Team:Bielefeld-Germany/Results/substrate

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==Dilution series==
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[[File:Bielefeld2012_detectionlimit.JPG|200px|thumb|right|'''Figure 1.1:'''The detection range of ethinyl estradiol. Concentrations between 0.1 µg ml <sup>-1</sup> and 3 µg ml <sup>-1</sup> a/partinfo> in a Bioengineering NFL22 with a total volume of 6re detectable.]]
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[[File:Bielefeld2012_detectionlimit.JPG|200px|thumb|right|'''Figure 1.1:''' The detection range of ethinyl estradiol. Concentrations between 0.1 µg ml <sup>-1</sup> and 3 µg ml <sup>-1</sup> a/partinfo> in a Bioengineering NFL22 with a total volume of 6re detectable.]]
At first we measured the dilution series of all different substrates. While we were able to measure estradiol and ethinyl estradiol, we did not succeed  with the estrone calibration curves. This was probably 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 emission values: Ex <sub>230</sub>, Em<sub>310</sub>.
At first we measured the dilution series of all different substrates. While we were able to measure estradiol and ethinyl estradiol, we did not succeed  with the estrone calibration curves. This was probably 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 emission values: Ex <sub>230</sub>, Em<sub>310</sub>.
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Revision as of 21:40, 25 October 2012

Substrate Analysis

Contents


Introduction

Spectrofluorophotometer Analysis

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.

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 detectable.

At first we measured the dilution series of all different substrates. While we were able to measure estradiol and ethinyl estradiol, we did not succeed with the estrone calibration curves. This was probably 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 emission values: Ex 230, Em310.

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 were the analgesics. For the three analgesics substrates we used we have different optimal extinction and emission values. Additional, difficulties occurred 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 emission values are to use. We found different values and additionally we analyzed it with the spectrofluorophotometer but this has also shown no clear peak for diclofenac.

Three of the four PAHs have the same extinction and emission values. Similar to the estrogens, the PAH calibration curves were generated. 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.

With more time granted, we want measure the anthracene.

Figure 1.4:BPUL degraded estradiol. Overlayed 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 start concentration was 1 µg mL -1 for all PAH´s. After 1 hour 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 measurements (polycyclic aromatic hydrocarbon) showed the PAHs decay without laccase at high speed. So we take a closer look at the three PAHs dissolved 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 which buffer substance may cause the decay. In this experiment we dissolved naphthalene in acetonitrile and in methanol and compared the pure solvents with the influence of the BR-buffer with and without ABTS. Using pure methanol or acetonitrile naphthalene decays slow in comparison to pure 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 non preferable choice to test if our laccases degrade PAHs.

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 chemically with the substrates, explains that all of our active laccases have the ability to degrade ethinyl 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 naproxene need an improvement of the calculated calibration curve. For naphthalene, acenaphthene and phenantrene we have to test different buffers or lower the temperature too keep it more stable. We have only tested BPUL and ECOL so far. "Team Activity Test" showed that TTHL is also active so we can test this laccase for the different substrates. Further there will be the Trametis versicolor laccases TVEL5, TVEL10, TVEL13 and TVEL20. Additional we want to determine the values kcat and km for the degradable substances. Using the LC-MS we did not measure our substrates with our self produced laccases. If there will be more time granted, we would test our self produced laccases with the substrates mentioned above.


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