Part:BBa_K863000

bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag

bpul (Laccase from Bacillus pumilus) with T7, RBS and HIS tag

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

Sequence and Features

Assembly Compatibility:

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  Illegal BglII site found at 156
  
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  COMPATIBLE WITH RFC[23]
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  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 523
  Illegal NgoMIV site found at 991
  Illegal AgeI site found at 279
  Illegal AgeI site found at 1156
  
• 1000
  COMPATIBLE WITH RFC[1000]

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

The first trials to produce the CotA-laccase from [http://www.dsmz.de/catalogues/details/culture/DSM-27.html 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 tested 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 ([http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium 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: [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_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 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).

12 L Fermentation E. coli KRX with BBa_K863000

Figure 7: Fermentation of E. coli KRX with BBa_K863000 (BPUL) in an Bioengineering NFL 22, scale: 12 L, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#HSG_Autoinduction_medium HSG autoinduction medium] + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold pO₂ at 50 %, OD₆₀₀ measured every hour.

Finally another scale-up was made and E. coli KRX with BBa_K863000 was fermented in a Bioengineering NLF 22 fermenter with a total volume of 12 L to produce a high amount of the enzyme for further characterizations. This time [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#HSG_Autoinduction_medium HSG autoinduction medium] was used to get a higher biomass. Agitation speed, pO₂ and OD₆₀₀ were determined and the glycerin concentration of the samples analyzed. The data are illustrated in Figure 7. At the beginning of the cultivation, the cells were in lag phase, in which they adapt to the medium. During their growth the cells consumed glycerin as well as O₂, which leads to an increase of agitation speed to hold a minimal pO₂ of 50 %. After 11 hours, the glycerin was completely consumed and the pO₂ increased up to 100 %, which indicates that the cells entered the stationary phase. The maximal OD₆₀₀ of 12.6 was reached after 12 hours of cultivation. The cells were harvested after 19 hours of cultivation.

Since Regionals: 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- equilibration buffer], mechanically disrupted 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, microfiltration as well as diafiltration to concentrate the protein concentration in the cell lysate solution. This solution 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) [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA equilibration buffer]. The bound proteins were eluted by an increasing [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography 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 70 mL, to 80 % (equates to 400 mM imidazol) and finally to 100 % (equates to 500 mM imidazol) with a length of 100 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. A typical chromatogram of purified laccases is illustrated here. Unfortunately, the data of this procedure are not available due to a computer crash after the purification step. All Fractions were analysed to detect BPUL.

SDS-PAGE of protein purification

Figure 8: SDS-PAGE of purification from the 12 L fermentations from 10/11 (BBa_K863000). Purification of the supernatant via microfiltration, diafiltration and Ni-NTA column (step gradient with 5 %, 50 % and 100 % elution buffer).

In Figure 8 the SDS-PAGE of the Ni-NTA purification of the lysed E.coli KRX culture containing BBa_K863000 is illustrated. It shows the permeate and retentate of microfiltration and diafiltration respectively, several fractions of flow-through, wash and the elutions with different buffer concentrations respectively. The selected samples were taken where peaks were seen in the chromatogram. The His-tagged BPUL has a molecular weight of 58.6 kDa. BPUL could not be attributed exactly to any band. There are some other non-specific bands, wich could not be identified because the MALDI was broken-down for the last two weeks.

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 9 and 10 the chromatogram of the peptide mass fingerprint and the single masses is shown.

Figure 9: MALDI-TOF spectrum

Figure 10: MALDI-TOF spectrum results of the analysis

Activity analysis of BPUL

Since Regionals: Initial activity tests of purified fractions

After the Regional Jamboree in Amsterdam further BPUL was produced. The most comprising fractions of the purification were analyzed for [http://2012.igem.org/Team:Bielefeld-Germany/Amsterdam/Labjournal#Tuesday_October_16th/ protein content] (10/16), re-buffered into deionized H₂O and incubated in 0.4 mM CuCl₂. Again, the [http://2012.igem.org/Team:Bielefeld-Germany/Amsterdam/Labjournal#Wednesday_October_17th/ protein content] (10/17) of each fraction was determined because of the loss of proteins through re-buffering. Initial activity tests were done in Britton-Robinson buffer with 0.1 mM ABTS. The protein content of each fraction was adjusted for comparison of the resulting activity (see Fig. 11).

Figure 11: Activity assay of each purified fraction of recent produced BPUL. Samples were re-buffered into H₂O and the protein amount in each fraction was adjusted. The measurement was done using the [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#General_setup_of_enzyme_activity_measurements/ standard activity assay protocol] over night. The first number indicates the percentage of used elution buffer, whereas the second number stands for the fraction number of this elution.

Fraction 50% 2 showed the highest activity. The first number indicates the percentage of used elution buffer, whereas the second number stands for the fraction number of this elution. The saturation was reached at ~1 h. For comparison it was stated that this fraction contains 90 % laccase and therefore the BPUL concentration is 25,1 µg mL^-1.

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 depending on different ABTS concentrations

Figure 14: 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 14). 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 15) or acteonitrile (Figure 16), 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_acetonitrile_on_ECOL Figure 20 and 21] 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 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.

