Team:Bielefeld-Germany/Results/pumi

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<h1>Laccase CotA from [http://www.dsmz.de/catalogues/details/culture/DSM-27.html ''Bacillus pumilus'' DSM 27 ( ATCC7061)]</h1>
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<a href="http://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#3"><img src="http://2012.igem-bielefeld.de/includes/wiki/images/Pfeil_links2.png"></a>
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              Laccase CotA from <a href="http://www.dsmz.de/catalogues/details/culture/DSM-27.html"> <i>Bacillus pumilus</i> DSM 27 ( ATCC7061)</a>
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<h1>Summary</h1>
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First some trials of shaking flask cultivations were made with different parameters to define the best conditions for production of the His tagged CotA from [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&nbsp;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&nbsp;L and also up to 6&nbsp;L that enables an intense screening afterwards. A further scale up to 12 L with a optimized medium (HSG) was implemented to enable additional experiments to characterize BPUL. With this a total specific enzyme activity of ~40 U mg<sup>-1</sup> was determined for BPUL at pH 5 at 25°C with ABTS as a substrate. Additional scale up experiments will be important for further real world applications.
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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 [http://www.dsmz.de/catalogues/details/culture/DSM-27.html ''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&nbsp;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&nbsp;L and also up to 6&nbsp;L.
 
