Difference between revisions of "Part:BBa K2286006"

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This part is an improvement of [https://parts.igem.org/Part:BBa_K1199044 BBa_K1199044] (W249P) for its catalytic activity to 2,3-DCP. We obtain 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.
 
This part is an improvement of [https://parts.igem.org/Part:BBa_K1199044 BBa_K1199044] (W249P) for its catalytic activity to 2,3-DCP. We obtain 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.
  
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===Catalytic activity towards to 2，3-DCP===
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===Catalytic activity towards 2，3-DCP===
  
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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.
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In order to clarify its activity level towards 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.
  
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[[File:T--UESTC-China--P84A-2,3-DCP.png|500px|thumb|center|'''Figure 1.''' The catalytic activity of mutants towards to 2,3-DCP.
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[[File:T--UESTC-China--P84A-2,3-DCP.png|500px|thumb|center|'''Figure 1.''' The catalytic activity of mutants towards 2,3-DCP.
 
       Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.]]
 
       Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.]]
  
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===Catalytic activity towards CPD===
  
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===Catalytic activity towards to CPD===
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Haloalcohol dehalogenase (HheC) could catalyze o-halide transferred into epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanism. It has 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.  
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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.  
+
  
 
[[File:T--UESTC-China--P84A-CPD.png|500px|thumb|center|'''Figure 2.''' The catalytic activity of mutants on CPD.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.]]
 
[[File:T--UESTC-China--P84A-CPD.png|500px|thumb|center|'''Figure 2.''' The catalytic activity of mutants on CPD.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.]]
  
  
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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.  
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The above results show that P84A not only has a higher catalytic activity towards to 2,3-DCP, but maintains its high catalytic activity for CPD.  
  
  
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<!-- Uncomment this to enable Functional Parameter display
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<!-- Uncomment this to enable Funct
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===Functional Parameters===
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<partinfo>BBa_K2286006 parameters</partinfo>
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Latest revision as of 13:59, 1 November 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) for its catalytic activity to 2,3-DCP. We obtain 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 2，3-DCP

In order to clarify its activity level towards 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 2,3-DCP. Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.

Catalytic activity towards CPD

Haloalcohol dehalogenase (HheC) could catalyze o-halide transferred into epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanism. It has 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 high catalytic activity for CPD.