Summary

First some trials of shaking flask cultivations were made with various parameters to identify the best conditions for production of the His tagged laccase Lbh1 from [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5 Bacillus halodurans C-125 ] named BHAL. Due to inactivity of the enzyme in the cell lysate a purification method was established (using Ni-NTA-Histag resin). BHAL could not be detected by SDS-PAGE (theoretical molecular weight of 56 kDa) or activity test by using the BioBrick <partinfo>BBa_K863020</partinfo> and E. coli KRX as expression system. Due to this results the new BioBrick <partinfo>BBa_K863022</partinfo> was constructed and expressed E. coli Rossetta-Gami 2. With this expression system the laccase could be produced and analysed via SDS-PAGE. A small scale Ni-NTA-column was used to purify the laccase. The fractionated samples were tested regarding their activity with ABTS and showed ability in oxidizing ABTS. A scale up to 12 L with a optimized medium (HSG) and a labscale Ni-NTA-Purification were implemented to enable additional experiments to characterize BHAL. A total specific enzyme activity of 8 U mg^-1 was determined for BHAL at pH 4 at 25°C with ABTS as a substrate.

Cultivation, Purification and SDS-PAGE

Cultivation

• flask design: shaking flask without baffles
• medium: LB-Medium
• antibiotics: 60 µg mL^-1 chloramphenicol and 300 µg mL^-1 ampicillin
• temperature: 37 °C
• cultivation time: 24 h

Purification

SDS-PAGE

Figure 1: SDS-PAGE of purified lysate derived from a flask cultivation of E. coli Rosetta-Gami 2 carrying <partinfo>BBa_K863022</partinfo>. Lanes 2 to 7 show the flow-through, the wash and the elution fractions 1 to 4. BHAL has a molecular weight of 56 kDa and is marked with an arrow.

In Figure 1 the different fractions of the purified cell lysate of E. coli Rosetta-Gami 2 with <partinfo>BBa_K863022</partinfo> are shown in a SDS-PAGE. BHAL has a molecular weight of 56 kDa. In lane 5, which corresponds to the elution fraction 2, a faint band of 56 kDa is visible. Therefore the fractions were further analysed by activity test and MALDI-TOF.

Since Regionals: 12L Fermentation of E. coli Rosetta-Gami 2 with <partinfo>BBa_K863022</partinfo>

Figure 2: Fermentation of E. coli Rosetta-Gami 2 with <partinfo>BBa_K863022</partinfo> (BHAL) in a Bioengineering NFL22. Conditions: 12 L of HSG autoinduction medium + 60 µg mL ^-1 chloramphenicol at 37 °C, pH 7. Agitation increased when pO₂ was below 50 % and OD₆₀₀ was measured each hour. The glycerin concentration was measured on important points of the cultivation with HPLC.

Since Regionals: Purification of BHAL

The harvested cells were resuspended in Ni-NTA equilibration buffer and mechanically disrupted by homogenization. The cell debris were removed by centrifugation and microfiltration via [http://www.millipore.com/catalogue/module/C7493 Millipore Pellicon XL 50]. The supernatant of the cell lysate was concentrated with [http://www.millipore.com/catalogue/module/C7493 Millipore Pellicon XL 50] with 10 kDa and 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 80 mL, to 50 % (equates to 250 mM imidazol) with a length of 80 mL and finally to 100 % (equates to 500 mM imidazol) with a length of 90 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. In figure 3 only the UV-detection signal of the wash step and the elution are shown, this is because of the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak. A typical chromatogram of purified laccases is illustrated here. The chromatogram of the BHAL elution is shown in Figure 5:

