Team:Bielefeld-Germany/Results/halo

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<h1>Laccase Lbh1 from [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5 ''Bacillus halodurans'' C-125] </h1>
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              Laccase Lbh1 from <a href="http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5"> <i>Bacillus halodurans</i>  C-125 </a>
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<h1>Summary</h1>
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First some trials of shaking flask cultivations were made with various parameters to identify the best conditions for the production of the His-tagged laccase Lbh1 from [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5 ''Bacillus halodurans'' C-125 ] named BHAL. Because of no measured activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). BHAL could not be detected by SDS-PAGE (theoretical molecular weight of 56&nbsp;kDa) or activity test by using the BioBrick <partinfo>BBa_K863020</partinfo> and ''E. coli'' KRX as expression system. Due to this results the new BioBrick <partinfo>BBa_K863022</partinfo> was constructed and expressed ''E. coli'' Rossetta-Gami&nbsp;2. With this expression system the laccase could be produced and analysed via SDS-PAGE. To prove the activity of the produced laccase we used a small scale Ni-NTA-column to purify our laccase. The fractionated samples were tested concerning their activity with ABTS and showed ability in oxidizing ABTS. A scale up was not yet performed.
First some trials of shaking flask cultivations were made with various parameters to identify the best conditions for the production of the His-tagged laccase Lbh1 from [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5 ''Bacillus halodurans'' C-125 ] named BHAL. Because of no measured activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). BHAL could not be detected by SDS-PAGE (theoretical molecular weight of 56&nbsp;kDa) or activity test by using the BioBrick <partinfo>BBa_K863020</partinfo> and ''E. coli'' KRX as expression system. Due to this results the new BioBrick <partinfo>BBa_K863022</partinfo> was constructed and expressed ''E. coli'' Rossetta-Gami&nbsp;2. With this expression system the laccase could be produced and analysed via SDS-PAGE. To prove the activity of the produced laccase we used a small scale Ni-NTA-column to purify our laccase. The fractionated samples were tested concerning their activity with ABTS and showed ability in oxidizing ABTS. A scale up was not yet performed.
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__TOC__
==Cultivation==
==Cultivation==
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The resulting fractions of the cultivation and pruification of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] (fraction 1 to 5) were analysed with activity tests. After rebuffering into deionized H<sub>2</sub>O and icubation with 0.4 mM CuCl<sub>2</sub> for 2 hours, the samples were measured with 100 mM sodium acetate buffer, 140&nbsp;µL sample, 0.1 mM ABTS, ad 200 µL deionized  H<sub>2</sub>O. The change in optical density was measured at 420 nm, reporting the oxidization of ABTS through laccases for 5 hours at 25°C. An increase in ABTS<sub>ox</sub> can be seen (figure 2), indicating produced [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] laccase in each fraction. Fraction 2 shows the highest amount of ABTS<sub>ox</sub> (55%) after reaching its saturation after 3 hours. Compared to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] is capable to reach a saturation after 3 hours with approximately oxidizing 55% of used ABTS. Therefore [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] is a interesting laccase for the project and is going to be characterized further.
The resulting fractions of the cultivation and pruification of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] (fraction 1 to 5) were analysed with activity tests. After rebuffering into deionized H<sub>2</sub>O and icubation with 0.4 mM CuCl<sub>2</sub> for 2 hours, the samples were measured with 100 mM sodium acetate buffer, 140&nbsp;µL sample, 0.1 mM ABTS, ad 200 µL deionized  H<sub>2</sub>O. The change in optical density was measured at 420 nm, reporting the oxidization of ABTS through laccases for 5 hours at 25°C. An increase in ABTS<sub>ox</sub> can be seen (figure 2), indicating produced [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] laccase in each fraction. Fraction 2 shows the highest amount of ABTS<sub>ox</sub> (55%) after reaching its saturation after 3 hours. Compared to [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccases [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] is capable to reach a saturation after 3 hours with approximately oxidizing 55% of used ABTS. Therefore [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] is a interesting laccase for the project and is going to be characterized further.
[[File:Bielefeld2012_17_09_BHAL1.jpg|thumbnail|center|450px|'''Figure 2''': Activity test of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] fractions after purification. Reaction setup includes 100 mM sodium actetate buffer (pH 5), 140 µL fraction sample (CuCl2 incubated), 0.1 mM ABTS, ad 200 µL deionized H2O. Measurements were done at 25°C and over a time period of 5 hours. Each fraction shows activity, especially fraction 2, which therefore contains most [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] laccase. (n=4)]]
[[File:Bielefeld2012_17_09_BHAL1.jpg|thumbnail|center|450px|'''Figure 2''': Activity test of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] fractions after purification. Reaction setup includes 100 mM sodium actetate buffer (pH 5), 140 µL fraction sample (CuCl2 incubated), 0.1 mM ABTS, ad 200 µL deionized H2O. Measurements were done at 25°C and over a time period of 5 hours. Each fraction shows activity, especially fraction 2, which therefore contains most [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863022 BHAL] laccase. (n=4)]]
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{{Team:Bielefeld/Sponsoren}}

