Summary

First some trials of shaking flask cultivations were made with different parameters to define the best conditions for production of the His tagged CotA aus [http://www.dsmz.de/catalogues/details/culture/DSM-27.html Bacillus pumilus DSM 27 ( ATCC7061)] named BPUL. Due to inactivity of the enzyme in the cell lysate a purification method was established (using Ni-NTA-Histag resin and Syringe or ÄKTA method). The purified BPUL could be detected by SDS-PAGE (molecular weight of 58.6 kDa) as well as by MALDI-TOF. To improve the purification strategies the length of the linear elution gradient was increased up to 200 mL . The fractionated samples were also tested concerning their activity and revealed high activity. Optimal conditions for activity were identified. After measuring activity of BPUL a successful scale up was made up to 3 L and also up to 6 L that enables an intense screening afterwards. The scale up will be important for a further application.

Cultivation, Purification and SDS-PAGE

Shaking Flask Cultivation

3 L Fermentation E. coli KRX with <partinfo>BBa_K863000</partinfo>

Figure 1: Fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in a Braun Biostat B, 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 measurement of BPUL activity we made a scale-up and fermented E. coli KRX with <partinfo>BBa_K863000</partinfo> in aBraun Biostat B fermenter with a total volume of 3 L. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 1. We got a long lag phase of 2 hours due to a relatively old preculture. The cell growth caused a decrease in pO₂ and after 3 hours the value fell below 50 %, so that the agitation speed increased automatically. After 8.5 hours the deceleration phase started and therefore the agitation speed was decreased. The maximal OD₆₀₀ of 3.53 was reached after 10 hours, which means a decrease in comparison to the fermentation of E. coli KRX under the same conditions (OD_600,max =4.86 after 8.5 hours, time shift due to long lag phase). The cells were harvested after 11 hours.

Purification of BPUL

The harvested cells were resuspended in Ni-NTA-equilibrationbuffer, 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 flowrate of 1 mL min^-1 cm^-2. The column was washed with 10 column volumes (CV) Ni-NTA-equilibrationbuffer. The bound proteins were eluted by an increasing Ni-NTA-elutionbuffer gradient from 0 % to 100 % with a total volume of 100 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 BPUL-elution is shown in Figure 2:

Figure 2: Chromatogram of wash and elution from FLPC Ni-NTA-His tag purification of BPUL produced by 3 L fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo>. BPUL was eluted between a process volume of 460 mL to 480 mL with a maximal UV-detection signal of 69 mAU

The chromatogram shows a remarkable widespread peak between the process volume of 460 mL to 480 mL with the highest UV-detection signal of 69 mAU, which can be explained by the elution of bound proteins. The corresponding fractions were analyzed by SDS-PAGE analysis. During the elution, a steady increase of the UV-signal caused by the increasing imidazol concentration during the elution gradient. Between the process volume of 550 and 580 mL there are several peaks (up to a UV-detection-signal of 980 mAU) detectable. These results are caused by an accidental detachment in front of the UV-detector. Just to be on the safe side, the corresponding fractions were analyzed by SDS-PAGE analysis. The corresponding SDS-PAGE is shown in Figure 3.

SDS-PAGE of purified BPUL

Figure 3: SDS-PAGE of purified E. coli KRX lysate containing <partinfo>BBa_K863000</partinfo> (fermented in a 3 L Biostat Braun B fermenter). The flow-through, wash and the elution fractions 7 and 8 are shown. The arrow marks the BPUL band with a molecular weight of 58.6 kDa.

Figure 3 shows the purified ECOL including flow-through, wash and the elution fractions 7 and 8. These two fractions were chosen due to a high peak in the chromatogram. BPUL has a molecular weight of 58.6 kDA and was marked with a red arrow. The band appears in both fractions. There are also some other non-specific bands, which could not be identified. To improve the purification the elution gradient length should be longer and slower the next time.

The appearing bands were analyzed by MALDI-TOF and could be identified as CotA (BPUL).

6 L Fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo>

Figure 4: Fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in aBioengineering NFL22 fermenter, scale: 6 L, autoinduction medium + 60 µg mL^-1 chloramphenicol, 37 °C, pH 7, agitation increased when pO₂ was below 30 %, OD₆₀₀ measured every hour.

Another scale-up for E. coli KRX with <partinfo>BBa_K863000</partinfo> was made up to a final working volume of 6 L in a Bioengineering NFL22. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 4. There was no noticeable lag phase. Agitation speed was increased up to 425 rpm after one hour due to problems caused by the control panel. The pO₂ decreased until a cultivation time of 4.75 hours. The increasing pO₂ level indicates the beginning of the deceleration phase. There is no visible break in cell growth caused by an induction of protein expression. A maximal OD₆₀₀ of 3.68 was reached after 8 hours of cultivation, which is similar to the 3 L fermentation (OD₆₀₀ = 3.58 after 10 hours, time shift due to long lag phase). The cells were harvested after 12 hours.

