Team:Bielefeld-Germany/Results/pumi

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[ '''' ] Laccase CotA from Bacillus pumilus DSM 27 ( ATCC7061)

Summary

First some trials of shaking flask cultivations were made with different parameters to define the best conditions for the production of the His-tagged CotA aus Bacillus pumilus DSM 27 ( ATCC7061) named BPUL. Because of no measured activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). The purified BPUL could be detected by SDS-PAGE (molecular weight of 58.6 kDa) as well as MALDI-TOF. To improve the purification strategies the length of the elution gradient was increased. The fractionated samples were also tested concerning their activity. A maximal activity of X was reached. After measuring activity of BPUL a scale up was made up to 3 L and also up to 6 L.


Contents


Shaking Flask Cultivation

The first trials to produce the CotA-laccase from Bacillus pumilus DSM 27 (ATCC7061, named BPUL) were performed in shaking flasks with various designs (from 100 mL-1 to 1 L flasks, with and without baffles) and under different conditions. The parameters we have changed during our screening experiments were temperature (27 °C,30 °C and 37 °C), the concentration of chloramphenicol (20 to 170 µg mL-1), induction strategy (autoinduction and manual induction with 0,1 % rhamnose) and cultivation time (6 to 24 h). Further we 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. Due to the screening experiments we identified the best conditions for expression of BPUL (see below). The addition of CuCl2 did not lead to better results, 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 and repeatability of the measured data and results were investigated for the shaking flask and bioreactor cultivation.

3 L Fermentation E. coli KRX with BBa_K863000

Figure 1: Fermentation of E. coli KRX with BBa_K863000 (BPUL) in Braun Biostat B, scale: 3 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold a pO2 of 50 %, OD600 measured every 30 minutes.

After the measurement of BPUL activity we made a scale-up and fermented E. coli KRX with BBa_K863000 in Braun Biostat B 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 (OD600,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 centrifuged. 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-Histag purification of BPUL produced by 3 L fermentation of E. coli KRX with BBa_K863000. 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. Afterwards the UV-signal increased caused by the changing 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-PAGES of purified BPUL

Figure 3: SDS-PAGE of purified E. coli KRX lysate containing BBa_K863000 (fermented in 3 L Biostat Braun B). 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 E. coli KRX with BBa_K863000 lysates (fermented in 3 L Braun Biostat B) including flow-through, wash and the elution fractions 7 and 8. These two fractions were chosen due to a remarkable 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 analysed by MALDI-TOF and could be identified as CotA (BPUL).

6 L Fermentation of E. coli KRX with BBa_K863000

Figure 4: Fermentation of E. coli KRX with BBa_K863000 (BPUL) in Bioengineering NFL22, 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 BBa_K863000 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 centrifuged. 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 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 BBa_K863000. 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-elutionbuffer 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-PAGES of purified BPUL

Figure 7: SDS-PAGE of purified E. coli with BBa_K863000 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 7 the SDS-PAGE of the Ni-NTA purification of the lysed culture of E. coli KRX containing BBa_K863000 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.

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


Activity Analysis of BPUL

Initial activity tests of purified fractions

Initial tests were done with elution fractions 1 to 4 to determine the activity of the purified BPUL laccases. The fractions were rebuffered into deionized H2O using 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), ad 198 deionized H2O and 0.1 mM ABTS 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 reactivity in laccases oxidizing ABTS (see figure 8). After 15 minutes the saturation took place with ~60 µM oxidized ABTS. After 5 hours ~5 µM ABTS got reduced again. This behavior has been seen in the activity plot of TVEL0 before indicating, that this laccase seems to cannot hold the level of oxidized ABTS and reduction of ABTS set in. 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, having 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 BPUL measurements.

Figure 8: BPUL 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 3.5 hours. Each tested fraction reveals activity reaching the saturation after 15 minutes with ~60 µM ABTSox. (n=4)


BPUL pH optimum

To determine at which pH the BPUL laccase has its optimum in activity, a gradient of sodium acetate buffer pHs was prepared. Starting with pH 1 to pH 9 BPUL 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 3 hours with 50% oxidization of ABTS through the BPUL laccase at pH 5 (55 µM oxidized ABTS) . The other tested pHs only led to a odization of 18% of added ABTS. Figure 10 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 BPUL 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 5hours. The optimal pH for BPUL is pH 5 with the most ABTSox.
Figure 10: 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.

BPUL CuCl2 concentration

Another test of BPUL was done to survey the best CuCl2 concentration for the activity of the purified BPUL laccase. 0.03 mg mL-1 of protein were incubated in different CuCl2 concentration ranging from 0 to 0.7 mM CuCl2. Activity tests were performed with the incubated samples, 100 mM sodium actetate buffer (pH 5), 0.1 mM ABTS, ad 200 µL H2O. 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 11). The differences in the activity of BPUL laccases incubated in different CuCl2 differ minimal. The most percentage is oxidized with BPUL laccases incubated 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 BPUL laccase reactivity but concentrations exceeding this value of CuCl2 may have a negative impact on the ability of oxidizing ABTS. Without any CuCl2 application BPUL laccases do not show any activity in oxidizing ABTS. This fits the expections knowing that 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 to detect the oxidization of ABTS through copper (see figure 12). The more CuCl2 was applicated, the more ABTS was oxidzied after 5 hours. Still the maximal change accounts ~6% oxidized ABTS after 5 hours.

Figure 11: Activity measurement using 0.1 mM ABTS of BPUL incubated in different CuCl2 concentrations. Without CuCl2 incubation BPUL is not active. Incubation with 0.1 mM CuCl2 or higher concentrations leas to an increase in ABTSox.
Figure 12: 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 them. This reduces the cost factor and risks.

BPUL activity at different temperatures

Figure 13: Standard activity test for 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 in the activity behaviour of BPUL at lower tempertaure conditions activity tests as described above were done at 10°C and 25°C. A small decrease in the activity can be observed considering the temperature shift from 25°C to 10°C. 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 theBPUL laccase but 0.4 mM CuCl2 at 10°C shows a negligible effect on the status of ABTS. A decrease in the reactivity of BPUL laccases was expected. Since it is not reduced in a high manner it is applicable for usage in waste water treatment plants where the temperature differs from 8.1°C to 20.8°C.

BPUL activity depending on different ABTS concentrations

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

Furthermore BPUL laccases were tested using different amounts of ABTS to calculate KM and Kcat values. The same measurement setup as described above was used only with different amounts of ABTS. As anticipated the amount of oxidized ABTS increased dependent on the amount of used ABTS (figure 14). Especially using 16 µL showed an increase in the reactivity until 1 hour (reaching 50 µM ABTSox but the amount of oxidized ABTS decreased afterward.

Impact of MeOH and acteonitrile on BPUL

For substrate analytic tests the influence of MeOH and acetonitrile on BPUL laccases had to be determined, because substrates have to be dissolved in these reagents. The experiment setup included 0.03 mg mL-1 BPUL laccase, 100 mM sodium actetate buffer, different amounts of MeOH (figure 15) or acteonitrile (figure 16), 0.1 mM ABTS, ad 200 µL deionized H2O. The observed reactivity of 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.

Figure 15: Standard BPUL activity test applying different amounts of MeOH. No considerable impact on the activity can be detected.
Figure 16: Standard BPUL activity test applying different amounts of acetonitrile. No considerable impact on the activity can be detected.



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