Difference between revisions of "Part:BBa K2286006"

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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2286006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2286006 SequenceAndFeatures</partinfo>
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==Characterization==
 
==Characterization==
  
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This part is an improvement of [https://parts.igem.org/Part:BBa_K1199044 BBa_K1199044] (W249P) in its catalytic activity for 2,3-DCP. We obtained P84A by molecular simulation and single-site saturation mutation. P84A not only had a higher catalytic activity for 2,3-DCP than W249P, but maintained its high catalytic activity for CPD.
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This part is an improvement of [https://parts.igem.org/Part:BBa_K1199044 BBa_K1199044] (W249P) in its catalytic activity for 2,3-DCP. We obtained P84A by molecular simulation and single-site saturation mutation. P84A not only has a higher catalytic activity for 2,3-DCP than W249P, but maintains its high catalytic activity for CPD.
  
 
===Catalytic activity towards to 2，3-DCP===
 
===Catalytic activity towards to 2，3-DCP===

Revision as of 09:26, 31 October 2017


HheC-P84A: halohydrin dehalogenase site-directed mutant

This is the site-directed mutant of halohydrin dehalogenase. We change the alanine to proline in the 84 site. And this sequence is codon-optimized for E.coli.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 718
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

This part is an improvement of BBa_K1199044 (W249P) in its catalytic activity for 2,3-DCP. We obtained P84A by molecular simulation and single-site saturation mutation. P84A not only has a higher catalytic activity for 2,3-DCP than W249P, but maintains its high catalytic activity for CPD.

Catalytic activity towards to 2，3-DCP

In order to clarify its activity level towards to 2,3-DCP, we tested its activity by the chloride ion method. The results showed that the catalytic activity of P84A to 2,3-DCP was 2.42 times that of W249P.

Figure 1. The catalytic activity of mutants towards to 2,3-DCP. Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.


Catalytic activity towards to CPD

Haloalcohol dehalogenase (HheC) could catalyze o-halide transferred into epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanism. It had a wide range of catalytic substrates. So in order to verify whether P84A is suitable for other substrates under the same conditions, we chose 3-chloropropane-1,2-diol (CPD) as the substrate for further detection respectively. The results show that the catalytic activity of P84A towards to CPD increased a little.

Figure 2. The catalytic activity of mutants on CPD.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.


The above results show that P84A not only has a higher catalytic activity towards to 2,3-DCP, but maintains its catalytic activity for CPD.