Summary

First some trials of shaking flask cultivations were made with changing parameters to identify the best conditions for the production of the laccase CueO from E. coli BL21 (DE3) named ECOL fused to a His tag. Because of no measured activity in the cell lysate a purification method was established (using Ni-NTA His tag resin and Syringe or ÄKTA method). The purified ECOL could be identified by SDS-PAGE (molecular weight of 53.4 kDa) as well as by MALDI-TOF. The fractionated samples were also tested concerning their activity. A maximal activity of 55% was reached, measured in ABTS_ox [µM]. After measuring activity of ECOL a scale up was made up to 3 L and then also up to 6 L that enables an intense screening afterwards. A further scale up to 12 L with a optimized medium was implemented to enable additional experiments to characterize ECOL. Additional scale up experiments will be important for further application. The enzyme was characterized regarding its temperature and pH optimum and concerning the influence of different concentrations of CuCl₂, ABTS, MeOH and acetonitrile.

Cultivation, Purification and SDS-PAGE

Shaking Flask Cultivations

3 L Fermentation E. coli KRX with <partinfo>BBa_K863005</partinfo>

Figure 1: Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in an Infors Labfors Bioreactor, scale: 3 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold pO₂ at 50 %, OD₆₀₀ measured every 30 minutes.

After the positive measurement of activity of ECOL we made a scale-up and fermented E. coli KRX with <partinfo>BBa_K863005</partinfo> in an Infors Labfors fermenter with a total volume of 3 L. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 1. The exponential phase started after 1.5 hours of cultivation. The cell growth caused a decrease in pO₂. After 2 hours of cultivation the agitation speed increased up to 629 rmp (5.9 hours) to hold the minimal pO₂ level of 50 %. Then, after 4 hours there was a break in cell growth due to induction of protein expression. The maximal OD₆₀₀ of 2.78 was reached after 5 hours. In comparison to E. coli KRX (OD_600,max =4.86 after 8.5 hours) and to E. coli KRX with <partinfo>BBa_K863000</partinfo> (OD_600,max =3.53 after 10 hours, time shift due to long lag phase) the OD_{600 max} is lower. In the following hours, the OD₆₀₀ and the agitation speed decreased and the pO₂ increased, which indicates the death phase of the cells. This is caused by the cell toxicity of ECOL (reference: [http://www.dbu.de/OPAC/ab/DBU-Abschlussbericht-AZ-13191.pdf DBU final report]). Hence, cells were harvested after 12 hours.

Purification of ECOL

The harvested cells were resuspended in Ni-NTA- equilibration buffer, mechanically disrupted by homogenization and cell debris were removed by centrifugation. The supernatant of the cell lysate was loaded on the Ni-NTA column (15 mL Ni-NTA resin) with a flow rate of 1 mL min^-1 cm^-2. Then the column was washed with 10 column volumes (CV) Ni-NTA equilibration buffer. The bound proteins were eluted by an increasing Ni-NTA elution buffer step elution from 5 % (equates to 25 mM imidazol) with a length of 50 mL, to 50 % (equates to 250 mM imidazol) with a length of 60 mL, to 80 % (equates to 400 mM imidazol) with a length of 40 mL and finally to 100 % (equates to 500 mM imidazol) with a length of 80 mL. This strategy was chosen to improve the purification caused by a step by step increasing Ni-NTA-elution buffer concentration. The elution was collected in 10 mL fractions. Due to the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak only the UV-detection signal of the wash step and the elution are shown. A typical chromatogram of purified laccases is illustrated here. The chromatogram of the ECOL elution is shown in Figure 2:

Figure 2: Chromatogram of wash and elution fractions from FLPC Ni-NTA His tag Purification of ECOL produced by 3 L fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo>. ECOL was eluted by a concentration of 50 % (equates to 250 mM imidazol) with a maximal UV-detection signal of 292 mAU.

