Summary

Spectrofluorophotometer Anaylses

We analyzed the degradation of our subtrates 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 ethiny 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 the peak does not appear after the degradation and new peaks appear indicating that thoose are new degradation products.

Figure 1: Ethinyl estradiol control without laccases

Figure 2: Ethinyl estradiol degradation. There are some peaks probabyl degradation products. The ethinyl estradiol peak disapeard

Figure 3: Estradiol control without laccases

Figure 4: Degradation of estradiol. We can see that some estradiol is left but probabyl degradation products could still be seen

Liquid chromatography–mass spectrometry

Dilution series

Our substrates are soluble in methanol. We set the standardts to a concentration of 1 mg mL^-1. The upper detection limit for the LC-MS was evaluated at concentration of 10µg/l for the substrates esteron and estradiol. The same limit of detection was used for ethinyl estradiol and anthracen. We only used thoose 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: Anthracen calibration curve

Figure 2: Estrone calibration curve

Degradation results

The TVEL0 was able to degrade the synthetic estradiol (fig. 1) and propably anthracen. (fig 3.). The ethinylestradiol calibration curve showed that it is stable in the used media (fig 2.). Anthracen disintegrates in the Britton Puffer. But it could be observed, that there is less anthracen measurable with the LC-MS. The results indicate, that thelaccase is able to degrade anthracene(fig. 4). Estrone (fig. 5) and estradiol (fig. 6) were degraded as well. Using estrone we could not identify any degradation products. The reason for this could be that the products are not detectable with LC-MS or with methods applied. We could identify peaks in the degradation of estradiol but were not able to identify them. In the following figures one can see the results of the LC-MS measurements. The controls in different media showed no changes in concentration.

Figure 1: Ethinylestradiol + TVEL0 measured by LC-MS. You can see that the over night sample has onyl half sub>2of the substrate left.

Figure 3: Anthracen + TVEL0. In the over night sample there is undetacable less Anthracen.

Figure 4: The negative control for Anthracen without Laccases. As you can see the concentration of Anthracen goes down. This is caused by the Britton Puffer

Figure 2: Our Ethinylestradiol 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 degradated but still the over night sample is still in the detection

Figure 6: Estradiol degradation analyses with mass-spectrometry. On the X-axis is listed the retention time. Y-axis the mass/charge ratio. From white to red is the intensity of the measured samples. On the figure above you can see the t0 Estradiol while the figure below shows the degradation. The analytes retendet in min 1 are the media soilling. 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 anaylzed 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.

High performance liquid chromatography

Dilution series

Figure 1.1:The detection range of ethinyl estradiol. Concentrations between 0.1 µg ml ^-1 and 3 µg ml ^-1 a/partinfo> in a Bioengineering NFL22 with a total volume of 6re detecable.

At first we mesured dilution series of all different substrates. While estradiol and ethinyl estradiol where easy to mesure, we didnt succed often with the estrone calibration curves. This was caused by its bad solubility. The retention time for estradiol is 5.8 minutes, for estrone 4.7 minutes and for ethinyl estradiol 5.2 minutes. For all estrogens we could use the same extinction and emmission values: Ex 230, Em 310.

Figure 1.2:The calibration curve of ethinyl estradiol. Concentrations between 0.1 µg ml ^-1 and 3 µg ml ^-1.

Figure 1.3:Calibration curve for estradiol.

The next substrate class where the analgesics. These three substrates have different optimal extinction and emission values. Additional difficulties occured with naproxene and ibuprofen. Instead of one single peak we found two for each substrate, and none of them correlated with the used concentration. With diclofenac we are still not sure which extinction and emmission values are to use. We found different values and aditionaly we analysed it with a spectrofluorometer but this has also shown no clear peak for diclofenac.

Three of the four PAH´s have the same extinction and emmission values. Simular to the estrogens, the PAH calibration curves are easy to generate. Naphthalene has a retention time of 9.6 minutes and its detection range is also 0.1 to 2.5 µg*mL^-1. Acenaphthene with a retention time of 15.1 minutes and phenantrene with a retention time of 17 minutes have maximal detectable concentration of 1.5 µg*mL^-1.

Anthracene will be mesured later.

Figure 1.4:BPUL degradated estradiol. Overleyed chromatograms from different timepoints. The first peak caused by the solvent.

Negative controls

Figure 2.1:The PAH´s naphthalene, acenaphthene and phenantrene in BR-buffer at 30 °C. The startconcentration was 1 µg mL ^-1 for all PAH´s. After 1 houre nearly all PAH´s decayed completely.

Figure 2.2:Naphthalene decay in four different conditions. Dissolved in methanol, dissolved in acetonitrile, with BR-buffer and with BR-buffer together with ABTS

The results of our negative controls of the first Polycyclic aromatic hydrocarbon, PAH, degradations showed, that PAH´s decay without laccase with a high speed. So we take a closer look at the three PAH´s in Britton Robinson (BR)-buffer. The result can be seen in figure 2.1. After one hour most of the used substrates decayed.

The next step was to check if and which substance cause the decay. In this test we dissolved naphthalene in acetonitrile and in methanol and compared the pure solvents with the influence of the BR-buffer and BR-buffer with ABTS. With pure methanol or acetonitrile naphthalene decays slow in comparison to BR-buffer. In BR-buffer with or without ABTS the decay happens faster and under nearly the same velocity. So BR-buffer seems to be a bad choice to test if our laccases degrade PAH´s.

Estradiol and ethinyl estradiol did not decay in BR-buffer, as shown by the negative controls in degradation experiments.

Degradation

Figure 3.1:Comparison between two of our laccases BPUL and ECOL with the purchased laccase TVEL0 under ABTS influence.

Figure 3.2:Purchased laccase TVEL0 degrades ethinyl estradiol. Estradiol in BR-buffer without laccase as negative control.

Degradation reactions with ABTS showed the expected results. "Team Activity Test" already showed, that the laccases have the ability to oxidise ABTS. The fact that oxidised ABTS reacts chemicaly with the substrates, explains that all of our active laccases have the ability to degrade ethinly estradiol and other substrates. The potential of the purchased laccase TVEL0 is much higher, shown in figure 3.2. BPUL has not the same potential as the purchased laccase. But BPUL degrades estradiol without the influence of ABTS.

Figure 3.3:Our laccase BPUL in unknown concentration degrades estradiol. Estradiol in BR-buffer without laccase as negative control.

Outlook

Anthracene, lindane and diclofenac are to be detected with the HPLC. Ibuprofen and naproxen need a good calibration curve. For naphthalene, acenaphthene and phenantrene we have to test different buffers or lower the temperature too keep ist more stable. We have only tested BPUL and ECOL so far. "Team Activity Test" showed that TTHL is active so we can test this laccase for the different substrates. Also there will be the trametis versicolor laccases TVEL5, TVEL10, TVEL13 and TVEL20. Additional we want to determine kcat/km for the degradable substances.
On the LC-MS we did not measure our Substrates with our self produced Laccases. If we had the opportunity, the would test our self produced Laccases with the Substrates. Besides we did not measure all Substrates they need to be measured as well to identify degradation products.