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We analyzed the degradation of our substrates with the spectrofluorophotometer. As you can see it in the figures below the ethinyl estradiol and estradiol are degraded over night. Figure 3 shows the ethinyl estradiol without laccase treatment, Figure 4 shows that no more ethinyl estradiol can be detected in the sample after the degradation and new peaks appear, which might be represent possibly degradation products. In Figure 5 you can see the estradiol control without laccases. Like ethinyl estradiol our estradiol peak does not appear after the degradation and new peaks appear indicating that those are new degradation products.

Figure 3: Ethinyl estradiol control without laccases. Ethinyl estradiol was measured in spectrofluorophotometer without laccase treatment to have a control.

Figure 4: Ethinyl estradiol degradation (with TVEL0). The ethinyl estradiol peak disappeared and some new peaks, probable degradation products, occurred.

Figure 5: Estradiol control without laccases.

Figure 6: Degradation (with TVEL0) of estradiol. It is shown that some estradiol is left but probable degradation products appeared.

Liquid chromatography–mass spectrometry

Dilution series

Our substrates are soluble in methanol. We set the standards to a concentration of 1 mg mL^-1. The upper detection limit for the LC-MS was evaluated at concentration of 10 µg L^-1 for the substrates estrone and estradiol. The same limit of detection was used for ethinyl estradiol and anthracene. We only used those four substrates. For all LC-MS sample preparations we used the T. versicolor laccases. The dilution series was prepared in methanol and 50 % acetonitril-water (v/v).

Figure 7: Anthracene calibration curve.

Figure 8: Estrone calibration curve.

Degradation results

The TVEL0 was able to degrade the synthetic estradiol (Fig. 9) and probably anthracene (Fig. 11). The ethinyl estradiol control showed that it is stable in the used media (Fig. 10). Anthracene disintegrates in the Britton Puffer. But it could be observed, that there is less anthracene measurable with the LC-MS. The results indicate, that the laccase is able to degrade anthracene (Fig. 12). Estrone (Fig. 13) and estradiol (Fig. 14) were degraded as well. Using estrone it could not be identify any degradation products. The reason for this could be that the products are not detectable with LC-MS or with the applied methods. Peaks in the degradation of estradiol have been shown but we were not able to identify them. It could be degradation products. In the following figures the results of the LC-MS measurements are presented.

Figure 9: Ethinyl estradiol + TVEL0 measured by LC-MS. It is shown that the over night sample has only half of the substrate left.

Figure 11: Anthracene + TVEL0. In the over night sample there are no detectable amounts of anthracene left.

Figure 12: The negative control for anthracene without laccases. Like it is shown the concentration of anthracene decreases. This is caused by the Britton Puffer.

Figure 10: Our ethinyl estradiol negative control without laccase. Variation on the peaks is probably caused by an pipetting mistake.

Figure 13: Estrone + TVEL0. The peaks shows that estrone is degraded but after incubation over night it is still estrone left.

Figure 14: Estradiol degradation analyses with mass-spectrometry. On the X-axis the retention time is listed. The Y-axis shows the mass/charge ratio. From white to red the intensity of the measured samples is presented. On the figure above you can see the t₀ estradiol while the figure below shows the degradation. The analytes retended in the first minute are the media soillings. Since we know that the retention time of estradiol is on min 5 we could see that over night no more estradiol is left and some other peaks appear which are probably degradation products

We also tried to measure the degradation using mass-spectrometry. Since quantification via mass-spectrometry is difficult regarding the ionization of the analytes, we quantified our substrates by UV-light. Nevertheless, mass spectrometry enables identification of possible degradation products. We analyzed estradiol degradation in detail (Fig. 14), resulting in the detection of possible chemical compounds generated during the (enzymatic) degradation. Until now we are not sure how estradiol degradation works, but with more time granted, the degradation products can be identified.
Since we have seen some possible degradation products, we used more estradiol, ethinyl estradiol and more laccase for the reaction to the LC-MS measurement. We found out that after the laccase treatment two new peaks appears in both estradiol and ethinyl estradiol. A check against databases could not identify those components so we did an MS-MS on component A. Since component B is fewer in concentration then component A, we could not find out anything about it (Fig. 15 and Fig. 16). To note the chemical formulares are M+H values. For the real chemical formular you have to deduct this H.

Figure 15: Massspectromerty measurement of estradiol. On the X-axis you can see the Time in minutes and on the Y-axis the relative intensity. You can see two new peaks component A and B after laccase treatment. Component A was taken for MS-MS

Figure 16: Massspectromerty measurement of ethinyl estradiol. On the X-axis you can see the Time in minutes and on the Y-axis the relative intensity. You can see two new peaks component C and D after laccase treatment. Component C was taken for MS-MS

The Massspectrometry-Massspectrometry results showed us interesting peaks. Compare to the native estradiol component A shows peaks with 2 H atoms difference respectively component C for ethinly estradiol. Since we wanted to know more about our products we went to Prof. Dr. Dietmar Kuck, an organic chemister, to discuss our results. He suggest us two modells for the producs. First of them was that our laccases builds ketone reacting with the hydroxy group not in the phenolic ring. But this idea was incorrect because we could see 2 H less molecule also in ethinyl estradiol and the ethin group could not react with oxygen (Fig 17). Our next idea was, as described in the literature, that our laccase dehydrates the hydroxy group on the phenolic compond of the estradiol and ethinyl estradiol. This would make sence because it would build a chinon which you can see in Figure 18.

Figure 17: Chemical structure of the ketone after laccase treatment. This structure was designed with ChemBioDraw Ultra 12.0 after discussion with Mr. Kuck on our MS/MS data.

Figure 18: Suggested Chemical structure of the oxydised estradiol. This structure was designed with ChemBioDraw Ultra 12.0 after discussion with Mr. Kuck on our MS/MS data.

Further substrate analysis via HPLC

HPLC analysis of polycylic aromatic hydrocarbons

The results of our negative control measurements of polycyclic aromatic hydrocarbons showed that the PAHs decayed without laccase treatment after one hour in Britton Robinson (BR)-buffer. This is shown in Figure X.
The next step was to check which substances in the reaction approach may cause the decay. Therefore naphthalene was dissolved in methanol (which is the solvent for the substrate and used for stopping the reaction), acetonitrile (which could be used as alternative solvent) and BR-buffer with and without ABTS. The results are shown in Figure Y. Using pure methanol and acetonitrile naphthalene is not decayed. In BR-buffer with and without ABTS the decay is nearly completed after one hour treatment. So BR-buffer seems a bad choice to test the degradation of naphtalene under laccase treatment.

Figure : Decay of the PAHs naphthalene, acenaphthene and phenantrene in BR-buffer at 30 °C after one hour. The initial concentration was 1 µg mL ^-1 for all PAHs. After one hour nearly all PAHs decayed completely. (n=2)

Figure : Naphthalene decay in four different approaches at 30 °C after one hour. Dissolved in methanol, dissolved in acetonitrile, with BR-buffer and with BR-buffer together with ABTS. (n=2)

HPLC analysis of analgesics

Another class of substrates were analgesics. For the three analgesics substrates we used we have different optimal extinction and emission values. Additional, difficulties occurred with ibuprofen. Instead of one single peak we found two and they didn't correlate with the used concentrations. With diclofenac the extinction and emission values were not found. Therefore the substrate was analyzed with the spectrofluorophotometer but this has also shown no clear peak for diclofenac and made it therefore not measurable.

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