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Laccase CueO from Escherichia coli BL21 (DE3)

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 X was reached. 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. The scale up will be important for further application.

Cultivation, Purification and SDS-PAGE

Shaking Flask Cultivations

The first trials to produce ECOL were produced in shaking flask with various designs (from 100 mL^-1 to 1 L flasks, with and without baffles) and under different conditions. The parameters tested during our screening experiments were temperature (27 °C,30 °C and 37 °C), concentrations of chloramphenicol (20-170 µg mL^-1), various induction strategies (autoinduction and manual induction) and cultivation time (6 - 24 h). Furthermore it was cultivated with and without 0.25 mM CuCl₂ to provide a sufficient amount of copper, which is needed for the active center of the laccase. Based on the screening experiments we identified the best conditions under which ECOL was expressed. The addition of CuCl₂ did not increase the activity, so it was omitted.

flask design: shaking flask without baffles
medium: autoinduction medium
antibiotics: 60 µg mL^-1 chloramphenicol
temperature: 37 °C
cultivation time: 12 h

The reproducibility of the measured data and results were investigated for the shaking flask and bioreactor cultivation.

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 Infors Labfors). 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 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 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 cytotoxity of the laccases 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 lysed by homogenization and cell debris were removed by centrifugation. 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 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 culture of E. coli KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 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 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).

MALDI-TOF Analysis of ECOL

We have the posibility to identify our possible E. coli laccase with the Maldi-TOF. Therefore we use the software

FlexControl
Flexanalysis and
Biotools

from Brunker Daltronics. We identified the E. coli laccase P36649 with a Scroe of 108 with an automatic run. In the pictures you can see the chromatogram of the peptid mass fingerprint and the single masses. There we get a sequence coverage of 26,1%. So we can assume that you isolated protein is the E. coli laccase.

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

Figure 8: Part of MALDI-TOD Evaluation

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

Initial activity tests of purified fractions

Initial tests were done with elution fractions 2,3,6,7 and 8 to determine the activity of the purified [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase. The fractions were rebuffered into deionized H₂O using [http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Supelco/Product_Information_Sheet/4774.pdf HiTrap Desalting Columns] and incubated with 0.4 mM CuCl₂. The reaction setup included 140 µL of a elution fraction, 100 mM sodium acetate buffer (pH 5), and 198 deionized H₂O and 0.1 mM ABTS and the absorption was measured at 420 nm to detect oxidization over a time period of 12 hours at 25°C. Each fraction contained active laccase able to oxidize ABTS (see Figure 9). After 1 hour saturation was observed with ~52 µM oxidized ABTS. After 12 hours ~10 µM ABTS got reduced again, if referred to fraction 6. This behavior has been observed in the activity plot ofTVEL0 before, indicating, that the oxidation catalyzed by this laccase is reversible. Additionally protein concentrations of each fraction were identified using the Bradford protocol. The tested fractions showed different amounts of protein after rebuffering, ranging from 0.2 to 0.6 mg mL^-1. Fraction 7, containing the most protein and also most of active laccase was chosen for subsequent activity tests of [http://partsregistry.org/Part:BBa_K863005 ECOL]. The protein concentration was reduced to 0.03 mg mL^-1 for each measured sample to allow a comparison between TVEL0 measurements and [http://partsregistry.org/Part:BBa_K863005 ECOL] measurements.

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.

To investigate the activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] at lower temperatures activity tests as described above were done at 10°C and 25°C (Figure 15). A small decrease in the activity can be observed upon reducing the temperature from 25°C to 10°C. After 2 hours when samples at 25°C reached the saturation samples at 10°C had not, but nonetheless the difference is minimal. After 3,5 hours 2% difference in oxidized ABTS is observable. The negative control without the [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase but 0.4 mM CuCl₂ at 10°C shows a negligible oxidation of ABTS. As a decrease in the reactivity of [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase was expected the observed small reduction in enzyme activity is excellent news for the possible application in waste water treatment plants where the temperature differs from 8.1°C to 20.8°C.

[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.

Furthermore [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase were tested using different amounts of ABTS to calculate K_M and K_cat values. The same measurement setup as described above was used only with different amounts of ABTS. As anticipated the amount of oxidized ABTS increased in dependence of the amount of ABTS used (Figure 16). The results of the measurements of the samples tested with 16 µL could not be detected longer than 1.5 h because the values were higher than the detection spectrum of the used device (TecanReader).

Impact of MeOH and acteonitrile 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 actetate buffer, different amounts of MeOH (Figure 17) or acteonitrile (Figure 18), 0.1 mM ABTS, ad 200 µL deionized H₂O. The observed activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] shows that the use of less MeOH or acetonitrile leads to an higher amount of oxidized ABTS after 5 hours. However, the negative control was observed to show an amount of 12 µM oxidized ABTS for acetonitrile and 15 µM of oxidized ABTS for MeOH. Considering these results it can be concluded that there is an impact of MeOH and acetonitrile on [http://partsregistry.org/Part:BBa_K863005 ECOL] activity. The enzyme activity is thus reduced to 2% (~ 2 µM of oxidized ABTS) regarding the influence of 16 µL acetonitrile and showing even less values than the negative controls of the samples incubated with 16 µL MeOH. These results show that for further measurements in substrate analytics it is recommended to not use high concentrations of MeOH or acetonitrile to ensure the functionatlity 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.

Substrate Analytic

Figure 19: Comparison of degradation reactions of ethinyl estradiole with ABTS between the laccases BPUL and ECOL with the purchased laccase TVEL0, measured by HPLC

In Figure 19 the degradation reactions of ethinyl estradiole with ABTS of BPUL and ECOL are shown. The data was measured by HPLC. Since it was already determined that both are able to oxidize ABTS by the activity test, the results were as expected. ABTS works as mediator, that means oxidized ABTS reacts chemically with a broad range of substrates, which explains the ability to degrade ethinyl estradiol and probably other substrates. In comparison, ECOL has a lower potential for degradation of ethinyl with ABTS than TVEL0 and BPUL. Until now, no further experiments were made.

Immobilisation

Fig.7: 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 20: 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.

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