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__NOTOC__
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==Shaking Flask Cultivation==
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==Cultivation, Purification and SDS-PAGE==
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===Shaking Flask Cultivation===
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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&nbsp;mL<sup>-1</sup> to 1&nbsp;L flasks, with and without baffles) and under different conditions. The parameters we have changed during our screening experiments were temperature (27&nbsp;°C,30&nbsp;°C and 37&nbsp;°C), different concentrations of chloramphenicol (20 to 170&nbsp;µg&nbsp;mL<sup>-1</sup>), induction strategies ([http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction] and manual induction with 0,1&nbsp;% rhamnose) and cultivation time (6 to 24&nbsp;h). Further we cultivated with and without 0,25&nbsp;mM CuCl<sub>2</sub>, 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 CuCl<sub>2</sub> did not lead to better results, so it was omitted.
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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&nbsp;mL<sup>-1</sup> to 1&nbsp;L flasks, with and without baffles) and under different conditions. The parameters tested during the screening experiments were temperature (27&nbsp;°C,30&nbsp;°C and 37&nbsp;°C), the concentration of chloramphenicol (20 to 170&nbsp;µg&nbsp;mL<sup>-1</sup>), induction strategy ([http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction] and manual induction with 0,1&nbsp;% rhamnose) and cultivation time (6 to 24&nbsp;h). Furthermore it was cultivated with and without 0.25&nbsp;mM CuCl<sub>2</sub>, 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 CuCl<sub>2</sub> did not increase activity, so it was omitted.
* flask design: shaking flask without baffles
* flask design: shaking flask without baffles
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* cultivation time: 12&nbsp;h
* cultivation time: 12&nbsp;h
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The reproducibility and repeatability of the measured data and results were investigated for the shaking flask and bioreactor cultivation.
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The reproducibility of the measured data and results were investigated for the shaking flask and bioreactor cultivation.
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==3&nbsp;L Fermentation ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> ==
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===3&nbsp;L Fermentation ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> ===
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[[File:Bielefeld2012_BPUL3LFermentation.jpg|450px|thumb|left|'''Figure 1:''' Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Biostat_B_Bioreactor_.283_L.29_by_Braun Braun Biostat&nbsp;B], scale: 3&nbsp;L, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation on cascade to hold a pO<sub>2</sub> of 50&nbsp;%, OD<sub>600</sub> measured every 30&nbsp;minutes.]]
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[[File:Bielefeld2012_BPUL3LFermentation.jpg|450px|thumb|left|'''Figure 1:''' Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in a [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Biostat_B_Bioreactor_.283_L.29_by_Braun Braun Biostat&nbsp;B], scale: 3&nbsp;L, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation on cascade to hold pO<sub>2</sub> at 50&nbsp;%, OD<sub>600</sub> measured every 30&nbsp;minutes.]]
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After the measurement of BPUL activity we made a scale-up and fermented ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> in [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Biostat_B_Bioreactor_.283_L.29_by_Braun Braun Biostat&nbsp;B] with a total volume of 3&nbsp;L. Agitation speed, pO<sub>2</sub> and OD<sub>600</sub> were determined and illustrated in figure 1. We got a long lag phase of 2&nbsp;hours due to a relatively old preculture. The cell growth caused a decrease in pO<sub>2</sub> and after 3&nbsp;hours the value fell below 50&nbsp;%, so that the agitation speed increased automatically. After 8.5&nbsp;hours the deceleration phase started and therefore the agitation speed was decreased. The maximal OD<sub>600</sub> of 3.53 was reached after 10&nbsp;hours, which means a decrease in comparison to the fermentation of ''E.&nbsp;coli'' KRX under the same conditions (OD<sub>600,max</sub> =4.86 after 8.5&nbsp;hours, time shift due to long lag phase). The cells were harvested after 11&nbsp;hours.
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After the measurement of BPUL activity we made a scale-up and fermented ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> in a[http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Biostat_B_Bioreactor_.283_L.29_by_Braun Braun Biostat&nbsp;B] fermenter with a total volume of 3&nbsp;L. Agitation speed, pO<sub>2</sub> and OD<sub>600</sub> were determined and illustrated in Figure 1. We got a long lag phase of 2&nbsp;hours due to a relatively old preculture. The cell growth caused a decrease in pO<sub>2</sub> and after 3&nbsp;hours the value fell below 50&nbsp;%, so that the agitation speed increased automatically. After 8.5&nbsp;hours the deceleration phase started and therefore the agitation speed was decreased. The maximal OD<sub>600</sub> of 3.53 was reached after 10&nbsp;hours, which means a decrease in comparison to the fermentation of ''E.&nbsp;coli'' KRX under the same conditions (OD<sub>600,max</sub> =4.86 after 8.5&nbsp;hours, time shift due to long lag phase). The cells were harvested after 11&nbsp;hours.
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== Purification of BPUL ==
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=== Purification of BPUL ===
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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 centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15&nbsp;mL Ni-NTA resin) with a flowrate of 1&nbsp;mL min<sup>-1</sup> cm<sup>-2</sup>. The column was washed with 10&nbsp;column&nbsp;volumes (CV) Ni-NTA-equilibrationbuffer. The bound proteins were eluted by an increasing Ni-NTA-elutionbuffer gradient from 0&nbsp;% to 100&nbsp;% with a total volume of 100&nbsp;mL and the elution was collected in 10&nbsp;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 [http://2012.igem.org/wiki/images/4/49/Bielefeld2012_Chromatogram_examplegrafik.jpg here]. The chromatogram of the BPUL-elution is shown in figure 2:
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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&nbsp;mL Ni-NTA resin) with a flowrate of 1&nbsp;mL min<sup>-1</sup> cm<sup>-2</sup>. The column was washed with 10&nbsp;column&nbsp;volumes (CV) Ni-NTA-equilibrationbuffer. The bound proteins were eluted by an increasing Ni-NTA-elutionbuffer gradient from 0&nbsp;% to 100&nbsp;% with a total volume of 100&nbsp;mL and the elution was collected in 10&nbsp;mL fractions. In Figure 2 only the UV-detection signal of the wash step and the elution are shown, this is because of the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak.A typical chromatogram of purified laccases is illustrated [http://2012.igem.org/wiki/images/4/49/Bielefeld2012_Chromatogram_examplegrafik.jpg here]. The chromatogram of the BPUL-elution is shown in Figure 2:
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[[File:Bielefeld2012_BPUL3LChromatogramm.jpg|450px|thumb|left|'''Figure 2:''' Chromatogram of wash and elution from FLPC Ni-NTA-Histag purification of BPUL produced by 3&nbsp;L fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo>. BPUL was eluted between a process volume of 460&nbsp;mL to 480&nbsp;mL with a maximal UV-detection signal of 69 mAU]]
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[[File:Bielefeld2012_BPUL3LChromatogramm.jpg|450px|thumb|left|'''Figure 2:''' Chromatogram of wash and elution from FLPC Ni-NTA-His tag purification of BPUL produced by 3&nbsp;L fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo>. BPUL was eluted between a process volume of 460&nbsp;mL to 480&nbsp;mL with a maximal UV-detection signal of 69 mAU]]
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The chromatogram shows a remarkable widespread peak between the process volume of 460&nbsp;mL to 480&nbsp;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&nbsp;mL there are several peaks (up to a UV-detection-signal of 980&nbsp;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.
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The chromatogram shows a remarkable widespread peak between the process volume of 460&nbsp;mL to 480&nbsp;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&nbsp;mL there are several peaks (up to a UV-detection-signal of 980&nbsp;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.
</p>
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==SDS-PAGES of purified BPUL==
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===SDS-PAGE of purified BPUL===
<div style="text-align:justify;">
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[[File:Bielefeld2012_0906.jpg|height={50px}|weight={400px}|thumb|left|'''Figure 3:''' SDS-PAGE of purified ''E. coli'' KRX lysate containing <partinfo>BBa_K863000</partinfo> (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.]]
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[[File:Bielefeld2012_0906.jpg|height={50px}|weight={400px}|thumb|left|'''Figure 3:''' SDS-PAGE of purified ''E. coli'' KRX lysate containing <partinfo>BBa_K863000</partinfo> (fermented in a 3 L Biostat Braun B fermenter). The flow-through, wash and the elution fractions 7 and 8 are shown. The arrow marks the BPUL band with a molecular weight of 58.6 kDa.]]
