Team:Bielefeld-Germany/Results/substrate

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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 1 shows the ethinyl estradiol without laccase treatment, Figure 2 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 3 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 1: Ethinyl estradiol control without laccases.

Figure 2: Ethinyl estradiol degradation (with TVEL0). The ethinyl estradiol peak disappeared and some new peaks, probable degradation products, occurred.

Figure 3: Estradiol control without laccases.

Figure 4: 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 1: Anthracene calibration curve.

Figure 2: Estrone calibration curve.

Degradation results

The TVEL0 was able to degrade the synthetic estradiol (Fig. 1) and probably anthracene (Fig 3). The ethinyl estradiol control showed that it is stable in the used media (Fig. 2). 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. 4). Estrone (Fig. 5) and estradiol (Fig. 6) 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 1: Ethinyl estradiol + TVEL0 measured by LC-MS. It is shown that the over night sample has only half of the substrate left.

Figure 3: Anthracene + TVEL0. In the over night sample there are no detectable amounts of anthracene left.

Figure 4: The negative control for anthracene without laccases. Like it is shown the concentration of anthracene decreases. This is caused by the Britton Puffer.

Figure 2: Our ethinyl estradiol negative control without laccase. Variation on the peaks is probably caused by an pipetting mistake.

Figure 5: Estrone + TVEL0. The peaks shows that estrone is degraded but after incubation over night it is still estrone left.

Figure 6: 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. 6), 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.

Degradation measurements with high performance liquid chromatography

The measurements were made to test if the produced laccases were able to degrade different hormones. Therefore the produced laccases were inserted in the same concentrations (3 µg mL^-1) to the different measurement approaches. For the correct pH value (which were measured by the Team Activity Test) Britton Robinson buffer at pH 5 was used for all measurements. The initial substrate concentration was 5 µg mL^-1. The results of the reactions without ABTS are shown in Figure 1. On the Y-axis the percentages of degraded estradiol (blue) and ethinyl estradiol (red) are indicated. The X-axis displays the different tested laccases. The degradation was measured at t₀ and after five hours of incubation at 30 °C. The negative control was the substrate in Britton Robinson buffer and showed no degradation of the substrates. The bought laccase TVEL0 which is used as positive control is able to degrade 94.7 % estradiol and 92.7 % ethinyl estradiol. The laccase BPUL (from Bacillus pumilus) degraded 35.9 % of used estradiol after five hours. ECOL was able to degrade 16.8 % estradiol. BHAL degraded 30.2 % estradiol. The best results were determined with TTHL (laccase from Thermus thermophilus). Here the percentage of degradation amounted 55.4 %.

Figure 1: Degradation of estradiol (blue) and ethinyl estradiol (red) with the different laccases after 5 hours without ABTS. In the graph it is shown that the bought laccase TVEL0 which was used as positive control is able to degrade more than 90 percent of the used substrates. None of the bacterial laccases are able to degrade ethinyl estradiol without ABTS but estradiol is degraded in a range from 16 %(ECOL) to 55 % (TTHL). (n = 4)

The results of the reactions of the laccaes with addition of ABTS are shown in Figure 2. On the Y-axis the percentages of degraded estradiol (blue) and ethinyl estradiol (red) are indicated. The X-axis displays the different tested laccases. The degradation was measured at t₀ and after five hours of incubation at 30 °C. The negative control was the substrate in Britton Robinson buffer and showed no degradation of the substrates. The bought laccase TVEL0 which is used as positive control is able to degrade 94.7 % estradiol and 92.7 % ethinyl estradiol. The laccase BPUL (from Bacillus pumilus) degraded 35.9 % of used estradiol after five hours. ECOL was able to degrade 16.8 % estradiol. BHAL degraded 30.2 % estradiol. The best results were determined with TTHL (laccase from Thermus thermophilus). Here the percentage of degradation amounted 55.4 %.

Figure 2: Degradation of estradiol (blue) and ethinyl estradiol (red) with the different laccases after 10 minutes hours with ABTS added. The commercial laccase TVEL0 which was used as positive control is able to degrade all of the used substrates. The bacterial laccase BPUL degraded 100 % of ethinyl estradiol and estradiol. ECOL the laccase from E. coli degraded 6,7 % estradiol and none of the used ethinyl estradiol. BHAL degraded 46.9 % of estradiol but no ethinyl estradiol. The laccase TTHL from Thermus thermophilus degraded 29.5 % of estradiol and 9.8 % ethinyl estradiol.(n = 4)