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bpul laccase from Bacillus pumilus

bpul (Laccase from Bacillus pumilus)

Sequence and Features

Usage and Biology

In the last few years a lot of attention has been drawn to laccases due to their ability to oxidize both phenolic and nonphenolic lignin related compounds as well as highly recalcitrant environmental pollutants. This makes them very useful for applications concerning several biotechnological processes. This includes the detoxification of industrial effluents, for example from the paper and pulp, textile and petrochemical industries. Laccases are also valuable as a tool as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Furthermore these enzymes are also used as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics^[1]. Their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. In our project laccases are used as cleaning agents for a water purification system. Laccases are copper-containing polyphenol oxidase enzymes (EC 1.10.3.2) that can be found in many plants, insects, microorganisms and mainly in fungi. These enzymes fulfill several functions in different metabolic pathways. Laccases are able to oxidize a broad range of substrates due to the contained copper-cluster, by reducing oxygen to water. The active site of the enzyme includes a four-copper-ion-cluster, which can be distinguished by spectroscopic analyses. This cluster consists of one blue copper-ion (type 1), one type 2 and two type 3 copper-ions. Because of the blue copper-ion, the laccases belong to the big family of the blue copper proteins. This specific blue copper ion is essential for the enzyme mediated radical oxidation of the phenolic groups. In this reaction the electron from the oxidation is transferred to the other three copper ions. These ions form a trinuclearic cluster, which transfers electrons to the terminal electron acceptor oxygen. By receiving four electrons the molecular oxygen is finally reduced to water.
[1] Susana Rodríguez Couto & José Luis Toca Herrera;Industrial and biotechnological applications of laccases: A review; 2006; Biotechnology Advances 24 500–513

Cultivation, Purification and SDS-PAGE

Shaking Flask Cultivation

3 L Fermentation E. coli KRX with BBa_K863000

Figure 1: Fermentation of E. coli KRX with BBa_K863000 (BPUL) in a [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Biostat_B_Bioreactor_.283_L.29_by_Braun Braun Biostat B], scale: 3 L, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium 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 a[http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Biostat_B_Bioreactor_.283_L.29_by_Braun Braun 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 [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer], mechanically lysed by [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Mechanical_lysis_of_the_.28bio-reactor.29_cultivation 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, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium 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 [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer], mechanically lysed by [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Mechanical_lysis_of_the_.28bio-reactor.29_cultivation 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) [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography 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 [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography 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 [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography 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 a Bioengineering NFL22 fermenter, 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 E. coli KRX culture 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

The E. coli laccase was identified using the following software

• FlexControl
• Flexanalysis and
• Biotools

from Brunker Daltronics. The peptid mass fingerprints were compared with the measured fingerprint gotten from the Maldi. With a sequence coverage of 21,9% BPUL was identified. In Figure 7 and 8 the chromatogram of the peptide mass fingerprint and the single masses is shown.

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 [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 [http://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#7 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 ABTS_ox 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 [http://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#7 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 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.

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 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.

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 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 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 [http://2012.igem.org/Team:Bielefeld-Germany/Results/coli#Impact_of_MeOH_and_acteonitrile_on_ECOL 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 ABTS_ox.

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 Analysis

The measurements were made to test if the produced laccases were able to degrade different hormones. Therefore the produced laccases were inserted in the same concentrations (3 µg mL^-1) to the different measurement approaches. To work with the correct pH value (which were measured by the Team Activity Test) Britton Robinson buffer at pH 5 was used for all measurements. The initial substrate concentration was 5 µg mL^-1. The results of the reactions without ABTS are shown in Figure 2. On the Y-axis the percentages of degraded estradiol (blue) and ethinyl estradiol (red) are indicated. The X-axis displays the different tested laccases. The degradation was measured at t₀ and after five hours of incubation at 30 °C. The negative control was the substrate in Britton Robinson buffer and showed no degradation of the substrates. The bought laccase TVEL0 which is used as positive control is able to degrade 94.7 % estradiol and 92.7 % ethinyl estradiol. The laccase BPUL (from Bacillus pumilus) degraded 35.9 % of used estradiol after five hours. ECOL was able to degrade 16.8 % estradiol. BHAL degraded 30.2 % estradiol. The best results were determined with TTHL (laccase from Thermus thermophilus). Here the percentage of degradation amounted 55.4 %.

The results of the reactions of the laccases with addition of ABTS are shown in Figure 3. The experimental set ups were the same as the reaction approach without ABTS described above. The X-axis displays the different tested laccases. On the Y-axis the percentages of degraded estradiol (blue) and ethinyl estradiol (red) are shown. The degradation was measured at t₀ and after five hours of incubation at 20 °C. The negative control showed no degradation of estradiol. 6.8 % of ethinyl estradiol was decayed. The positive control TVEL0 is able to degrade 100 % estradiol and ethinyl estradiol. The laccase BPUL (from Bacillus pumilus) degraded 46.9 % of used estradiol after ten minutes incubation. ECOL was able to degrade 6.7 % estradiol. BHAL degraded 46.9 % estradiol. With TTHL (laccase from Thermus thermophilus)a degradation 29.5 % were determined.

Figure 2: Degradation of estradiol (dark green) and ethinyl estradiol (light green) with the different laccases after 5 hours without ABTS. In the graph it is shown that the bought laccase TVEL0 which was used as positive control is able to degrade more than 90 percent of the used substrates. None of the bacterial laccases are able to degrade ethinyl estradiol without ABTS but estradiol is degraded in a range from 16 %(ECOL) to 55 % (TTHL). The original concentrations of substrates were 2 µg per approach. (n = 4)

Figure 3: Degradation of estradiol (blue) and ethinyl estradiol (red) with the different laccases after 10 minutes hours with ABTS added. The commercial laccase TVEL0 which was used as positive control is able to degrade all of the used substrates. The bacterial laccase BPUL degraded 100 % of ethinyl estradiol and estradiol. ECOL the laccase from E. coli degraded 6.7 % estradiol and none of the used ethinyl estradiol. BHAL degraded 46.9 % of estradiol but no ethinyl estradiol. The laccase TTHL from Thermus thermophilus degraded 29.5 % of estradiol and 9.8 % ethinyl estradiol. The original concentrations of substrates were 2 µg per approach. (n = 4)

Immobilization

Figure 21: 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 22: 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 slight decrease in the activity of BPUL in the supernatant

Figure 23: Enzymatic activity of immobilized laccases compared to nontreated laccases.

Figure 20 shows the percentage of laccases in the supernatant after incubation with CPC-beads, relative to the original concentration . The concentration of laccases in the supernatant after incubation was measured using Roti®-Nanoquant. The results show that 75.2% of BPUL remained in the supernatant. This indicates a relatively low binding capacity of BPUL on CPC-beads.

In figure 21, the enzymatic activity of BPUL in the supernatant is compared to the activity of nontreated BPUL. Although an activity can already be detected in the supernatant, this activity is lower compared to the original.

Figure 22 presents the enzymatic activity of immobilized laccases compared to nontreated laccases. The activity of bound BPUL is higher than the activity of ECOL, even if BPUL binds worse to the CPC-beads than ECOL.