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Figure 3 shows the purified ''E. coli'' KRX with <partinfo>BBa_K863000</partinfo> lysates (fermented in 3&nbsp;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.
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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.
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The appearing bands were analysed by MALDI-TOF and could be identified as CotA (BPUL).
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The appearing bands were analyzed by MALDI-TOF and could be identified as CotA (BPUL).
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==6&nbsp;L Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo>==
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===6&nbsp;L Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo>===
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[[File:Bielefeld2012_BPUL6LFermentation.jpg|450px|thumb|left|'''Figure 4:''' Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in Bioengineering NFL22, scale: 6&nbsp;L, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60&nbsp;µg&nbsp;mL<sup>-1</sup> chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation increased when pO<sub>2</sub> was below 30&nbsp;%, OD<sub>600</sub> measured every hour. ]]
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[[File:Bielefeld2012_BPUL6LFermentation.jpg|450px|thumb|left|'''Figure 4:''' Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in aBioengineering NFL22 fermenter, scale: 6&nbsp;L, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60&nbsp;µg&nbsp;mL<sup>-1</sup> chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation increased when pO<sub>2</sub> was below 30&nbsp;%, OD<sub>600</sub> measured every hour. ]]
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Another scale-up for ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> was made up to a final working volume of 6&nbsp;L in a Bioengineering NFL22. Agitation speed, pO<sub>2</sub> and OD<sub>600</sub> were determined and illustrated in figure 4. There was no noticeable lag phase. Agitation speed was increased up to 425&nbsp;rpm after one hour due to problems caused by the control panel. The pO<sub>2</sub> decreased until a cultivation time of 4.75&nbsp;hours. The increasing pO<sub>2</sub>-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 OD<sub>600</sub> of 3.68 was reached after 8&nbsp;hours of cultivation, which is similar to the 3&nbsp;L fermentation (OD<sub>600</sub> = 3.58 after 10 hours, time shift due to long lag phase). The cells were harvested after 12&nbsp;hours.
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Another scale-up for ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> was made up to a final working volume of 6&nbsp;L in a Bioengineering NFL22. Agitation speed, pO<sub>2</sub> and OD<sub>600</sub> were determined and illustrated in Figure 4. There was no noticeable lag phase. Agitation speed was increased up to 425&nbsp;rpm after one hour due to problems caused by the control panel. The pO<sub>2</sub> decreased until a cultivation time of 4.75&nbsp;hours. The increasing pO<sub>2</sub> 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 OD<sub>600</sub> of 3.68 was reached after 8&nbsp;hours of cultivation, which is similar to the 3&nbsp;L fermentation (OD<sub>600</sub> = 3.58 after 10 hours, time shift due to long lag phase). The cells were harvested after 12&nbsp;hours.
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==Purification of BPUL==
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===Purification of BPUL===
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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 centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15&nbsp;mL Ni-NTA resin) with a flowrate of 1&nbsp;mL min<sup>-1</sup> cm<sup>-2</sup>. The column was washed with 5&nbsp;column&nbsp;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&nbsp;% (equates to 20&nbsp;mM imidazol) to 100&nbsp;% (equates to 500&nbsp;mM imidazol) with a length of 200&nbsp;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&nbsp;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 [http://2012.igem.org/wiki/images/4/49/Bielefeld2012_Chromatogram_examplegrafik.jpg here]. The chromatogram of the BPUL-elution is shown in figure 5.
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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&nbsp;mL Ni-NTA resin) with a flow rate of 1&nbsp;mL min<sup>-1</sup> cm<sup>-2</sup>. The column was washed with 5&nbsp;column&nbsp;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&nbsp;% (equates to 20&nbsp;mM imidazol) to 100&nbsp;% (equates to 500&nbsp;mM imidazol) with a length of 200&nbsp;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&nbsp;mL fractions. In Figure 5 only the UV-detection signal of the wash step and the elution are shown, this is because of the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak. A typical chromatogram of purified laccases is illustrated [http://2012.igem.org/wiki/images/4/49/Bielefeld2012_Chromatogram_examplegrafik.jpg here]. The chromatogram of the BPUL elution is shown in Figure 5.
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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&nbsp;mL and 900&nbsp;mL there is remarkable widespread peak with a UV-detection signal of 115&nbsp;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-elutionbuffer] concentration between 10&nbsp;% and 20&nbsp;% (equates to 50-100&nbsp;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.
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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&nbsp;mL and 900&nbsp;mL there is remarkable widespread peak with a UV-detection signal of 115&nbsp;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&nbsp;% and 20&nbsp;% (equates to 50-100&nbsp;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.
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==SDS-PAGES of purified BPUL==
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===SDS-PAGE of purified BPUL===
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[[File:Bielefeld2012 0914.jpg|450px|thumb|left|'''Figure 7:''' SDS-PAGE of purified ''E.&nbsp;coli'' with <partinfo>BBa_K863000</partinfo> lysate (fermented in Bioengineering, 6 L). The flow-through, wash and elution fraction 1 to 9 are shown. The arrow marks the BPUL band with a molecular weight of 58.6 kDa. ]]
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[[File:Bielefeld2012 0914.jpg|450px|thumb|left|'''Figure 6:''' SDS-PAGE of purified ''E.&nbsp;coli'' with <partinfo>BBa_K863000</partinfo> 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. ]]
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In figure 7 the SDS-PAGE of the Ni-NTA  purification of the lysed culture of ''E. coli'' KRX containing <partinfo>BBa_K863000</partinfo> is illustrated. It shows the flow-through, wash and elution fractions 1 to 9. The His-tagged BPUL has a molecular weight of 58.6 kDA and was marked with a red arrow. The band appears in all fractions from 2 to 9 with varying strength, the strongest ones in fractions 7 to 9. There are also some other non-specific bands, which could not be identified. Therefore the purification method could moreover be improved.
+
In Figure 6 the SDS-PAGE of the Ni-NTA  purification of the lysed ''E. coli'' KRX culture containing <partinfo>BBa_K863000</partinfo> is illustrated. It shows the flow-through, wash and elution fractions 1 to 9. The His-tagged BPUL has a molecular weight of 58.6 kDA and was marked with a red arrow. The band appears in all fractions from 2 to 9 with varying strength, the strongest ones in fractions 7 to 9. There are also some other non-specific bands, which could not be identified. Therefore the purification method could moreover be improved.
 +
In summary, the scale up was successful, improving protein production and purification method once again.  
-
Furthermore the bands were analysed by MALDI-TOF and identified as CotA (BPUL).  
+
Furthermore the bands were analyzed by MALDI-TOF and identified as CotA (BPUL).  
</div>
</div>
 +
<br style="clear: both" />
 +
===Since Regionals: 12&nbsp;L Fermentation ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> ===
 +
 +
 +
[[File:Bielefeld2012_BPUL_Fermentation_12L.jpg|450px|thumb|left|'''Figure 7:''' Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in an Bioengineering NFL 22, scale: 12&nbsp;L, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#HSG_Autoinduction_medium HSG autoinduction medium] + 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation on cascade to hold pO<sub>2</sub> at 50&nbsp;%, OD<sub>600</sub> measured every hour.]]
 +
 +
<p align="justify">
 +
Finally another scale-up was made and ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> was fermented in a Bioengineering NLF 22 fermenter with a total volume of 12&nbsp;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, pO<sub>2</sub> and OD<sub>600</sub> 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 O<sub>2</sub>, which leads to an increase of agitation speed to hold a minimal pO<sub>2</sub> of 50 %. After 11 hours, the glycerin was completely consumed and the pO<sub>2</sub> increased up to 100 %, which indicates that the cells entered the stationary phase. The maximal OD<sub>600</sub> of 12.6 was reached after 12 hours of cultivation. The cells were harvested after 19 hours of cultivation.
 +
 +
</p>
 +
 +
 +
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==Activity Analysis of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL]==
+
=== Since Regionals: Purification of BPUL ===
 +
 
