MALDI-TOF Analysis of ECOL:
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Revision as of 19:15, 28 September 2012
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[[Image:Bielefeld2012_Massemspektroskopie_Ecoli.png|thumb|left|350px|'''Figure 7:''' The MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) spectrum.]][[
File
:Bielefeld2012_Massenspektrometrische_Ecoli_Auswertung.png|thumb|right|350px|'''Figure 8:''' Part of MALDI-TOD Evaluation]]
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[[Image:Bielefeld2012_Massemspektroskopie_Ecoli.png|thumb|left|350px|'''Figure 7:''' The MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) spectrum.]][[
Image
:Bielefeld2012_Massenspektrometrische_Ecoli_Auswertung.png|thumb|right|350px|'''Figure 8:''' Part of MALDI-TOD Evaluation]]
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pH. Therefore it has the same pH optimum as [http://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#7 TVEL0].
pH. Therefore it has the same pH optimum as [http://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#7 TVEL0].
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[[
File
:Bielefeld2012 E.colipHmitCuOX.jpg|thumbnail|500px|center|'''Figure 10:'''
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[[
Image
:Bielefeld2012 E.colipHmitCuOX.jpg|thumbnail|500px|center|'''Figure 10:'''
[http://partsregistry.org/Part:BBa_K863005 ECOL] laccase activity measured in 100 mM sodium acetate buffer with a
[http://partsregistry.org/Part:BBa_K863005 ECOL] 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 12 hours.
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 12 hours.
The optimal pH for [http://partsregistry.org/Part:BBa_K863005 ECOL] is pH 5 with the most ABTSox.]]
The optimal pH for [http://partsregistry.org/Part:BBa_K863005 ECOL] is pH 5 with the most ABTSox.]]
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[[
File
:Bielefeld2012 E.colipHohneCuOX.jpg|thumbnail|500px|center|'''Figure 11:'''
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[[
Image
:Bielefeld2012 E.colipHohneCuOX.jpg|thumbnail|500px|center|'''Figure 11:'''
[http://partsregistry.org/Part:BBa_K863005 ECOL] laccase activity measured in 100 mM sodium acetate buffer with a
[http://partsregistry.org/Part:BBa_K863005 ECOL] 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 12 hours.
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 12 hours.
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The optimal pH for [http://partsregistry.org/Part:BBa_K863005 ECOL] is pH 5 with the most ABTSox.]]
The optimal pH for [http://partsregistry.org/Part:BBa_K863005 ECOL] is pH 5 with the most ABTSox.]]
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[[
File
:Bielefeld2012_PH_neg_control1.jpg|thumbnail|500px|center|'''Figure 12:''' Negative control for pH activity test using 0.04 mM
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[[
Image
:Bielefeld2012_PH_neg_control1.jpg|thumbnail|500px|center|'''Figure 12:''' Negative control for pH activity test using 0.04 mM
CuCl2 H2O instead of laccase to determine the potential of ABTS getting oxidized by CuCl2.]]
CuCl2 H2O instead of laccase to determine the potential of ABTS getting oxidized by CuCl2.]]
With regard to our project knowledge of the optimal pH is useful. Since waste water in waste water treatment plants has an average
With regard to our project knowledge of the optimal pH is useful. Since waste water in waste water treatment plants has an average
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Another test of [http://partsregistry.org/Part:BBa_K863005 ECOL] was done to survey the best CuCl2 concentration for the activity of the purified [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase. 0.03 mg mL-1 protein were incubated with different CuCl2 concentration ranging from 0 to 0.7 mM CuCl2. Activity tests were performed with the incubated samples, in 100 mM sodium actetate buffer (pH 5), 0.1 mM ABTS, to a final volume of 200 µL. The activity was measured at 420 nm, 25°C and over a time period of 10 hours. As expected the saturation takes place after 5 hours (see Figure 13). The differences in the activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase incubated in different CuCl2 differ minimal. The highest activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase is observed after incubation with 0.4 mM CuCl2 (42% of added ABTS). With a higher concentration of 0.7 mM CuCl2 the activity seems to be reduced (only 41% ABTS got oxidized). This leads to the assumption that CuCl2 supports the [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase activity but concentrations exceeding this value of CuCl2 may have a negative impact on the ability of oxidizing ABTS. Without any CuCl2 application [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase show less activity in oxidizing ABTS (see Figure 12). 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 but no laccase was added to detect the oxidization of ABTS through copper (see Figure 14). The more CuCl2 was present, the more ABTS was oxidized after 5 hours. Still the maximal change accounts only for ~6% oxidized ABTS after 5 hours.