Figure 3: Chromatogram of wash and elution fractions from FLPC Ni-NTA His-tag Purification of BHAL produced by 12 L fermentation of E. coli Rosetta Gami 2 with <partinfo>BBa_K863022</partinfo>. BHAL was eluted by a concentration of 50 % (equates to 250 mM imidazol) with a maximal UV-detection signal of 123 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. Contrary to our expectations, the chromatogram shows the second distinguished peak. This peak was detected at a Ni-NTA-equilibration buffer concentration of 100 % (equates to 500 mM imidazol) and resulted from the elution of bound protein. Earlier measurements of other bacterial laccases showed that the elution of these laccases begins with a elution buffer concentration of 50 %(equates to 250 mM imidazol). One explanation of this result could be a low concentration of the produced BHAL. Consequently all elution fractions were analyzed by SDS-PAGE to detect BHAL. In the chromatogram no further peaks were detected. The following increasing UV detection signal by increasing concentration of the eltutionbuffer results from the rising imidazol concentration of the Ni-NTA elution buffer. The corresponding SDS-PAGES are shown in Figure 4.

Since Regionals: SDS-PAGE of protein purification

Figure 4: SDS-PAGE of purification from the 12 L fermentations from 10/11 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BBa_K863022]). Purification of the supernatant via microfiltration, diafiltration and Ni-NTA column (step gradient with 5 %, 50 % and 100 % elution buffer).

In Figure 4 the SDS-PAGE of the Ni-NTA purification of the lysed E.coli Rosetta-Gami 2 culture containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BBa_K863022] is illustrated. It shows the permeate and retentate of microfiltration and diafiltration respectively, several fractions of flow-through, wash and the elutions with different buffer concentrations respectively. The selected samples were taken where peaks were seen in the chromatogram. The His-tagged BHAL has a molecular weight of 56 kDa. Apparently the concentration of BHAL is too low to see a band.

Activity Analysis of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL]

Initial activity tests of purified fractions

The resulting fractions of the cultivation and purification of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] (fraction 1 to 5) were analysed with activity tests. After rebuffering into deionized H₂O and incubation with 0.4 mM CuCl₂ for 2 hours, the samples were measured with 140 µL sample, 0.1 mM ABTS, 100 mM sodium acetate buffer to a final volume of 200 µL. The change in optical density was measured at 420 nm, reporting the oxidation of ABTS for 5 hours at 25°C. An increase in ABTS_ox can be seen (Figure 4), indicating produced [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] laccase in each fraction. Fraction 2 shows the highest amount of ABTS_ox (55%) reaching saturation after 3 hours. Similar to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] is capable to reach saturation after 3 hours with approximately oxidizing 55% of the supplied ABTS. Therefore [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] is going to be characterized further.

Figure 4: Activity test of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] fractions after purification. Reaction setup includes 140 µL fraction sample (CuCl2 incubated), 0.1 mM ABTS and 100 mM sodium actetate buffer (pH 5) to a final volume of 200 µL. Measurements were done at 25°C and over a time period of 5 hours. Each fraction shows activity, especially fraction 2, which therefore contains most [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] laccase. (n=4)

Since Regionals: Initial activity tests of purified fractions

Different fractions of the purification of a new cultivation since the Regional Jamborees in Amsterdam were tested regarding their activity of the produced BHAL. Before and after re-buffering the protein concentration was determined. The initial activity tests were done in Britton-Robinson buffer (pH 5) with 0.1 mM ABTS at 25 °C. The protein amount was adjusted in each sample for a comparison. One distinct fraction showed the highest activity: fraction 5% 3 (Fig. 5). The contained laccase amount was calculated by assuming that the most active fraction contains 90 % laccase. This leads to a BHAL concentration of 10,9 ng mL^-1.