 +
<p align="justify">
 +
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&nbsp;mL Ni-NTA resin) with a flow rate of 1&nbsp;mL min<sup>-1</sup> cm<sup>-2</sup>. Then the column was washed with 10&nbsp;column&nbsp;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&nbsp;% (equates to 25&nbsp;mM imidazol) with a length of 50&nbsp;mL, to 50&nbsp;% (equates to 250&nbsp;mM imidazol) with a length of 70&nbsp;mL, to 80&nbsp;% (equates to 400&nbsp;mM imidazol) and finally to 100&nbsp;% (equates to 500&nbsp;mM imidazol) with a length of 100&nbsp;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&nbsp;mL fractions.  A typical chromatogram of purified laccases is illustrated [http://2012.igem.org/wiki/images/4/49/Bielefeld2012_Chromatogram_examplegrafik.jpg 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.
 +
</p>
 +
 
 +
<br style="clear: both" />
 +
 
 +
===Since Regionals: SDS-PAGE of protein purification===
 +
[[File:Bielefeld2012_1019pumi.jpg|300px|thumb|left|'''Figure 8:''' SDS-PAGE of purification from the 12&nbsp;L fermentations from 10/11 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000]). Purification of the supernatant via microfiltration, diafiltration and Ni-NTA column (step gradient with 5&nbsp;%, 50&nbsp;% and 100&nbsp;% elution buffer).]]
 +
 
 +
In Figure 8 the SDS-PAGE of the Ni-NTA purification of the lysed ''E.coli'' KRX culture containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 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.
 +
 
 +
 
 +
<br style="clear: both" />
 +
 
 +
===MALDI-TOF Analysis of BPUL===
 +
 
 +
The ''E. coli'' laccase was identified using the following software
 +
*FlexControl
 +
*Flexanalysis and
 +
*Biotools
 +
from Brunker Daltronics.
 +
The in silico- tryspinated created peptide mass fingerprints were compared with the measured masses 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 are shown.
 +
<br style="clear: both" />
 +
[[File:Bielefeld2012_Massemspektroskopie_BPumi_.png|thumb|400px|left|center|'''Figure 9:''' MALDI-TOF spectrum]] [[File:Bielefeld2012_Massenspektrometrische_Auswertung_BPumi.png|400px|thumb|right|'''Figure 10:''' MALDI-TOF spectrum results of the analysis]]
 +
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 +
 