Another test of [http://partsregistry.org/Part:BBa_K863005 ECOL] was done to survey the best CuCl2 concentration for the activity of the purified [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase. 0.03 mg mL-1 protein were incubated with different CuCl2 concentration ranging from 0 to 0.7 mM CuCl2. Activity tests were performed with the incubated samples, in 100 mM sodium actetate buffer (pH 5), 0.1 mM ABTS, to a final volume of 200 µL. The activity was measured at 420 nm, 25°C and over a time period of 10 hours. As expected the saturation takes place after 5 hours (see Figure 13). The differences in the activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase incubated in different CuCl2 differ minimal. The highest activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase is observed after incubation with 0.4 mM CuCl2 (42% of added ABTS). With a higher concentration of 0.7 mM CuCl2 the activity seems to be reduced (only 41% ABTS got oxidized). This leads to the assumption that CuCl2 supports the [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase activity but concentrations exceeding this value of CuCl2 may have a negative impact on the ability of oxidizing ABTS. Without any CuCl2 application [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase show less activity in oxidizing ABTS (see Figure 12). 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 but no laccase was added to detect the oxidization of ABTS through copper (see Figure 14). The more CuCl2 was present, the more ABTS was oxidized after 5 hours. Still the maximal change accounts only for ~6% oxidized ABTS after 5 hours.
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[[
File
:Bielefeld2012 ColicoppergradientOX.jpg|thumbnail|500px|center|'''Figure 13:''' Activity measurement using 0.1 mM ABTS of [http://partsregistry.org/Part:BBa_K863005 ECOL] incubated in different CuCl2 concentrations. Incubation with 0.1 mM CuCl2 or higher concentrations leads to an increase in ABTSox.]]
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[[
Image
:Bielefeld2012 ColicoppergradientOX.jpg|thumbnail|500px|center|'''Figure 13:''' Activity measurement using 0.1 mM ABTS of [http://partsregistry.org/Part:BBa_K863005 ECOL] incubated in different CuCl2 concentrations. Incubation with 0.1 mM CuCl2 or higher concentrations leads to an increase in ABTSox.]]
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[[
File
:Bielefeld2012_Pumi_Cu_NegControl1.jpg|thumbnail|500px|center|'''Figure 14:''' 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.]]
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[[
Image
:Bielefeld2012_Pumi_Cu_NegControl1.jpg|thumbnail|500px|center|'''Figure 14:''' 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 enzymes. This reduces the cost factor for the needed CuCl2 to incubate the laccases before application.
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 enzymes. This reduces the cost factor for the needed CuCl2 to incubate the laccases before application.
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=== [http://partsregistry.org/Part:BBa_K863005 ECOL] activity at different temperatures ===
=== [http://partsregistry.org/Part:BBa_K863005 ECOL] activity at different temperatures ===
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[[
File
:Bielefeld2012 10und25GradOX.jpg|thumbnail|450px|left|'''Figure 15:''' Standard activity test for [http://partsregistry.org/Part:BBa_K863005 ECOL] 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.]]
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[[
Image
:Bielefeld2012 10und25GradOX.jpg|thumbnail|450px|left|'''Figure 15:''' Standard activity test for [http://partsregistry.org/Part:BBa_K863005 ECOL] 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 [http://partsregistry.org/Part:BBa_K863005 ECOL] at lower temperatures activity tests as described above were done at 10°C and 25°C (Figure 15). A significant decrease in the activity can be observed upon reducing the temperature from 25°C to 10°C. While the activity at 10 °C is reduced, final saturation levels are still comparable: after 3,5 hours, only 2% difference in oxidized ABTS is observable. The negative control without the [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase and only 0.4 mM CuCl2 at 10°C shows a negligible oxidation of ABTS.
To investigate the activity of [http://partsregistry.org/Part:BBa_K863005 ECOL] at lower temperatures activity tests as described above were done at 10°C and 25°C (Figure 15). A significant decrease in the activity can be observed upon reducing the temperature from 25°C to 10°C. While the activity at 10 °C is reduced, final saturation levels are still comparable: after 3,5 hours, only 2% difference in oxidized ABTS is observable. The negative control without the [http://partsregistry.org/Part:BBa_K863005 ECOL] laccase and only 0.4 mM CuCl2 at 10°C shows a negligible oxidation of ABTS.
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[[
File
:Bielefeld2012_Massemspektroskopie_Ecoli.png|thumb|left|400px|'''Figure 7:''' The MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) spectrum.]][[
File
:Bielefeld2012_Massenspektrometrische_Ecoli_Auswertung.png|thumb|right|400px|'''Figure 8:''' Part of MALDI-TOD Evaluation]]
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[[
Image
:Bielefeld2012_Massemspektroskopie_Ecoli.png|thumb|left|400px|'''Figure 7:''' The MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) spectrum.]][[
Image
:Bielefeld2012_Massenspektrometrische_Ecoli_Auswertung.png|thumb|right|400px|'''Figure 8:''' Part of MALDI-TOD Evaluation]]
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== Substrate Analytic==
== Substrate Analytic==
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[[
File
:Bielefeld2012_EBT_ABTS.png|450px|thumb|left|'''Figure 19:''' Comparison of degradation reactions of ethinyl estradiole with ABTS between the laccases BPUL and ECOL with the purchased laccase TVEL0, measured by HPLC]]
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[[
Image
:Bielefeld2012_EBT_ABTS.png|450px|thumb|left|'''Figure 19:''' Comparison of degradation reactions of ethinyl estradiole with ABTS between the laccases BPUL and ECOL with the purchased laccase TVEL0, measured by HPLC]]