The chromatogram shows two distinguished peaks. The first peak was detected at a Ni-NTA-equilibration buffer concentration of 5 % (equates to 25 mM imidazol) and resulted from the elution of weakly bound proteins. After increasing the Ni-NTA elution buffer concentration to 50 % (equates to 250 mM imidazol), an UV-detection signal peak of 292 mAU was measured. The area of this peak indicates that a high amount of protein was eluted. The corresponding fractions were analyzed by SDS-PAGE to detect ECOL. There were no further peaks detectable. The following increasing UV detection signal results from the rising imidazol concentration of the Ni-NTA elution buffer. The corresponding SDS-PAGES are shown in Figure 3.

SDS-PAGE of ECOL purification

Figure 3: SDS-Pages of purified E. coli KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] lysate (fermented in 3 L an Infors Labfors fermenter). The flow-through and elution fraction 2-9 are shown. The arrow marks the ECOL band with a molecular weight of 53.4 kDa.

In Figure 3 the SDS-PAGE of the Ni-NTA His tag purification of the lysed culture (E. coli KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]) is shown including the flow-through and the fractions 2 to 9. The red arrow indicates the band of ECOL with a molecular weight of 53.4 kDa, which appears in all fractions. The strongest bands appear in fractions 6 and 7. These were the first two fractions (each 10 mL) eluted with 50 % Ni-NTA elution buffer (equates to 250 mM imidazol), in which the distinguished peak appeared. These bands were analyzed by MALDI-TOF and identified as CueO (ECOL). In contrast, the second, faint band with a lower molecular weight could not be identified.

6 L Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo>

Figure 4: Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in a Bioengineering NFL22 fermenter, scale: 6 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation increased when pO₂ was below 30 %, OD₆₀₀ taken every hour.

Another scale-up of the fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> was made up to a final working volume of 6 L in a Bioengineering NFL 22 fermenter. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 3. There was no noticeable lag phase and the cells immediately began to grow. The cells were in an exponential phase between 2 and 4 hours of cultivation, which results in a decrease of pO₂ value and therefore in an increase of agitation speed. After 4 hours of cultivation the maximal OD₆₀₀ of 2.76 was reached, which is comparable to the 3 L fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo>. Due to induction of protein expression there is a break in cell growth. The death phase started, which is indicated by an increasing pO₂ and a decreasing OD₆₀₀. This demonstrates the cytotoxicity of the laccase for E. coli, which was reported by the [http://www.dbu.de/OPAC/ab/DBU-Abschlussbericht-AZ-13191.pdf DBU]. In comparison to the fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> under the same conditions (OD_600,max= 3.53), the OD_600,max was lower. Cells were harvested after 12 hours.

Purification of ECOL

The harvested cells were resuspended in Ni-NTA-equilibration buffer, mechanically disrupted by homogenization and cell debris were removed by centrifugation. The supernatant of the cell lysate was loaded on the Ni-NTA column (15 mL Ni-NTA resin) with a flow rate of 1 mL min^-1 cm^-2. The column was washed by 10 column volumes (CV) Ni-NTA- equilibration buffer. The bound proteins were eluted by an increasing Ni-NTA- elution buffer gradient from 0 % to 100 % with a length of 200 mL and the elution was collected in 10 mL fractions. Due to the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak only the UV-detection signal of the wash step and the eluate are shown. A typical chromatogram of purified laccases is shown here. The chromatogram of the ECOL elution is shown in Figure 5:

Figure 5: Chromatogram of wash and elution from FLPC Ni-NTA His tag purification of ECOL produced by 3 L fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo>. ECOL was eluted between a process volume 670 mL to 750 mL with a maximal UV-detection signal of 189 mAU.

After washing the column with 10 CV Ni-NTA-elution buffer the elution process was started. At a process volume of 670 mL to 750 mL the chromatogram shows a remarkable widespread peak (UV-detection signal 189 mAU) caused by the elution of a high amount of proteins. The run of the curve show a fronting. This can be explained by the elution of weakly bound proteins, which elutes at low imidazol concentrations. A better result could be achieved with a step elution strategy (see purification of the 3 L Fermentation above). To detect ECOL the corresponding fractions were analyzed by SDS-PAGE.