Purification of BPUL

The harvested cells were prepared in Ni-NTA-equilibrationbuffer, 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 with 5 column volumes (CV) Ni-NTA-equilibrationbuffer. The bound proteins were eluted by an increasing elutionbuffer gradient from 0 % (equates to 20 mM imidazol) to 100 % (equates to 500 mM imidazol) with a length of 200 mL. This strategy was chosen to improve the purification by a slower increase of Ni-NTA-elutionbuffer 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 BPUL elution is shown in Figure 5.

Figure 5: Chromatogram of wash and elution from FLPC Ni-NTA-Histag Purification of BPUL produced by 6 L fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo>. BPUL was eluted between a process volume of 832 mL and 900 mL with a maximal UV-detection signal of 115 mAU.

The chromatogram shows a peak at the beginning of the elution. This can be explained by pressure fluctuations upon starting the elution procedure. In between the processing volumes of 832 mL and 900 mL there is remarkable widespread peak with a UV-detection signal of 115 mAU. This peak corresponds to an elution of bound proteins at a Ni-NTA elution buffer concentration between 10 % and 20 % (equates to 50-100 mM imidazol). The corresponding fractions were analyzed by SDS-PAGE. The ensuing upwards trend of the UV-signal is caused by the increasing imidazol concentration during the elution gradient. Towards the end of the elution procedure there is a constant UV-detection signal, which shows, that most of the bound proteins was already eluted. Just to be on the safe side, all fractions were analyzed by SDS-PAGE to detect BPUL. The SDS-PAGE is shown in Figure 6.

SDS-PAGE of purified BPUL

Figure 6: SDS-PAGE of purified E. coli with <partinfo>BBa_K863000</partinfo> lysate (fermented in Bioengineering, 6 L). The flow-through, wash and elution fraction 1 to 9 are shown. The arrow marks the BPUL band with a molecular weight of 58.6 kDa.

In Figure 6 the SDS-PAGE of the Ni-NTA purification of the lysed culture of E. coli KRX containing <partinfo>BBa_K863000</partinfo> is illustrated. It shows the flow-through, wash and elution fractions 1 to 9. The His-tagged BPUL has a molecular weight of 58.6 kDA and was marked with a red arrow. The band appears in all fractions from 2 to 9 with varying strength, the strongest ones in fractions 7 to 9. There are also some other non-specific bands, which could not be identified. Therefore the purification method could moreover be improved. In summary, the scale up was successful, improving protein production and purification method once again.

Furthermore the bands were analyzed by MALDI-TOF and identified as CotA (BPUL).

MALDI-TOF Analysis of BPUL

This laccase we identified with a manual maesure using again the Maldi-TOF and the software Biotools. We insert our sequenc from the laccase into a sequnce editor and simulate a trypsin digest. After this we compare these peptid mass fingerprints with the measured fingerprint we get from the Maldi. With a sequence coverage of 21,9% we identified the B. pumilus laccase. In the figures you can see the chromatogram of the peptide mass fingerprint and the single masses.

Figure 8: MALDI-TOF spectrum results of the analysis

Activity analysis of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL]

Initial activity tests of purified fractions

Initial tests were done with elution fractions 1 to 4 to determine the activity of the purified [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] 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, 0.1 mM ABTS and 100 mM sodium acetate buffer (pH 5) to a final volume of 200 µL and the absorption was measured at 420 nm to detect oxidization over a time period of 5 hours at 25°C. Each fraction did show contained active laccase able to oxidize ABTS (see Figure 9). After 15 minutes, saturation was observed with ~60 µM oxidized ABTS. After 5 hours ~5 µM ABTS got reduced again. This behavior has been observed in the activity plot of the positive control TVEL0 before, indicating, that the oxidation catalyzed by this laccase seems is reversible. Additionally, protein concentrations of each fraction were identified using the Bradford protocol. The four tested fractions showed approximately the same amount of protein after rebuffering, namely 0.5 mg mL^-1. Fraction 4, containing the most protein and also most of active laccase was chosen for subsequent activity tests of BPUL. 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/wiki/index.php?title=Part:BBa_K863000 BPUL] measurements.

Figure 9: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase activity measured in 0.1 mM ABTS and 100 mM sodium acetate buffer (pH 5) to a final volume of 200 µL at 25°C over a time period of 3.5 hours. Each tested fraction reveals activity reaching the saturation after 15 minutes with ~60 µM ABTS_ox after 0.4 mM CuCl₂ incubation. (n=4)

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] pH optimum

To determine at which pH the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] 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/wiki/index.php?title=Part:BBa_K863000 BPUL] 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. The saturation is reached after 3 hours with 50% oxidization of ABTS through the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase at pH 5 (55 µM oxidized ABTS). The other tested pHs only led to a oxidation of 18% of added ABTS. Figure 11 represents the negative control showing the oxidation of ABTS through 0.4 mM CuCl₂ at the chosen pHs. The highest increase in oxidized 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase at pH 5, which is still the optimal pH. Therefore it has the same pH optimum as TVEL0.