Figure 5: Activity assay of each purified fraction of recent produced BHAL. Samples were re-buffered into H₂O and the protein amount in each fraction had 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/wiki/index.php?title=Part:BBa_K863022 BHAL] activity depending on different ABTS concentrations

To be able to calculate the activity in Units mg^-1, measurements had to be done under substrate saturation. This allows the comparison of Units mg^-1 with other laccase activities and data found in literature. For this purpose ABTS concentrations ranging from 0.1 mM to 8 mM were applied in an experimental setup containing Britton-Robinson buffer (pH) and a temperature of 25 °C. For measurements with 0.1 mM to 5 mM ABTS 616 ng BHAL were used (Fig. 6). For measurements with 5 mM to 8 mM ABTS only 308 ng BHAL were applied (Fig. 7). Applying less than 7 mM ABTS a static increase in oxidized ABTS was given. Measurements with 8 mM ABTS showed a slower increase in oxidized ABTS as with 7 mM ABTS (Fig. 7). This may be due to a substrate toxication. The most compromising ABTS concentration was 7 mM with the highest increase in oxidized ABTS. Therefore a substrate saturation was reached with 7 mM ABTS.

Figure 6: Activity assay to determine the substrate saturation with ABTS as a substrate. Measurements were done with 616 ng BHAL laccase in Britton-Robinson buffer (pH 5) at 25 °C. ABTS concentrations ranged from 0.1 mM to 5 mM.

Figure 7: Activity assay to determine the substrate saturation with ABTS as a substrate. Measurements were done with 308 ng BHAL laccase in Britton-Robinson buffer (pH 5) at 25 °C. ABTS concentrations ranged from 5 mM to 8 mM. An ABTS concentration of 7 mM was determined as substrate saturated.

Since Regionals: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] pH optimum

Figure 8: 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 was detected.

To determine the optimal experimental setup for BHAL activity measurements, the best pH had to be determined. Using Britton-Robinson buffer pHs between pH 4 and pH 9 had been adjusted. 308 ng BHAL per well had been tested under these pH conditions using 7 mM ABTS. The CuCl₂ incubated and therefor activated BHAL showed a high activity at pH 4 and pH 5, where most of ABTS was oxidized (compared to Fig. 8 and 9). The calculated specific enzyme activity of BHAL showed high activity at both mentioned pHs (Fig. 10). While BHAL had an activity of ~8 U mg^-1 at pH 4 and pH 5, the enzyme activity decreased at higher pHs. At a pH of 6 only 1/3 of enzyme activity could be detected compared to the activity at pH 4 and pH 5. While still active at pH 7, the BHAL is not as suitable as thought for an application at a waste water treatment plant because of its high activity in acidic environments.

Figure 9: Oxidized ABTS by BHAL at different pH adjustments. The experimental setup included CuCl₂ incubated BHAL (308 ng), Britton Robinson buffer adjusted to the tested pHs and 5 mM ABTS. Measurements were done at 25 °C for 30 minutes. The highest amount of oxidzed ABTS could be detected at pH 4 and pH 5.

Figure 10: Calculated specific enzyme activity of BHAL at different pH conditions. The highest specific enzyme activity for ABTS was under pH 4 and pH 5 conditions. The higher the pH, the less ABTS got oxidzed. One unit is defined as the amount of laccase that oxidizes 1 μmol of ABTS substrate per minute.

Since Regionals: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] activity at different temperatures

Figure 11: Standard activity test for BHAL 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 was analyzed.

Figure 12: 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 3 U mg^-1 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 BHAL 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 revealed a lower activity of BHAL at 10 °C in comparison to 25 °C (see Fig. 11). The obtained results were used to calculate the specific enzyme activity which was at 4.2 and 7.2 U mg^-1, respectively (see Figure 12). The negative control without BHAL but 0.4 mM CuCl₂ at 10 °C and 25 °C showed a negligible oxidation of ABTS. The activity of BHAL was increased to about 60 % at 10 °C but nevertheless the observed activity at both conditions was great news for the possible application in waste water treatment plants.

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 immobilized to CPC-Beads. 99 % of ECOL, 97 % of BPUL and 79 % of BHAL and TTHL laccases were bound to the beads.

Figure 22: Illustration of ABTS oxidation by BHAL with time compared to the negative control. The increase in ABTS oxidized proves laccase activity.