 +
==Activity analysis of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL]==
===Initial activity tests of purified fractions===
===Initial activity tests of purified fractions===
 +
<p align="justify">
<p align="justify">
-
Initial tests were done with elution fractions 1 to 4 to determine the activity of the purified [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases. The fractions were rebuffered into deionized H<sub>2</sub>O  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 CuCl<sub>2</sub>. The reaction setup included 140 µL of a elution fraction, 100 mM sodium acetate buffer (pH 5), ad 198 deionized H<sub>2</sub>O 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 [http://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#7 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<sup>-1</sup>. 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<sup>-1</sup> for each measured sample to allow a comparison between TVEL0 measurements and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] measurements.
+
Initial tests were done with elution fractions 1 to 4 to determine the activity of the purified [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase. The fractions were rebuffered into deionized H<sub>2</sub>O  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 CuCl<sub>2</sub>. 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 11). 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<sup>-1</sup>. 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<sup>-1</sup> for each measured sample to allow a comparison between TVEL0 measurements and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] measurements.
</p>
</p>
-
[[File:Bielefeld2012_Party_Pumi_bestimmung2.jpg|thumbnail|600px|center|'''Figure 8:''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase activity measured in 100 mM sodium acetate buffer (pH 5), 0.1 mM ABTS, ad 200 µL deionized H<sub>2</sub>O 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<sub>ox</sub>. (n=4)]]
+
[[File:Bielefeld2012_Party_Pumi_bestimmung2.jpg|thumbnail|600px|center|'''Figure 11:''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase activity measured in 0.1 mM ABTS and 100 mM sodium acetate buffer (pH 5) to a final volume of 200 µL at 25°C over a time period of 3.5 hours. Each tested fraction reveals activity reaching the saturation after 15 minutes with ~60 µM ABTS<sub>ox</sub> after 0.4 mM CuCl<sub>2</sub> incubation. (n=4)]]
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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] pH optimum===
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] pH optimum===
 +
<p align="justify">
<p align="justify">
-
To determine at which pH the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase has its optimum in activity, a gradient of sodium acetate buffer pHs was prepared. Starting with pH 1 to pH 9 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity was tested using the described conditions above and 0.03 mg mL<sup>-1</sup> 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase at pH 5 (55 µM oxidized ABTS) . The other tested pHs only led to a odization of 18% of added ABTS. Figure 10 represents the negative control showing the oxidization of ABTS through 0.4 mM CuCl<sub>2</sub> 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 CuCl<sub>2</sub>. Nevertheless this result does not have an impact on the reactivity of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase at pH 5, which is still the optimal pH. Therefore it has the same pH optimum as [http://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#7 TVEL0].
+
<br>
-
[[File:Bielefeld2012_PH_Pumi1.jpg|thumbnail|500px|center|'''Figure 9:''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase activity measured in 100 mM sodium acetate buffer with a range of different pHs from pH 1 to pH 9, 0.1 mM ABTS, ad 200 µL deionized H<sub>2</sub>O at 25°C over a time period of 5hours. The optimal pH for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] is pH 5 with the most ABTS<sub>ox</sub>.]]
+
''Note: The experimental setup for the pH acticity assay was not well chosen. The buffering capacity of sodium acetate buffer is restricted to a smaller pH range than used in this experiment. The activity assay was optimized after the Regionals in Amsterdam to ensure correct measurements and values.''<br><br>
-
[[File:Bielefeld2012_PH_neg_control1.jpg|thumbnail|500px|center|'''Figure 10:''' Negative control for pH activity Tests using 0.04 mM CuCl<sub>2</sub> H<sub>2</sub>O instead of Laccase to determine the potential of ABTS getting oxidized through CuCl<sub>2</sub>.]]
+
 
