Difference between revisions of "Part:BBa K3588003"

(→‎Function)
 
Line 8: Line 8:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3588003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3588003 SequenceAndFeatures</partinfo>
−
 
+
===Synthesis===
 +
When trying to order this code it was far too complex for IDT to synthesis, even with codon optimisation.
  
 
<!-- Add more about the biology of this part here -->
 
<!-- Add more about the biology of this part here -->

Latest revision as of 03:08, 28 October 2020


Cocaine Esterase mutant enzyme (E172-173)

The mutant enzyme of part (part:BBa_K3588001). It is able to breakdown cocaine, as well as benzoylecogonine and cocaethylene, through hydrolysis into benzoate and ecgonine methyl ester. With a dramatically increased in vitro half life from the wildtype enzyme it is currently in phase II trials as a cocaine overdose treatment,[1] though in our project we were intending to use it to breakdown cocaine in the sewers.

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 1179
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Synthesis

When trying to order this code it was far too complex for IDT to synthesis, even with codon optimisation.

Function

The enzyme was designed in order to deal with the problem of drug overdosing, by administering an enzyme with a high catalysis rate into the bloodstream of patients who are overdosing, the effects can be rapidly reduced, however the problem posed by the usage of naturally occurring CocE is its lack of thermal stability (half life at 37°C = ~12mins), so by mutating the original enzyme and thus effecting the bonding of the protein chain a more thermally stable version of the enzyme was created. As well as this, its ability to catabolise coca-ethylene whilst not producing coca-ethylene when in the presence of alcohol means it can be used to treat those who co-abuse cocaine and alcohol. Thereby increasing its potential for in vivo usage, thus the current trials it is undergoing.

The enzyme CocE catalyses the breakdown of cocaine into benzoate and ecgonine methyl ester [2]

properties

Cocaine Substrate Kinetics

Kcat = 2600 min-1 KM = 2.9 μM

other details

half life (at 37°C) = ~ 6hrs

design considerations

This enzyme has two major changes from the wildtype, denoted by the E172-173 notation. For more on the details of the mutations and the methods that were used can be found in the paper: 'Rational Design, Preparation, and Characterization of a Therapeutic Enzyme Mutant with Improved Stability and Function for Cocaine Detoxification' [3]

usage

  1. ↑ https://pubs.acs.org/doi/10.1021/cb500257s
  2. ↑ http://dx.doi.org/10.1128/aem.66.3.904-908.2000
  3. ↑ https://pubs.acs.org/doi/10.1021/cb500257s