automated protein structure and function prediction”, Nature Protocols, vol 5, no 4, bll 725–738, Apr 2010.

[8] T. Bertrand et al., “Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics”, Biochemistry, vol 41, no 23, bll 7325–7333, Jun 2002.

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[9] Z. Asadgol, H. Forootanfar, S. Rezaei, A. H. Mahvi y M. A. Faramarzi, "Removal of phenol and bisphenol-A catalyzed by laccase in aqueous solution", Journal of Environmental Health Science and Engineering, vol. 12, n.º 1, junio de 2014. Accedido el 21 de octubre de 2021. [Online]. Available: https://doi.org/10.1186/2052-336x-12-93 

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[10] S. Zeng, J. Zhao y L. Xia, "Simultaneous production of laccase and degradation of bisphenol A with Trametes versicolor cultivated on agricultural wastes", Bioprocess and Biosystems Engineering, vol. 40, n.º 8, pp. 1237–1245, mayo de 2017. Accedido el 21 de octubre de 2021. [Online]. Available: https://doi.org/10.1007/s00449-017-1783-1 

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[11] H. Ali, S. Mukhopadhyay y N. R. Jana. New Journal of Chemistry. [Online]. Available: https://pubs.rsc.org/en/content/articlelanding/2019/nj/c8nj05883k 

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[12] R. Abo, N.-A. Kummer y B. J. Merkel, "Optimized photodegradation of Bisphenol A in water using ZnO, TiO₂ and SnO₂ photocatalysts under UV radiation as a decontamination procedure", Drinking Water Engineering and Science, vol. 9, n.º 2, pp. 27–35, september 19 of 2016. [Online]. Available: https://doi.org/10.5194/dwes-9-27-2016  

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[13] Z. Jiao et al., "Degradation of Bisphenol A by CeCu Oxide Catalyst in Catalytic Wet Peroxide Oxidation: Efficiency, Stability, and Mechanism", International Journal of Environmental Research and Public Health, vol. 16, n.º 23, p. 4675, noviembre de 2019. Accedido el 21 de octubre de 2021. [Online]. Available: https://doi.org/10.3390/ijerph16234675