 +
To determine at which pH the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase has its optimum in activity, a gradient of sodium acetate buffer pHs was prepared. Starting with pH 1 to pH 9 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity was tested using the described conditions above and 0.03 mg mL<sup>-1</sup> protein. The results are shown in Figure 12. A distinct pH optimum can be seen at pH 5. The saturation is reached after 3 hours with 50% oxidization of ABTS through the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase at pH 5 (55 µM oxidized ABTS). The other tested pHs only led to a oxidation of 18% of added ABTS. Figure 13 represents the negative control showing the oxidation of ABTS through 0.4 mM CuCl<sub>2</sub> 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 CuCl<sub>2</sub>. Nevertheless this result does not have an impact on the reactivity of the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase at pH 5, which is still the optimal pH. Therefore it has the same pH optimum as [http://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#7 TVEL0].
 +
[[File:Bielefeld2012_PH_Pumi1.jpg|thumbnail|500px|center|'''Figure 12:''' [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase activity measured in 100 mM sodium acetate buffer with a range of different pHs from pH 1 to pH 9, 0.1 mM ABTS to a final volume of 200 µL at 25°C over a time period of 5 hours. Before the measurements samples were incubated with CuCl<sub>2</sub>. The optimal pH for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] is pH 5 with the most ABTS<sub>ox</sub>.]]
 +
[[File:Bielefeld2012_PH_neg_control1.jpg|thumbnail|500px|center|'''Figure 13:''' Negative control for pH activity Tests using 0.04 mM CuCl<sub>2</sub> H<sub>2</sub>O instead of Laccase to determine the potential of ABTS getting oxidized through CuCl<sub>2</sub>.]]
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.
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.
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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] CuCl<sub>2</sub> concentration===
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] CuCl<sub>2</sub> concentration===
<p align="justify">
<p align="justify">
-
Another test of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] was done to survey the best CuCl<sub>2</sub> concentration for the activity of the purified [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase. 0.03  mg mL<sup>-1</sup> of protein were incubated in different CuCl<sub>2</sub> concentration ranging from 0 to 0.7 mM CuCl<sub>2</sub>. Activity tests were performed with the incubated samples, 100 mM sodium actetate buffer (pH 5), 0.1 mM ABTS, ad 200 µL H<sub>2</sub>O. 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases incubated in different CuCl<sub>2</sub> differ minimal. The most percentage is oxidized with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases incubated with 0.6 mM CuCl<sub>2</sub> (52% of added ABTS). With a higher concentration of 0.7 mM CuCl<sub>2</sub> the activity seems to be reduced (only 48% ABTS got oxidized). This leads to the assumption that CuCl<sub>2</sub> supports the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase reactivity but concentrations exceeding this value of CuCl<sub>2</sub> may have a negative impact on the ability of oxidizing ABTS. Without any CuCl<sub>2</sub> application [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 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 CuCl<sub>2</sub> but no laccase to detect the oxidization of ABTS through copper (see figure 12). The more CuCl<sub>2</sub> was applicated, the more ABTS was oxidzied after 5 hours. Still the maximal change accounts ~6% oxidized ABTS after 5 hours.
+
Another test of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] was done to survey the best CuCl<sub>2</sub> concentration for the activity of the purified [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase. 0.03  mg mL<sup>-1</sup> of protein were incubated with different CuCl<sub>2</sub> concentrations ranging from 0 to 0.7 mM CuCl<sub>2</sub>. 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 14). The differences in the activity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases incubated in different CuCl<sub>2</sub> differ minimal. The highest activity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase is observed after incubation with 0.6 mM CuCl<sub>2</sub> (52% of added ABTS). With a higher concentration of 0.7 mM CuCl<sub>2</sub> the activity seems to be reduced (only 48% ABTS got oxidized). This leads to the assumption that CuCl<sub>2</sub> supports the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase reactivity but concentrations exceeding this value of CuCl<sub>2</sub> 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 CuCl<sub>2</sub> without applying laccase to detect the oxidization of ABTS through copper (see Figure 15). The more CuCl<sub>2</sub> was present, the more ABTS was oxidzied after 5 hours. Still the maximal change accounts only for ~6% oxidized ABTS after 5 hours.
-
[[File:Bielefeld2012_Pumi_Cu1.jpg|thumbnail|500px|center|'''Figure 11:''' Activity measurement using 0.1 mM ABTS of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] incubated in different CuCl<sub>2</sub> concentrations. Without CuCl<sub>2</sub> incubation [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] is not active. Incubation with 0.1 mM CuCl<sub>2</sub> or higher concentrations leas to an increase in ABTS<sub>ox</sub>.]]
+
[[File:Bielefeld2012_Pumi_Cu1.jpg|thumbnail|500px|center|'''Figure 14:''' Activity measurement using 0.1 mM ABTS of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] incubated in different CuCl<sub>2</sub> concentrations. Without CuCl<sub>2</sub> incubation the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase shows half of the activit as after CuCl<sub>2</sub> incubation. Incubation with 0.1 mM CuCl<sub>2</sub> or higher concentrations leas to an increase in ABTS<sub>ox</sub>.]]
-
[[File:Bielefeld2012_Pumi_Cu_NegControl1.jpg|thumbnail|500px|center|'''Figure 12:''' Negative control for CuCl<sub>2</sub> activity Tests using different concentrations of CuCl<sub>2</sub> H<sub>2</sub>O instead of Laccase to determine the potential of ABTS getting oxidized through CuCl<sub>2</sub>.]]
+
[[File:Bielefeld2012_Pumi_Cu_NegControl1.jpg|thumbnail|500px|center|'''Figure 15:''' Negative control for CuCl<sub>2</sub> activity Tests using different concentrations of CuCl<sub>2</sub> H<sub>2</sub>O instead of Laccase to determine the potential of ABTS getting oxidized through CuCl<sub>2</sub>.]]
-
In relation to apply the laccase in waste water treatment plants it is beneficial knowing, that small amounts of CuCl<sub>2</sub> are enough to activate them. This reduces the cost factor and risks.
+
In relation to apply the laccase in waste water treatment plants it is beneficial knowing, that small amounts of CuCl<sub>2</sub> are enough to activate the enzyme. Still it is expensive to be reliant on CuCl<sub>2</sub> and a potential risk using heavy metals for waste water purifcation.
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</p>
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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity at different temperatures===
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity at different temperatures===
-
[[File:Bielefeld2012_BPUL_Temp.jpg|thumbnail|450px|left|'''Figure 13:''' Standard activity test for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] measured at 10°C and 25°C resulting in a decreased activity at 10°C. As a negative control the impact of 0.4 mM CuCl<sub>2</sub> in oxidizing ABTS at 10°C were analyzed.]]
+
[[File:Bielefeld2012_BPUL_Temp.jpg|thumbnail|450px|left|'''Figure 16:''' Standard activity test for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] measured at 10°C and 25°C resulting in a decreased activity at 10°C. As a negative control the impact of 0.4 mM CuCl<sub>2</sub> in oxidizing ABTS at 10°C were analyzed.]]
<p align="justify">
<p align="justify">
-
To investigate in the activity behaviour of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 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 the[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase but 0.4 mM CuCl<sub>2</sub> at 10°C shows a negligible effect on the status of ABTS.
+
To investigate the activity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] at lower temperatures, activity tests as described above were performed at 10°C and 25°C. A small decrease in the activity can be observed upon reducing the temperature from 25°C to 10°C (see Fig. 16). After 3.5 hours when samples at 25°C reached the saturation samples at 10°C had not, but nonetheless the difference is minimal. After 3 hours 5% difference in oxidized ABTS is observable. The negative control without the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase but 0.4 mM CuCl<sub>2</sub> at 10°C shows a negligible oxidation of ABTS.
-
A decrease in the reactivity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 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.
+
A a decrease in the reactivity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase was expected. The observed small reduction in enzyme activity is excellent news for the possible application in waste water treatment plants where the temperature differs from 8.1°C to 20.8°C.
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</p>
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===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity depending on different ABTS concentrations===
===[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity depending on different ABTS concentrations===
-
[[File:Bielefeld2012_BPUL_ABTS.jpg|thumbnail|450px|left|'''Figure 14:''' Analysis of ABTS oxidation by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] Laccases tested with different amounts of ABTS. The higher the amount of ABTS the more oxidized ABTS can be detected.]]
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[[File:Bielefeld2012_BPUL_ABTS.jpg|thumbnail|450px|left|'''Figure 17:''' Analysis of ABTS oxidation by [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases incubated in 0.4 CuCl<sub>2</sub> tested with different amounts of ABTS. The higher the amount of ABTS the more oxidized ABTS can be detected.]]
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Furthermore [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases were tested using different amounts of ABTS to calculate K<sub>M</sub> and K<sub>cat</sub> 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 ABTS<sub>ox</sub> but the amount of oxidized ABTS decreased afterward.
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Furthermore, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase were tested using different amounts of ABTS to calculate K<sub>M</sub> and K<sub>cat</sub> 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 17). Especially using 16 µL showed an increase in the activity until 1 hour (reaching 50 µM ABTS<sub>ox</sub>), but the amount of oxidized ABTS decreased afterward.
