Coding
HerE

Part:BBa_K1615045

Designed by: Brooke Rothschild-Mancinelli   Group: iGEM15_Edinburgh   (2015-08-28)
Revision as of 22:51, 21 September 2015 by LiusaidhOwen (Talk | contribs)

Heroin Esterase in RFC25

Heroin esterase, an acetylmorphine carboxylesterase, was isolated from Rhodococcus erythropolis strain H1 in 1994 from the garden soil at Cambridge and is able to use heroin as its sole carbon and energy source by deacetylating the C-3 and C-6 groups to form morphine1. The gene her encodes this enzyme and can be expressed in the chassis Escherichia coli2. The pH optimum for this enzyme is pH8.5 in bicine buffer1.

How heroin esterase works.jpeg

The activity of heroin esterase can be tested using 4-nitrophenyl acetate which is hydrolysed by heroin esterase to form 4-nitrophenol and acetate3. This produces a yellow colour which can be read at 410 nm.

BBa K1615045.jpg



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

Heroin esterase kinetics.jpg



The following table summarises the kinetic analyses and statistics of the measurement for heroin esterase fused to a cellulose binding domain.

Heroin esterase CBD kinetics.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



1Cameron, G. W., Jordan, K. N., Holt, P. J., Baker, P. B., Lowe, C. R., & Bruce, N. C. (1994). Identification of a heroin esterase in Rhodococcus sp. strain H1. Applied and environmental microbiology, 60(10), 3881-3883.


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.

3Sigma-aldrich. 4-nitrophenyl acetate product information.


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 21
    Illegal BamHI site found at 777
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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