Revision as of 19:43, 26 September 2012

Laccase Lbh1 from Bacillus halodurans C-125

Summary

First some trials of shaking flask cultivations were made with various parameters to identify the best conditions for the production of the His-tagged laccase Lbh1 from Bacillus halodurans C-125 named BHAL. Because of no measured activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). BHAL could not be detected by SDS-PAGE (theoretical molecular weight of 56 kDa) or activity test by using the BioBrick BBa_K863020 and E. coli KRX as expression system. Due to this results the new BioBrick BBa_K863022 was constructed and expressed E. coli Rossetta-Gami 2. With this expression system the laccase could be produced and analysed via SDS-PAGE. To prove the activity of the produced laccase we used a small scale Ni-NTA-column to purify our laccase. The fractionated samples were tested concerning their activity with ABTS and showed ability in oxidizing ABTS. A scale up was not yet performed.


Contents


Cultivation

The first trials to produce the Lbh1 - laccase from Bacillus Halodurans (named BHAL) were performed in shaking flasks with various flask designs (from 100 mL-1 to 1 L flasks, with and without baffles) and under several conditions. The varied parameters in our screening experiments were temperature (27 °C,30 °C and 37 °C), concentration of chloramphenicol (20-170 µg mL-1), induction strategy (autoinduction and manual induction with 0,1 % rhamnose) and cultivation time (6 to 24 h). Furthermore we cultivated with and without 0.25 mM CuCl2 to provide a sufficient amount of copper, which is needed for the active center of the laccase. E.coli KRX was not able to produce active BHAL under the screened conditions, therefore another chassis was chosen. For further cultivations E. coli Rosetta-Gami 2 was transformed with BBa_K863012, because of its ability to translate rare codons. BHAL was produced under the following conditions:

  • flask design: shaking flask without baffles
  • medium: LB-Medium
  • antibiotics: 60 µg mL-1 chloramphenicol and 300 µg mL-1 ampicillin
  • temperature: 37 °C
  • cultivation time: 24 h

Just a small scale cultivation was performed so far.

Purification

The cells were harvested and resuspended in Ni-NTA-equilibrationbuffer, mechanically lysed by sonification and centrifuged. After preparing the cell paste the BHAL could not be purificate with the 15 mL column, because of its damage. For this reason a small scale purification (6 mL) of the supernatant of the lysate was made with a 1 mL Ni-NTA-column. The elution was collected in 1 mL fractions.

SDS-PAGE

Figure 1:SDS-PAGE of purified lysate of flask cultivation of E. coli Rosetta-Gami 2 with BBa_K863022. Lanes 2 to 7 show the flow-through, the wash and the elution fractions 1 to 4. BHAL has a molecular weight of 56 kDa and is marked with an arrow.

In figure 1 the different fractions of the purified cell lysate of E. coli Rosetta-Gami 2 with BBa_K863022 are shown in a SDS-PAGE. BHAL has a molecular weight of 56 kDa. In lane 5, which corresponds to the elution fraction 2, a light band of 56 kDa is visible. Therefore the fractions were further analysed by activity test and MALDI-TOF.


Activity Analysis of BHAL

The resulting fractions of the cultivation and pruification of BHAL (fraction 1 to 5) were analysed with activity tests. After rebuffering into deionized H2O and icubation with 0.4 mM CuCl2 for 2 hours, the samples were measured with 100 mM sodium acetate buffer, 140 µL sample, 0.1 mM ABTS, ad 200 µL deionized H2O. The change in optical density was measured at 420 nm, reporting the oxidization of ABTS through laccases for 5 hours at 25°C. An increase in ABTSox can be seen (figure 2), indicating produced BHAL laccase in each fraction. Fraction 2 shows the highest amount of ABTSox (55%) after reaching its saturation after 3 hours. Compared to BPUL laccases BHAL is capable to reach a saturation after 3 hours with approximately oxidizing 55% of used ABTS. Therefore BHAL is a interesting laccase for the project and is going to be characterized further.

Figure 2: Activity test of BHAL fractions after purification. Reaction setup includes 100 mM sodium actetate buffer (pH 5), 140 µL fraction sample (CuCl2 incubated), 0.1 mM ABTS, ad 200 µL deionized H2O. Measurements were done at 25°C and over a time period of 5 hours. Each fraction shows activity, especially fraction 2, which therefore contains most BHAL laccase. (n=4)

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