Difference between revisions of "Part:BBa K1615000"
 
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The structural gene morphine-6-dehydrogenase (<i>morA</i>) was first isolated from <i>Pseudomonas putida</i> M10 as it is capable of growth with morphine as its sole carbon source<sup>1</sup>. Morphine dehydrogenase catalyses the oxidation of both morphine and codeine to produce morphinone and codeinone respectively. During this process NADP<sup>+</sup> is reduced to NADPH which means that this enzyme is frequently used to detect morphine and codeine<sup>2</sup>.
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<br>The structural gene morphine-6-dehydrogenase (<i>morA</i>) was first isolated from <i>Pseudomonas putida</i> M10 as it is capable of growth with morphine as its sole carbon source<sup>1</sup>. Morphine dehydrogenase (MDH) catalyses the oxidation of both morphine and codeine to produce morphinone and codeinone respectively. During this process NADP<sup>+</sup> is reduced to NADPH which means that this enzyme is frequently used to detect morphine and codeine<sup>2</sup>.
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<br>[[File:morphine dehydrogenase activity.jpg]]
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<br>To test the morphine dehydrogenase activity it can be coupled with morphine and NADP<sup>+</sup> to produce morphinone and NADPH. The amount of NADPH produced can be measured at 340nm. Morphine dehydrogenase with t7 promoter characterised by Edinburgh iGEM team was provided by Prof Chris French. Michaelis Menten kinetic analysis was performed giving values of Vmax and Km, 61.22 and 140.5 uM respectively.
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[[File:Morphine dehydrogenase graph.jpg]]
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The following table summarises the kinetic analysis and statistics of the measurement.
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[[File:Morphine dehydrogenase kinetics table.jpg]]
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The graph below shows the production of NADPH in the presence of heroin due to the activity of heroin esterase and morphine dehydrogenase.
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[[File:heroin esterase + morphine dehydrogenase activity.jpg]]
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<sup>1</sup>Bruce, N. C., Wilmot, C. J., Jordan, K. N., Trebilcock, A. E., Stephens, L. D. G., & Lowe, C. R. (1990). Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. <i>Archives of microbiology</i>, 154(5), 465-470.
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<br><sup>2</sup>Rathbone, D. A., Holt, P. J., Lowe, C. R., & Bruce, N. C. (1997). Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. <i>Applied and environmental microbiology</i>, 63(5), 2062-2066.
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<br><sup>2</sup>Walker, E., et al. (2000). Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation. <i>Biochem. J</i> 345, 687-692.
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<sup>1</sup>Bruce, N. C., Wilmot, C. J., Jordan, K. N., Trebilcock, A. E., Stephens, L. D. G., & Lowe, C. R. (1990). Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. <i>Archives of microbiology</i>, 154(5), 465-470.
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<br><sup>2</sup>Rathbone, D. A., Holt, P. J., Lowe, C. R., & Bruce, N. C. (1997). Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. <i>Applied and environmental microbiology</i>, 63(5), 2062-2066.
 
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To test the morphine dehydrogenase activity it can be coupled with morphine and NADP<sup>+</sup> to produce morphinone and NADPH. The amount of NADPH produced can be measured at 340nm.
 
 
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===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 23:00, 21 September 2015

Morphine-6-dehydrogenase RFC25

The structural gene morphine-6-dehydrogenase (morA) was first isolated from Pseudomonas putida M10 as it is capable of growth with morphine as its sole carbon source1. Morphine dehydrogenase (MDH) catalyses the oxidation of both morphine and codeine to produce morphinone and codeinone respectively. During this process NADP+ is reduced to NADPH which means that this enzyme is frequently used to detect morphine and codeine2.


Morphine dehydrogenase activity.jpg



To test the morphine dehydrogenase activity it can be coupled with morphine and NADP+ to produce morphinone and NADPH. The amount of NADPH produced can be measured at 340nm. Morphine dehydrogenase with t7 promoter characterised by Edinburgh iGEM team was provided by Prof Chris French. Michaelis Menten kinetic analysis was performed giving values of Vmax and Km, 61.22 and 140.5 uM respectively.



Morphine dehydrogenase graph.jpg



The following table summarises the kinetic analysis and statistics of the measurement.



Morphine dehydrogenase kinetics table.jpg

The graph below shows the production of NADPH in the presence of heroin due to the activity of heroin esterase and morphine dehydrogenase.

Heroin esterase + morphine dehydrogenase activity.jpg

1Bruce, N. C., Wilmot, C. J., Jordan, K. N., Trebilcock, A. E., Stephens, L. D. G., & Lowe, C. R. (1990). Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Archives of microbiology, 154(5), 465-470.
2Rathbone, D. A., Holt, P. J., Lowe, C. R., & Bruce, N. C. (1997). Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. Applied and environmental microbiology, 63(5), 2062-2066.
2Walker, E., et al. (2000). Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation. Biochem. J 345, 687-692.

1Bruce, N. C., Wilmot, C. J., Jordan, K. N., Trebilcock, A. E., Stephens, L. D. G., & Lowe, C. R. (1990). Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Archives of microbiology, 154(5), 465-470.
2Rathbone, D. A., Holt, P. J., Lowe, C. R., & Bruce, N. C. (1997). Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. Applied and environmental microbiology, 63(5), 2062-2066.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 840
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]