Team:Bielefeld-Germany/Results/pumi

From 2012.igem.org

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===MALDI-TOF Analysis of BPUL===
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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.
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In the figures you can see the chromatogram of the peptide mass fingerprint and the single masses.
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[[File:Bielefeld2012_Massemspektroskopie_BPumi_.png|400px|thumb|center|Figure 7: MALDI-TOF spectrum]]
[[File:Bielefeld2012_Massemspektroskopie_BPumi_.png|400px|thumb|center|Figure 7: MALDI-TOF spectrum]]

Revision as of 02:13, 27 September 2012

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 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.


Contents


Cultivation, Purification and SDS-PAGE

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 changed parameters during the 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). 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 the best conditions for expression of BPUL were identified(see below). The addition of CuCl2 did not increase 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 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 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 BBa_K863000 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 (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 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 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. 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 BBa_K863000 (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 BBa_K863000

Figure 4: Fermentation of E. coli KRX with BBa_K863000 (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 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 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 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 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 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 6 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. 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 7: MALDI-TOF spectrum


Figure 8: MALDI-TOF spectrum results of the analysis

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 laccase. 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, 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 BPUL measurements.

Figure 9: 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 ABTSox after 0.4 mM CuCl2 incubation. (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 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 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 BPUL laccase at pH 5, which is still the optimal pH. Therefore it has the same pH optimum as TVEL0.

Figure 10: 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 BPUL is pH 5 with the most ABTSox.
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.

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 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 BPUL laccases incubated in different CuCl2 differ minimal. The highest activity of 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 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 BPUL incubated in different CuCl2 concentrations. Without CuCl2 incubation the 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 ABTSox.
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.

BPUL activity at different temperatures

Figure 14: 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 the activity of 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 BPUL laccase but 0.4 mM CuCl2 at 10°C shows a negligible oxidation of ABTS. A a decrease in the reactivity of 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.

BPUL activity depending on different ABTS concentrations

Figure 15: Analysis of ABTS oxidation by 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, BPUL laccase 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 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 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, 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 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 BPUL laccase show a distinct higher activity of 50 µM ABTSox.

Figure 16: Standard BPUL activity test applying different amounts of MeOH. No considerable impact on the activity can be detected.
Figure 17: Standard 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.

In Figure 18 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, BPUL has a lower potential for degration of ethinyl estradiol with ABTS than TVEL0, but a higher potential than ECOL.

In Figure 19 and 20 the degradation of estradiol by BPUL is shown. BPUL is able to degrade estradiol even without ABTS, but does not has the same potential as the purchased laccase TVEL0.


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.















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