SDS-PAGES of ECOL purification

Figure 6: SDS-Pages of lysed E. coli KRX culture containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] (fermented in a 6 L Bioengineering NFL22) after purification. The flow-through, wash and the elution fraction 1 to 15 are shown (except from fraction 11/12). The arrow marks the ECOL band with a molecular weight of 53.4 kDa.

In Figure 6 the SDS-PAGE of the Ni-NTA His tag purification of the lysed culture E. coli KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] (6 L fermentation) including the flow-through, wash and the fractions 1 to 15 (except from fraction 11/12) is shown. The red arrow indicates the band of ECOL with a molecular weight of 53.4 kDa, which appears in all fractions. The strongest bands appear from fractions 3 and 8 with a decreasing amount of other non-specific bands. In summary, the scale up was successful, improving protein production and purification once again. Furthermore the bands were analyzed by MALDI-TOF and identified as CueO (ECOL).

Since Regionals: 12 L Fermentation E. coli KRX with <partinfo>BBa_K863005</partinfo>

Figure 7: Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in an Bioengineering NLF 22, scale: 12 L, HSG autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold pO₂ at 50 %, OD₆₀₀ measured every hour.

Finally we made another scale-up and fermented E. coli KRX with <partinfo>BBa_K863005</partinfo> in an Bioengineering NLF 22 fermenter with a total volume of 12 L to produce a high amount of the enzyme for further characterizations. This time we used HSG autoinduction medium to get a higher biomass. Agitation speed, pO₂ and OD₆₀₀ were determined and we analyzed the glycerin concentration of the samples. The data are illustrated in Figure 7. For adaption to the medium, there is a lag phase of one hour. Between the 3 and 8 hours of cultivation the cells are in the exponential phase. During this phase the cells consumed O₂, so that the agitation speed was increased automatically, as well as glycerin. After 11 hours of cultivation the pO₂ increased, the glycerin is completely consumed and the cells are in the stationary phase. The maximal OD₆₀₀ of 11.1 was reached after 15 hours of cultivation. The cells were harvested after 19 hours of cultivation.

Since Regionals: Purification of ECOL

The harvested cells were resuspended in Ni-NTA- equilibration buffer, mechanically disrupted by homogenization and cell debris were removed by centrifugation, microfiltration as well as diafiltration to concentrate the protein concentration in the cell lysate solution. This solution of the cell lysate was loaded on the Ni-NTA column (15 mL Ni-NTA resin) with a flow rate of 1 mL min^-1 cm^-2. Then the column was washed with 10 column volumes (CV) Ni-NTA equilibration buffer. The bound proteins were eluted by an increasing Ni-NTA elution buffer step elution from 5 % (equates to 25 mM imidazol) with a length of 40 mL, to 50 % (equates to 250 mM imidazol) with a volume of 80 mL, to 80 % (equates to 400 mM imidazol) and finally to 100 % (equates to 500 mM imidazol) with a volume of 80 mL. This strategy was chosen to improve the purification caused by a step by step increasing Ni-NTA-elution buffer concentration. The elution was collected in 10 mL fractions. Due to the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak only the UV-detection signal of the wash step and the elution are shown. A typical chromatogram of purified laccases is illustrated here. The chromatogram of the ECOL elution is shown in Figure 2.

Figure 2: Chromatogram of wash and elution fractions from FLPC Ni-NTA His tag purification of ECOL produced by 12 L fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo>. ECOL was eluted at a concentration of 50 % (equates to 250 mM imidazol) with a maximal UV-detection signal of 292 mAU.

