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Team:Exeter/enzyme-kinetics
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<h1>Kinetic Analysis of NemA and XenB by HPLC</h1> | <h1>Kinetic Analysis of NemA and XenB by HPLC</h1> | ||
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<table align="center"><tr><td align="center">https://static.igem.org/mediawiki/2014/9/9b/TNT-Standard-Curve-scaled.gif</td></tr></table> | <table align="center"><tr><td align="center">https://static.igem.org/mediawiki/2014/9/9b/TNT-Standard-Curve-scaled.gif</td></tr></table> | ||
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<h2>Conclusion</h2> | <h2>Conclusion</h2> | ||
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<p> | <p> | ||
NemA and Xen B are capable of catalysing the conversion of TNT to various products using NADH and FMN as cofactors. The binding affinity of each protein for this substrate (the Michealis Menten constant, Km) and the maximum reaction velocity (Vmax), were determined and are comparable to the published values; shown in figure 1. NemA and XenB are therefore suitable enzymes for use in our system and have been shown to function at the physiologically relevant pH of 7. | NemA and Xen B are capable of catalysing the conversion of TNT to various products using NADH and FMN as cofactors. The binding affinity of each protein for this substrate (the Michealis Menten constant, Km) and the maximum reaction velocity (Vmax), were determined and are comparable to the published values; shown in figure 1. NemA and XenB are therefore suitable enzymes for use in our system and have been shown to function at the physiologically relevant pH of 7. | ||
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<table align="center" border="1"> | <table align="center" border="1"> | ||
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<td>Experimental Results</td> | <td>Experimental Results</td> | ||
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<td>Published Values</td> | <td>Published Values</td> | ||
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<td>8</td> | <td>8</td> | ||
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<td>15</td> | <td>15</td> | ||
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</p> | </p> | ||
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<table align="center"> | <table align="center"> | ||
<tr><td>https://static.igem.org/mediawiki/2014/8/8b/TNT_degradation.png</td></tr> | <tr><td>https://static.igem.org/mediawiki/2014/8/8b/TNT_degradation.png</td></tr> | ||
<tr><td align="center"><i>Figure 2</i></td></tr> | <tr><td align="center"><i>Figure 2</i></td></tr> | ||
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Various hydroxylamino derivatives may be produced, as well as ammonium ions which could be used as a nitrogen source by the E.coli for growth. | Various hydroxylamino derivatives may be produced, as well as ammonium ions which could be used as a nitrogen source by the E.coli for growth. | ||
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A standard curve of TNT concentration was determined first. Integration of the area below the absorbance peak of TNT at the concentrations described, resulted in the standard curve shown in figure 3. For further details see materials and methods. | A standard curve of TNT concentration was determined first. Integration of the area below the absorbance peak of TNT at the concentrations described, resulted in the standard curve shown in figure 3. For further details see materials and methods. | ||
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<table align="center"> | <table align="center"> | ||
<tr><td>https://static.igem.org/mediawiki/2014/thumb/9/97/Standard_Curve.png/800px-Standard_Curve.png</td></tr> | <tr><td>https://static.igem.org/mediawiki/2014/thumb/9/97/Standard_Curve.png/800px-Standard_Curve.png</td></tr> | ||
<tr><td align="center"><i>Figure 3: Standard curve demonstrating HPLC peak area response to different concentrations of TNT</i></td></tr> | <tr><td align="center"><i>Figure 3: Standard curve demonstrating HPLC peak area response to different concentrations of TNT</i></td></tr> | ||
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<h4>Determination of NemA activity</h4> | <h4>Determination of NemA activity</h4> | ||
Revision as of 13:57, 27 September 2014
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Contents
Kinetic Analysis of NemA and XenB by HPLC
Conclusion
NemA and Xen B are capable of catalysing the conversion of TNT to various products using NADH and FMN as cofactors. The binding affinity of each protein for this substrate (the Michealis Menten constant, Km) and the maximum reaction velocity (Vmax), were determined and are comparable to the published values; shown in figure 1. NemA and XenB are therefore suitable enzymes for use in our system and have been shown to function at the physiologically relevant pH of 7.
NemA Vmax (TNT) XenB Vmax (TNT) NemA Vmax (Nitroglycerin) XenB Vmax (Nitroglycerin) NemA Km (TNT) XenB Km (TNT) NemA Km (Nitroglycerin) XenB Km (Nitroglycerin) Experimental Results Published Values 8 15 Figure 1 </p>
Abstract
NemA and XenB are two proteins that the iGEM Exeter team propose will allow E.coli to degrade TNT, at concentrations above those normally toxic to the cell. Among many others proposed, NemA catalyses the reaction shown in figure 2.
Figure 2 Various hydroxylamino derivatives may be produced, as well as ammonium ions which could be used as a nitrogen source by the E.coli for growth. The following experimental account describes the protocol used to confirm the substrate target of NemA and XenB and analyse the respective kinetic capabilities of these enzymes.
Results
Choice of analytical procedure
NADH is a cofactor in the conversion of TNT to X. This opens up the possibility to simply measuring the catalytic rate of TNT degradation by following change in the absorbance at 340nm. However, very early in the process we realised that TNT also produces a significant absorbance at 340nm, a fact that would complicate our analytical procedure. We therefore chose to use High Performance Liquid Chromatography (HPLC) analysis. As can be seen (Fig X), HPLC separates TNT and NAD by elution time meaning that the overlap in absorbance values are not an issue.
TNT Standard Curve
A standard curve of TNT concentration was determined first. Integration of the area below the absorbance peak of TNT at the concentrations described, resulted in the standard curve shown in figure 3. For further details see materials and methods.
Figure 3: Standard curve demonstrating HPLC peak area response to different concentrations of TNT Determination of NemA activity
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