Since Regionals: Searching for substrate saturation of BPUL

In order to find the substrate saturation, laccase activity was measured with ABTS concentrations ranging from 0.1 mM to 8 mM. 616 ng BPUL were used for measurements with ABTS concentrations of 0.1 mM to 5 mM, 308 ng BPUL were used for measurements with ABTS concentrations of 5 mM to 8 mM. Measurements were done in Britton-Robinson buffer (pH 5) at 25 °C for 30 minutes taking the OD₄₂₀ every 5 minutes. Comparing the graphs in Figure 17 and Figure 18, both show a comprising substrate saturation with 5 mM ABTS. Higher concentrations of ABTS than 5 mM did not show any other effects on the activity of BPUL. For all following BPUL activity measurements after the Regional Jamborees in Amsterdam a concentration of 5 mM ABTS was applied.

Figure 17: Activity assay to determine the substrate saturation with ABTS as a substrate. Measurements were done with 616 ng BPUL in Britton-Robinson buffer (pH 5) at 25 °C. ABTS concentrations ranged from 0.1 mM to 5 mM.

Figure 18: Activity assay to determine the substrate saturation with ABTS as a substrate. Measurements were done with 308 ng BPUL in Britton-Robinson buffer (pH 5) at 25 °C. ABTS concentrations ranged from 5 mM to 8 mM. An ABTS concentration of 5 mM was determined as substrate saturation.

Since Regionals: BPUL pH optimum

Figure 19: Microtiter plate of the measurements for pH optimum determination. The more intensive the blue color the more ABTS got oxidized. At pH 5 and pH 4 the darkest colour has been reached.

The pH of the medium containing the enzyme is of high importance for its activity. The pH optima of BPUL are pH 4 and pH 5. This is the result of activity measurements using Britton-Robinson buffer with differently adjusted pHs. BPUL was re-buffered into H₂O and incubated with 0.4 mM CuCl₂. The range from pH 4 to pH 9 was tested under substrate saturation at 25 °C for 30 minutes. At pH 4 and pH 5 ABTS got oxidized the fastest (see Fig. 19 and Fig. 20). At higher pHs than pH 5, the activity of BPUL was decreased considerably. The resulting Units mg^-1 support the observed data (see Fig. 21). At pH 4 and pH 5 BPUL showed a specific enzyme activity of ~37 U mg^-1. The higher the pH, the less U mg^-1 could be calculated for BPUL. At pH 7 1/3 of the activity decreased, but still BPUL was active at this pH allowing an application of this laccase in a waste water treatment plant where the average pH is a pH of 6.9.

Figure 20: Oxidized ABTS by BPUL at different pH adjustments. The experimental setup included CuCl₂ incubated BPUL laccase (308 ng), Britton Robinson buffer adjusted to the tested pHs and 5 mM ABTS. Measurements were done at 25 °C for 30 minutes. The highest amount of oxidized ABTS was detected at pH 4 and pH 5.

Figure 21: Calculated specific enzyme activity of BPUL at different pH conditions. The highest specific enzyme activity for ABTS was under pH 4 and pH 5 conditions. The higher the pH, the less ABTS got oxidized. One unit is defined as the amount of laccase that oxidizes 1 μmol of ABTS substrate per minute.

Since Regionals: BPUL activity at different temperatures

Figure 22: Standard activity test for BPUL measured at 10 °C and 25 °C resulting in a comparable activity at the tested temperatures. As a negative control the impact of 0.4 mM CuCl₂ in oxidizing ABTS at 10 °C and 25 °C was analyzed.

Figure 23: Deriving from the obtained values of oxidized ABTS in time at 10 °C and 25 °C the specific enzyme activity was calculated. For the temperatures only a difference of 1 U mg^-1 could be detected. One unit is defined as the amount of laccase that oxidizes 1 μmol of ABTS substrate per minute.

To investigate the activity of BPUL at temperatures that will apply at a waste water treatment plant throughout the year, activity tests were performed at 10 °C and 25 °C as described above. The obtained results reveal a comparable activity of BPUL at high and low temperatures (see Fig. 22). The measurements were conducted for 30 minutes until saturation initiated. Both samples reached saturation after 15-20 minutes. The obtained results were used to calculate the specific enzyme activity which was in both cases at about 37 U mg^-1 (see Fig. 23). The negative control without BPUL laccase but 0.4 mM CuCl₂ at 10 °C and 25 °C show a negligible oxidation of ABTS. The observed activity at both conditions was good news for the possible application in waste water treatment plants where the temperature differs from 8.1 °C to 20.8 °C.

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 24. 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 25. 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 24: 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 25: 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 26: The percentage of laccases immobilized to CPC-Beads. 99 % of ECOL, 97 % of BPUL and 79 % of BHAL and TTHL laccases were bound to the beads.

Figure 26 shows the percentage of laccases bound 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 showed that only 3% of BPUL laccases was still present in the supernatant. This illustrates that BPUL was successfully immobilized on the CPC-beads.

Figure 27: Enzymatic activity of BPUL 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 BPUL in the Supernatant.

In figure 27, 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 low compared to the original.

Figure 28: Illustration of ABTS oxidation by BPUL with time compared to the negative control. The increase in ABTS oxidized proves laccase activity.

Figure 28 shows the illustration of ABTS oxidation by BPUL with time compared to the negative control. The increase in ABTS oxidized proves laccase activity even if a direct comparison with the original and not immobilized laccase solution was not possible due to the very high activity of BPUL, which could not be measured properly.

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