The chromatogram shows two distinguished peaks. The first peak was detected at a Ni-NTA-equilibration buffer concentration of 5 % (equates to 25 mM imidazol) and resulted from the elution of weakly bound proteins. After increasing the Ni-NTA elution buffer concentration to 50 % (equates to 250 mM imidazol), an UV-detection signal peak of 140 mAU was measured. The area of this peak indicates that a high amount of protein was eluted. In addition, a second peak right behind the first peak can be detected. At this point it is not clear which peak contains our product and which peak is caused by impurities. The corresponding fractions were analyzed by SDS-PAGE to detect ECOL. A last peak can be detected after increasing the elution buffer concentration to 100 % (equates to 500 mM imidazol). This peak could be explained by impurities which were strongly bound on the Ni-NTA-resin. All corresponding fractions with an UV-signal were analyzed by SDS-PAGES. The Results are shown in Figure 3.

SDS-Page of protein purification

Figure 1: SDS-Page of purification from the 12 L fermentations from 10/11 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] A, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] D) and 10/12 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012] B, <partinfo>BBa_K863022</partinfo> C). Purification of the supernatant via microfiltration, diafiltration and His-trap column (step gradient with 5 %, 50 % and 100 % elution buffer). The red arrow shows ECOL.

The SDS-Page showed ECOL after purification via His-trap column. Charged are selected samples where peaks were seen in the chromatogram.

MALDI-TOF Analysis of ECOL

The E. coli laccase was identified using the following software

• FlexControl
• Flexanalysis and
• Biotools

Figure 7: The MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) spectrum.

Figure 8: Part of MALDI-TOF Evaluation

Activity Analysis of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 ECOL]

Initial activity tests of purified fractions

Figure 9: [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase activity measured in 100 mM sodium acetate buffer (pH 5), 0.1 mM ABTS, to a final volume of 200 µL at 25 °C over a time period of 12 hours. Each tested fraction reveals activity reaching saturation after 2.5 to 4 hours with a maximum of ~52 µM ABTS_ox (fraction 7). (n=4)

[http://partsregistry.org/Part:BBa_K863005 ECOL] pH optimum

To determine at which pH the [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase has its optimum in activity, a gradient of sodium acetate buffer pHs was prepared. Starting with pH 1 to pH 9 [http://partsregistry.org/Part:BBa_K863005 ECOL] activity was tested using the described conditions above and 0.03 mg mL^-1 protein. The results are shown in Figure 10. A distinct pH optimum can be seen at pH 5. Saturation is reached after 2.5 hours with 53% oxidization of ABTS by the [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase at pH 5 (53 µM oxidized ABTS). The other tested pHs only led to a oxidation of up to 17% of added ABTS, respectively. Figure 11 shows the results of the analog experiments with laccase that was not incubated with CuCl₂ before the activity measurements. Again, a pH optimum at pH 5 can be determined with 24 µM ABTS (24%) oxidized by [http://partsregistry.org/Part:BBa_K863005 ECOL] after 8 hours under these conditions.

Figure 12 represents the negative control showing the oxidization of ABTS by 0.4 mM CuCl₂ at the chosen pHs. The greatest increase in oxidized ABTS can be seen at a pH of 5: after 5 hours 15% ABTS is oxidized by CuCl₂ alone. Nevertheless this result does not have an impact on the activity of the [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase at pH 5, which is still the optimal pH. Therefore it has the same pH optimum as TVEL0.

Figure 10: [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase activity measured in 100 mM sodium acetate buffer with a range of different pHs from pH 1 to pH 9, 0.1 mM ABTS, to a final volume of 200 µL at 25°C over a time period of 12 hours. The optimal pH for [http://partsregistry.org/Part:BBa_K863005 ECOL] is pH 5 with the most ABTS_ox.

Figure 11: [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase activity measured in 100 mM sodium acetate buffer with a range of different pHs from pH 1 to pH 9, 0.1 mM ABTS, to a final volume of 200 µL at 25°C over a time period of 12 hours. The tested enzymes were not incubated with CuCl₂ before activity measurements. The optimal pH for [http://partsregistry.org/Part:BBa_K863005 ECOL] is pH 5 with the most ABTS_ox.

Figure 12: Negative control for pH activity test using 0.04 mM CuCl₂ H₂O instead of laccase to determine the potential of ABTS getting oxidized by CuCl₂.