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===Impact of MeOH and acteonitrile on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL]===
===Impact of MeOH and acteonitrile on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL]===
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For substrate analytic tests the influence of MeOH and acetonitrile on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases had to be determined, because substrates have to be dissolved in these reagents. The experiment setup included 0.03 mg mL<sup>-1</sup> [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 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  H<sub>2</sub>O. The observed reactivity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] in regard of oxidizing ABTS did not reveal a huge decrease. The less MeOH or acetonitrile was used, the higher was the amount of oxidized ABTS after 3 hours. An application of 16 µL MeOH or acetonitrile led to a decrease of maximal 10% oxidized ABTS compared to 2 µL MeOH or acetonitrile.
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For substrate analytic tests the influence of MeOH and acetonitrile on [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases had to be determined, because substrates have to be dissolved in these reagents. The experiment setup included 0.03 mg mL<sup>-1</sup> [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase, different amounts of MeOH (Figure 18) or acteonitrile (Figure 19), 0.1 mM ABTS and 100 mM sodium actetate buffer to a final volume of 200 µL. The observed reactivity of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] in regard of oxidizing ABTS did not reveal a huge decrease. The less MeOH or acetonitrile was used, the higher was the amount of oxidized ABTS after 3 hours. An application of 16 µL MeOH or acetonitrile led to a decrease of maximal 10% oxidized ABTS compared to 2 µL MeOH or acetonitrile. Negative controls are shown in [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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase show a distinct higher activity of 50 µM ABTS<sub>ox</sub>.
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[[File:Bielefeld2012_Pumi_MeOH1.jpg|thumbnail|500px|center|'''Figure 15:''' Standard [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity test applying different amounts of MeOH. No considerable impact on the activity can be detected.]]
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[[File:Bielefeld2012_Pumi_MeOH1.jpg|thumbnail|500px|center|'''Figure 18:''' Standard [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity test applying different amounts of MeOH. No considerable impact on the activity can be detected.]]
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[[File:Bielefeld2012_Pumi_acetonitrile1.jpg|thumbnail|500px|center|'''Figure 16:''' Standard [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity test applying different amounts of acetonitrile. No considerable impact on the activity can be detected.]]
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[[File:Bielefeld2012_Pumi_acetonitrile1.jpg|thumbnail|500px|center|'''Figure 19:''' Standard [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity test applying different amounts of acetonitrile. No considerable impact on the activity can be detected.]]</p>
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===Since Regionals: Initial activity tests of purified fractions===
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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 H<sub>2</sub>O and incubated in 0.4 mM CuCl<sub>2</sub>. 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. 20).
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[[File:Bielefeld2012_new_BPUL_acitivity.jpg|500px|thumb|center|'''Figure 20:''' Activity assay of each purified fraction of recent produced BPUL. Samples were re-buffered into H<sub>2</sub>O 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.]]
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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<sup>-1</sup>.
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===Since Regionals: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity depending on different ABTS concentrations===
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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 OD<sub>420</sub> every 5 minutes. Comparing the graphs in Figure 21 and Figure 22, 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.
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[[File:Bielefeld2012_BPUL_klein_ABTS.jpg|thumb|left|360px|'''Figure 21:''' 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.]]
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[[File:Bielefeld2012_BPUL_hoch.jpg|thumb|right|360px|'''Figure 22:''' 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.]]
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===Since Regionals: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] pH optimum===
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[[File:Bielefeld2012_Pumi_pH_Foto.png|thumb|right|200px|'''Figure 23:''' 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.]]
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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 H<sub>2</sub>O and incubated with 0.4 mM CuCl<sub>2</sub>. 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. 23 and Fig. 24). At higher pHs than pH 5, the activity of BPUL was decreased considerably. The resulting Units mg<sup>-1</sup> support the observed data (see Fig. 25). At pH 4 and pH 5 BPUL showed a specific enzyme activity of ~37 U mg<sup>-1</sup>. The higher the pH, the less U mg<sup>-1</sup> 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.
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[[File:Bielefeld2012_BPUL_pH_new.jpg|thumb|left|360px|'''Figure 24:''' Oxidized ABTS by BPUL at different pH adjustments. The experimental setup included CuCl<sub>2</sub> 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.]]
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[[File:Bielefeld2012_BPUL_pH_Units.jpg|thumb|right|360px|'''Figure 25:''' 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.]]
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===Since Regionals: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] activity at different temperatures===
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[[File:Bielefeld2012 BPUL Temp ABTSox.jpg|left|200px|thumb|'''Figure 26:''' 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 CuCl<sub>2</sub> in oxidizing ABTS at 10 °C and 25 °C was analyzed.]]
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[[File:Bielefeld2012 BPUL Temp Units.jpg|right|200px|thumb|'''Figure 27:''' 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<sup>-1</sup> could be detected. One unit is defined as the amount of laccase that oxidizes 1 μmol of ABTS substrate per minute.]]
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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. 26). 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<sup>-1</sup> (see Fig. 27). The negative control without BPUL laccase but 0.4 mM CuCl<sub>2</sub> 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.
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== Substrate Analysis==
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[[File:Bielefeld2012_Ohne_ABTS.png|400px|thumb|right|'''Figure 28: 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&nbsp;%(ECOL) to 55&nbsp;% (TTHL). The original concentrations of substrates were 2 µg per approach. (n&nbsp;=&nbsp;4)]]
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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<sup>-1</sup>) 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&nbsp;5 was used for all measurements. The initial substrate concentration was 5 µg mL<sup>-1</sup>. The results of the reactions without ABTS are shown in Figure 28. 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 t<sub>0</sub> and after five hours of incubation at 30&nbsp;°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&nbsp;% estradiol and 92.7&nbsp;%  ethinyl estradiol. The laccase BPUL (from ''Bacillus pumilus'') degraded 35.9&nbsp;% of used estradiol after five hours. ECOL  was able to degrade 16.8&nbsp;% estradiol. BHAL degraded 30.2&nbsp;% estradiol. The best results were determined with TTHL (laccase from ''Thermus thermophilus''). Here the percentage of degradation amounted 55.4&nbsp;%. 
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[[File:Bielefeld2012_Mit_ABTS.png|400px|thumb|left|'''Figure 29: 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&nbsp;% estradiol and none of the used ethinyl estradiol. BHAL degraded 46.9&nbsp;% of estradiol but no ethinyl estradiol. The laccase TTHL from ''Thermus thermophilus'' degraded 29.5&nbsp;% of estradiol and 9.8&nbsp;% ethinyl estradiol. The original concentrations of substrates were 2 µg per approach. (n&nbsp;=&nbsp;4)]]
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The results of the reactions of the laccases with addition of ABTS are shown in Figure 29. 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 t<sub>0</sub> and after five hours of incubation at 20&nbsp;°C. The negative control showed no degradation of estradiol. 6.8&nbsp;% of ethinyl estradiol was decayed. The positive control TVEL0 is able to degrade 100&nbsp;% estradiol and ethinyl estradiol. The laccase BPUL (from ''Bacillus pumilus'') degraded 46.9&nbsp;% of used estradiol after ten minutes incubation. ECOL  was able to degrade 6.7&nbsp;% estradiol. BHAL degraded 46.9&nbsp;% estradiol. With TTHL (laccase from ''Thermus thermophilus'') a degradation 29.5&nbsp;% were determined.
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== Immobilization ==
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[[File:Bielefeld2012-Immobilized_proteins.jpg|500px|left|thumb|'''Figure 30''': 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.]]
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Figure 30 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.
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[[File:Bielefeld2012_bpumi.jpg|500px|left|thumb|'''Figure 31''': 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.]]
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In figure 31, 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.
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[[File:Bielefeld2012-Graphen_Bead_BPUL.jpg|500px|left|thumb|'''Figure 32''': Illustration of ABTS oxidation by BPUL with time compared to the negative control. The increase in ABTS oxidized proves laccase activity.]]
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Figure 32 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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{{Team:Bielefeld/Sponsoren}}