Figure 10: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 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 to a final volume of 200 µL at 25°C over a time period of 5 hours. Before the measurements samples were incubated with CuCl₂. The optimal pH for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] 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.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] CuCl₂ concentration

Another test of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] was done to survey the best CuCl₂ concentration for the activity of the purified [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase. 0.03 mg mL^-1 of protein were incubated with different CuCl₂ concentrations ranging from 0 to 0.7 mM CuCl₂. Activity tests were performed with the incubated samples, 0.1 mM ABTS and 100 mM sodium actetate buffer (pH 5) to a final volume of 200 µL. The reactivity was measured at 420 nm, 25°C and over a time period of 5 hours. As expected the saturation takes place after 3 hours (see Figure 12). The differences in the activity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases incubated in different CuCl₂ differ minimal. The highest activity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase is observed after incubation with 0.6 mM CuCl₂ (52% of added ABTS). With a higher concentration of 0.7 mM CuCl₂ the activity seems to be reduced (only 48% ABTS got oxidized). This leads to the assumption that CuCl₂ supports the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase reactivity but concentrations exceeding this value of CuCl₂ may have a negative impact on the ability of oxidizing ABTS. 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 without applying laccase to detect the oxidization of ABTS through copper (see Figure 13). The more CuCl₂ was present, the more ABTS was oxidzied after 5 hours. Still the maximal change accounts only for ~6% oxidized ABTS after 5 hours.

Figure 12: Activity measurement using 0.1 mM ABTS of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] incubated in different CuCl₂ concentrations. Without CuCl₂ incubation the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase shows half of the activit as after CuCl₂ incubation. Incubation with 0.1 mM CuCl₂ or higher concentrations leas to an increase in ABTS_ox.

Figure 13: 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 enzyme. Still it is expensive to be reliant on CuCl₂ and a potential risk using heavy metals for waste water purifcation.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity at different temperatures

Figure 14: Standard activity test for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] 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/wiki/index.php?title=Part:BBa_K863000 BPUL] at lower temperatures, activity tests as described above were performed at 10°C and 25°C. A small decrease in the activity can be observed upon reducing the temperature from 25°C to 10°C (see Fig. 14). After 3.5 hours when samples at 25°C reached the saturation samples at 10°C had not, but nonetheless the difference is minimal. After 3 hours 5% difference in oxidized ABTS is observable. The negative control without the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase but 0.4 mM CuCl₂ at 10°C shows a negligible oxidation of ABTS. A a decrease in the reactivity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] 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/wiki/index.php?title=Part:BBa_K863000 BPUL] activity depending on different ABTS concentrations

Figure 15: Analysis of ABTS oxidation by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases incubated in 0.4 CuCl₂ tested with different amounts of ABTS. The higher the amount of ABTS the more oxidized ABTS can be detected.

Furthermore, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] 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 15). Especially using 16 µL showed an increase in the activity until 1 hour (reaching 50 µM ABTS_ox), but the amount of oxidized ABTS decreased afterward.

Impact of MeOH and acteonitrile on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL]

For substrate analytic tests the influence of MeOH and acetonitrile on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases 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/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase, different amounts of MeOH (Figure 16) or acteonitrile (Figure 17), 0.1 mM ABTS and 100 mM sodium actetate buffer to a final volume of 200 µL. The observed reactivity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] in regard of oxidizing ABTS did not reveal a huge decrease. The less MeOH or acetonitrile was used, the higher was the amount of oxidized ABTS after 3 hours. An application of 16 µL MeOH or acetonitrile led to a decrease of maximal 10% oxidized ABTS compared to 2 µL MeOH or acetonitrile. Negative controls are shown in Figure 17 and 18 of the ECOL laccase. MeOH and acetonitril are able to oxidize ABTS. After 5 hours at 25°C ~15 µM ABTS get oxidized through MeOH or acetonitrile, but samples with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase show a distinct higher activity of 50 µM ABTS_ox.

Figure 16: Standard [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity test applying different amounts of MeOH. No considerable impact on the activity can be detected.

Figure 17: Standard [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity test applying different amounts of acetonitrile. No considerable impact on the activity can be detected.

Substrate Analytic

Figure 18: Comparison of degradation reactions of ethinyl estradiole with ABTS between the laccases BPUL and ECOL with the purchased laccase TVEL0, measured by HPLC.

Figure 19: Degradation of estradiol by BPUL in unknown concentration measured by HPLC Estradiol in BR-buffer without laccase was used as negative control.

Figure 20: Degradation of estradiol by BPUL measured by HPLC. Overleyed chromatograms from different timepoints. The first peak caused by the solvent.