With regard to our project knowledge of the optimal pH is useful. Since waste water in waste water treatment plants has an average pH of 6.9 it has to be kept in mind, that a adjustment of the pH is necessary for optimal laccase activity.

[http://partsregistry.org/Part:BBa_K863005 ECOL] CuCl₂ concentration

Another test of [http://partsregistry.org/Part:BBa_K863005 ECOL] was done to survey the best CuCl₂ concentration for the activity of the purified [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase. 0.03 mg mL^-1 protein were incubated with different CuCl₂ concentration ranging from 0 to 0.7 mM CuCl₂. Activity tests were performed with the incubated samples, in 100 mM sodium actetate buffer (pH 5), 0.1 mM ABTS, to a final volume of 200 µL. The activity was measured at 420 nm, 25°C and over a time period of 10 hours. As expected the saturation takes place after 5 hours (see Figure 13). The differences in the activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase incubated in different CuCl₂ differ minimal. The highest activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase is observed after incubation with 0.4 mM CuCl₂ (42% of added ABTS). With a higher concentration of 0.7 mM CuCl₂ the activity seems to be reduced (only 41% ABTS got oxidized). This leads to the assumption that CuCl₂ supports the [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase activity but concentrations exceeding this value of CuCl₂ may have a negative impact on the ability of oxidizing ABTS. Without any CuCl₂ application [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase show less activity in oxidizing ABTS (see Figure 12). This fits the expectations as laccases are copper reliant enzymes and gain their activity through the incorporation of copper. Additionally negative controls were done using the tested concentrations of CuCl₂ but no laccase was added to detect the oxidization of ABTS through copper (see Figure 14). The more CuCl₂ was present, the more ABTS was oxidized after 5 hours. Still the maximal change accounts only for ~6% oxidized ABTS after 5 hours.

Figure 13: Activity measurement using 0.1 mM ABTS of [http://partsregistry.org/Part:BBa_K863005 ECOL] incubated in different CuCl₂ concentrations. Incubation with 0.1 mM CuCl₂ or higher concentrations leads to an increase in ABTS_ox.

Figure 14: Negative control for CuCl₂ activity Tests using different concentrations of CuCl₂ H₂O instead of laccase to determine the potential of ABTS getting oxidized through CuCl₂.

In relation to apply the laccase in waste water treatment plants it is beneficial knowing, that small amounts of CuCl₂ are enough to activate the enzymes. This reduces the cost factor for the needed CuCl₂ to incubate the laccases before application.

[http://partsregistry.org/Part:BBa_K863005 ECOL] activity at different temperatures

Figure 15: Standard activity test for [http://partsregistry.org/Part:BBa_K863005 ECOL] measured at 10°C and 25°C resulting in a decreased activity at 10°C. As a negative control the impact of 0.4 mM CuCl₂ in oxidizing ABTS at 10°C were analyzed.

[http://partsregistry.org/Part:BBa_K863005 ECOL] activity depending on different ABTS concentrations

Figure 16: Analysis of ABTS oxidation by [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase tested with different amounts of ABTS. The higher the amount of ABTS the more oxidized ABTS can be detected.

Impact of MeOH and acetonitrile on [http://partsregistry.org/Part:BBa_K863005 ECOL]

For substrate analytic tests the influence of MeOH and acetonitrile on [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase had to be determined, because substrates have to be dissolved in these reagents. The experiment setup included 0.03 mg mL^-1 [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase, 100 mM sodium acetate buffer, different amounts of MeOH (Figure 17) or acteonitrile (Figure 18), 0.1 mM ABTS, to a final volume of 200 µL. The activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] was found to be increased in presence of low concentrations (1 % v/v) of either MeOH or acetonitrile resulting in an higher amount of oxidized ABTS after 5 hours. Increasing concentrations of either substance decrease this positive effect, resulting in a significantly decreased laccase activity in presence of 8 % (v/v) MeOH. These results indicate that for further measurements in substrate analytics it is recommended not to use high concentrations of MeOH or acetonitrile to ensure the functionality of [http://partsregistry.org/Part:BBa_K863005 ECOL].