Latest revision as of 03:37, 27 October 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 from 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. A further scale up to 12 L with a optimized medium (HSG) was implemented to enable additional experiments to characterize BPUL. With this a total specific enzyme activity of ~40 U mg-1 was determined for BPUL at pH 5 at 25°C with ABTS as a substrate. Additional scale up experiments will be important for further real world applications.


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 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 (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 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. In Figure 2 only the UV-detection signal of the wash step and the elution are shown, this is because of the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak.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. In Figure 5 only the UV-detection signal of the wash step and the elution are shown, this is because of the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak. 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 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).


Since Regionals: 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, 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 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 Ni-NTA- equilibration buffer, mechanically disrupted by 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) Ni-NTA equilibration buffer. The bound proteins were eluted by an increasing 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.


Since Regionals: 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 in silico- tryspinated created peptide mass fingerprints were compared with the measured masses 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 are shown.

Figure 9: MALDI-TOF spectrum
Figure 10: 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 11). 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 11: 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


Note: The experimental setup for the pH acticity assay was not well chosen. The buffering capacity of sodium acetate buffer is restricted to a smaller pH range than used in this experiment. The activity assay was optimized after the Regionals in Amsterdam to ensure correct measurements and values.

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 12. 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 13 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 12: 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 13: 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 14). 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 15). 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 14: 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 15: 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 16: 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. 16). 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 17: 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 17). 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 18) or acteonitrile (Figure 19), 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 18: Standard BPUL activity test applying different amounts of MeOH. No considerable impact on the activity can be detected.
Figure 19: Standard BPUL activity test applying different amounts of acetonitrile. No considerable impact on the activity can be detected.


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 protein content (10/16), re-buffered into deionized H2O and incubated in 0.4 mM CuCl2. Again, the 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. 20).

Figure 20: 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 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.


Since Regionals: BPUL activity depending on different ABTS concentrations

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 21 and Figure 22, 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 21: 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 22: 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 23: 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. 23 and Fig. 24). At higher pHs than pH 5, the activity of BPUL was decreased considerably. The resulting Units mg-1 support the observed data (see Fig. 25). 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 24: 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 25: 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 26: 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 27: 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. 26). 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. 27). 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

Figure 28: 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)


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 28. 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 %.

Figure 29: 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)

The results of the reactions of the laccases with addition of ABTS are shown in Figure 29. 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.

Immobilization

Figure 30: 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 30 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 31: 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 31, 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 32: Illustration of ABTS oxidation by BPUL with time compared to the negative control. The increase in ABTS oxidized proves laccase activity.

Figure 32 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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