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 Histag. Because of no measured activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). The purified ECOL could be identified by SDS-PAGE (molecular weight of 53.4 kDa) as well as MALDI-TOF. The fractionated samples were also tested concerning their activity. A maximal activity of X was reached. After measuring activity of ECOL a scale up was made up to 3 L and then also up to 6 L.

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 Infors Labfors Bioreactor, scale: 3 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold a pO₂ of 50 %, OD₆₀₀ taken 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 Infors Labfors with a total volume of 3 L. Agitation speed, pO₂ and 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 OD_600,max 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: DBU final report). Therefore they were harvested after 12 hours.

Purification of ECOL

The harvested cells were resuspended in Ni-NTA-equilibration buffer, mechanically lysed by homogenization and centrifuged. The supernatant of the lysed cell paste 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 by 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 strategies 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 from FLPC Ni-NTA-Histag 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 by 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-elutionbuffer concentration to 50 % (equates to 250 mM imidazol) a peak up to a UV-detection signal 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-signals equates to the 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 BBa_K863005 lysate (fermented in 3 L Infors Labfors). The flow-through, wash 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 BBa_K863005) is illustrated 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. The second small band with a lower molecular weight was not identified. Furthermore the bands were analyzed by MALDI-TOF and identified as CueO (ECOL).

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

Figure 4: Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in Bioengineering NFL22, 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 Bioengineering NFL 22. 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 cytotoxity of the laccases for E. coli, which was reported by the 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-equilibratio nbuffer, mechanically lysed by homogenization and centrifuged. The supernatant of the lysed cell paste 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 elution are shown. A typical chromatogram of purified laccases is illustrated here. The chromatogram of the ECOL elution is shown in figure 4:

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 culture of E. coli KRX containing BBa_K863005 (fermented in 6 L Bioengineering NFL22) after purification. The flow-through, wash and elution fraction 1-15 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 result of the Ni-NTA-Histag purification of the lysed culture E. coli KRX containing 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. Furthermore the bands were analyzed by MALDI-TOF and identified as CueO (ECOL).

Activity Analysis of ECOL

Initial activity tests of purified fractions

Figure 8: ECOL laccase activity measured in 100 mM sodium acetate buffer (pH 5), 0.1 mM ABTS, ad 200 µL deionized H₂O at 25°C over a time period of 12 hours. Each tested fraction reveals activity reaching the saturation after 2.5 to 4 hours with a maximum of ~52 µM ABTS_ox (fraction 7). (n=4)

ECOL pH optimum

To determine at which pH the ECOL laccase has its optimum in activity, a gradient of sodium acetate buffer pHs was prepared. Starting with pH 1 to pH 9 ECOL activity was tested using the described conditions above and 0.03 mg mL^-1 protein. The results are shown in figure 9. A distinct pH optimum can be seen at pH 5. The saturation is reached after 2.5 hours with 53% oxidization of ABTS through the ECOL laccase at pH 5 (53 µM oxidized ABTS) . The other tested pHs only led to a oxdization of up to 17% of added ABTS, respectively. Figure 10 shows the results of the analog experiments with laccase that was not incubated with CuCl₂ before the activity measurements. A pH optimum can as well be determined for pH 5. An amount of 24 µM ABTS (24%) is oxidized via ECOL after 8 hours.

Figure 11 represents the negative control showing the oxidization of ABTS through 0.4 mM CuCl₂ at the chosen pHs. The highest increase in oxidzied ABTS can be seen at a pH of 5. After 5 hours 15% ABTS are oxidized only through CuCl₂. Nevertheless this result does not have an impact on the reactivity of the ECOL laccase at pH 5, which is still the optimal pH. Therefore it has the same pH optimum as TVEL0.

Figure 9: BPUL 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, ad 200 µL deionized H₂O at 25°C over a time period of 12 hours. The optimal pH for ECOL is pH 5 with the most ABTS_ox.

Figure 10: 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, ad 200 µL deionized H₂O 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 ECOL is pH 5 with the most ABTS_ox.

Figure 11: Negative control for pH activity Tests using 0.04 mM CuCl₂ H₂O instead of Laccase to determine the potential of ABTS getting oxidized through CuCl₂.

In regard to our project an optimal pH of 5 is a helpful result. Since waste water in waste water treatment plants has a average pH of 6.9 it has to be kept in mind, that a adjustment of the pH is necessary.

ECOL CuCl₂ concentration

ECOL activity at different temperatures

Figure 13: 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 were analyzed.

ECOL activity depending on different ABTS concentrations

Figure 14: Analysis of ABTS oxidation by ECOL Laccases tested with different amounts of ABTS. The higher the amount of ABTS the more oxidized ABTS can be detected.

Impact of MeOH and acteonitrile on ECOL