Figure 17: Standard [http://partsregistry.org/Part:BBa_K863005 ECOL] activity test applying different amounts of MeOH. No considerable impact on the activity can be detected.

Figure 18: Standard [http://partsregistry.org/Part:BBa_K863005 ECOL] activity test applying different amounts of acetonitrile. No considerable impact on the activity can be detected.

Since Regionals: Initial activity tests of purified fractions

Another cultivation of ECOL has been done after the Regional Jamboree in Amsterdam. The fractions of the purifictaion were analyzed further on protein content and re-buffered subsequently into deionized H₂O. To determine the protein content afterwards because of loss of proteins through re-buffering, another protein concentration measurement has been done. The re-buffered fractions have been incubated with 0.4 mM CuCl₂ to gain higher activity of the laccases, because they are copper-dependent. Standard activity tests were done with all ECOL fractions with adjusted protein content for comparison. The experimental setup included the ECOL fractions, Britton-Robinson buffer (pH 5) and 0.1 mM ABTS. Measurements were done at 25 °C. Resulting, one fraction showed very high activity in comparison to the other fractions (Fig. x). This fraction, fraction 50% 2, oxidized up to 23 µM ABTS after 5 hours. This fraction was set as containing 90 % ECOL laccase of the whole protein content. Therefore a ECOL concentration of 63,9 µg mL^-1 was gained. This fraction was analyzed further on pH optimum, temperature dependency and ABTS saturation.

Figure x: Activity assay of each purified fraction of the cultivation with ECOL. Samples were re-buffered into H₂O and the protein amount in each fraction has been adjusted. The measurements were done using the standard activity assay protocol over night. The first number indicates the percentage of used elution buffer, whereas the second number stands for the fraction number of this elution.

Since Regionals: [http://partsregistry.org/Part:BBa_K863005 ECOL] activity depending on different ABTS concentrations

To calculate the activity in Units mg^-1, measurements had to be done under substrate saturation. With this the comparison of Units mg^-1 with other laccase activities and the literature is possible. To find the optimal substrate saturation ABTS concentrations ranging from 0.1 mM to 8 mM were applied in an experimental setup containing Britton-Robinson buffer (pH 5) and temperature conditions of 25 °C. For measurements with 0.1 mM to 5 mM ABTS, 616 ng BHAL laccase were used (Fig. x). For measurements with 5 mM to 8 mM ABTS only 308 ng BHAL laccase were applied (Fig. y). The amount of oxidized ABTS increased according to the increase of ABTS concentration. To make sure that the substrate saturation is given, 9 mM ABTS have been used in further tests.

Figure x: Activity assay to determine the substrate saturation with ABTS as a substrate. Measurements were done with 616 ng ECOL laccase in Britton-Robinson buffer (pH 5) at 25 °C. ABTS concentrations ranged from 0.1 mM to 5 mM.

Figure x: Activity assay to determine the substrate saturation with ABTS as substrate. Measurements were done with 308 ng ECOL in Britton-Robinson buffer (pH 5) at 25 °C. ABTS concentrations ranged from 5 mM to 8 mM. An ABTS concentration of 8 mM was determined as substrate saturated.

Since Regionals: [http://partsregistry.org/Part:BBa_K863005 ECOL] pH optimum

Figure x: Microtiter plate of the measurements for pH optimum determination. The more intensive the blue color the more ABTS got oxidized. At pH 4 and pH 5 the darkest colour has been reached.

Activity assay measurements for ECOL laccases were done to find the optimal pH for further analysis. Britton-Robinson buffer, adjusted to pHs ranging from pH 4 to pH 9, was used with 9 mM ABTS to detect the change in OD₄₂₀. The measurements were done with 308 ng ECOL laccase for each sample. The highest activity was reached when measured in Britton-Robinson buffer at pH 4 and pH 5 (Fig. x, Fig. y, Fig. z). More than 5 U mg^-1 of specific enzyme activity have calculated for these pHs (Fig. x). When testing the activity under basic conditions, the enzyme activity decreases. At pH 7 about 1 U mg^-1 was determined. This makes an application of the ECOL not feasible since the water in the waste water treatment plants is in average of pH 6.9.

Figure x: Oxidized ABTS by ECOL at different pH adjustments. The experimental setup included CuCl₂ incubated ECOL (308 ng), Britton-Robinson buffer adjusted to the tested pHs and 5 mM ABTS. Measurements were done at 25 °C for 30 minutes. The most amount of oxidized ABTS can be detected at pH 4 and pH 5.

Figure x: Calculated specific enzyme activity of ECOL at different pH conditions. The highest specific enzyme activity for ABTS is under pH 4 and pH 5 conditions. The higher the pH, the less ABTS gets oxidized. One unit is defined as the amount of laccase that oxidizes 1 μmol of ABTS substrate per minute.

Since Regionals: [http://partsregistry.org/Part:BBa_K863005 ECOL] activity at different temperatures

Standard activity test for ECOL measured at 10 °C and 25 °C resulting in a decreased activity at 10 °C. As a negative control the impact of 0.4 mM CuCl2 in oxidizing ABTS at 10 °C and 25 °C were analyzed.

Deriving from the obtained values of oxidized ABTS in time at 10 °C and 25 °C the specific enzyme activity was calculated. For the temperatures a difference of 9 U/mg could be detected. One unit is defined as the amount of laccase that oxidizes 1 μmol of ABTS substrate per minute.

To investigate the activity of ECOL at temperatures that will apply at a waste water treatment plant throughout the year, activity tests were performed at 10 °C and 25 °C as described above. The measurements were conducted for 30 minutes. The obtained results reveal a lower activity of ECOL at 10 °C in comparison to 25 °C (see Fig. X). The received values were used to calculate the specific enzyme activity which was between 1 and 12 U mg^-1 , respectively (see Figure X). The negative control without ECOL but 0.4 mM CuCl₂ at 10 °C and 25 °C show a negligible oxidation of ABTS. The activity of ECOL is decreased to about 90% at 10 °C. An application of ECOL at warm temperatures is therefore possible but during the cold seasons a more cryo stable enzyme would be preferable.

Substrate Analysis

Figure 2: Degradation of estradiol (dark green) and ethinyl estradiol (light green) 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). The original concentrations of substrates were 2 µg per approach. (n = 4)

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. To work with 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 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 3: 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. The original concentrations of substrates were 2 µg per approach. (n = 4)

The results of the reactions of the laccases with addition of ABTS are shown in Figure 3. The experimental set ups were the same as the reaction approach without ABTS described above. The X-axis displays the different tested laccases. On the Y-axis the percentages of degraded estradiol (blue) and ethinyl estradiol (red) are shown. The degradation was measured at t₀ and after five hours of incubation at 20 °C. The negative control showed no degradation of estradiol. 6.8 % of ethinyl estradiol was decayed. The positive control TVEL0 is able to degrade 100 % estradiol and ethinyl estradiol. The laccase BPUL (from Bacillus pumilus) degraded 46.9 % of used estradiol after ten minutes incubation. ECOL was able to degrade 6.7 % estradiol. BHAL degraded 46.9 % estradiol. With TTHL (laccase from Thermus thermophilus)a degradation 29.5 % were determined.

Immobilization

Figure 20: The percentage of laccases in the supernatant relative to the original concentration. The results show that only 0.2% of ECOL laccases are still present in the supernatant, whereas 75% of BPUL remained in the supernatant. This illustrate that almost all ECOL were bound to the beads. On the contrary, only 25% of BPUL laccases were able to bind.

Figure 21: Enzymatic activity of ECOL supernatant compared to the activity of nontreated laccases, measured using 0.1 mM ABTS at 25°C over a time period of 12hours. The results show a dramatic decrease of ECOL in the Supernatant.

Figure 22: Enzymatic activity of immobilized laccases compared to nontreated laccases.