Difference between revisions of "Part:BBa K1199044"

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<partinfo>BBa_K1199044 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1199044 SequenceAndFeatures</partinfo>
  
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==Improvement from UESTC-China==
 
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As a mutant with high activity toward 1,3-DCP, HheC-W249P has little change in the activity of 2,3-DCP. This year we UESTC-China used this part as the substrate to successfully obtain two mutants BBa_K2286006 (P84A) and BBa_K2286008 (P84-W249P) with improved 2,3-DCP activity by molecular simulation and single-site saturation mutagenesis.
 
  
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===Activity toward 2，3-DCP===
 
  
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Halogen ion release occurs when the haloalcohol dehalogenase catalyzes the the o-halanol to the epoxide. The interaction of the halide with Hg2+ allows Hg2+ to be separated from SCN-, which forms a colored complex with Fe3 +. This complex can be determined by absorption at 460 nm. Therefore, we determined these two mutants activity by the halide ion method.
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==Improvement from iGEM17-UESTC-China==
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As a mutant with high activity toward 1,3-DCP, HheC-W249P has few changes towards to 2,3-DCP. This year we UESTC-China used this part as the substrate to successfully obtain two mutants [https://parts.igem.org/Part:BBa_K2286006 BBa_K2286006] (P84A) and [https://parts.igem.org/Part:BBa_K2286008 BBa_K2286008] (P84A-W249P) with improved 2,3-DCP activity by molecular simulation and single-site saturation mutagenesis.
  
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===Catalytic activity towards to 2，3-DCP===
  
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Halogen ion release occurs when the haloalcohol dehalogenase catalyzes the the o-halanol to the epoxide. The interaction of the halide with Hg2+ allows Hg2+ to be separated from SCN-, which forms a colored complex with Fe3 +. This complex can be determined by absorption at 460 nm. Therefore, we determined these two mutants activity by the halide ion method.
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              <p>Figure 1. The activity of mutants on 2,3-DCP</p>
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              <p>Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃</p>
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[[File:T--UESTC-China--W249P-P84A-W.png|500px|thumb|center|'''Fig. 1''' The activity of mutants on 2,3-DCP.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.]]
  
 
The results show that the catalytic activity of mutant P84A to 2,3-DCP is 2.42 times that of W249P, and the catalytic activity of P84A-W249P to 2,3-DCP is 1.15 times that of W249P at 37℃ and pH 8.5.
 
The results show that the catalytic activity of mutant P84A to 2,3-DCP is 2.42 times that of W249P, and the catalytic activity of P84A-W249P to 2,3-DCP is 1.15 times that of W249P at 37℃ and pH 8.5.
  
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===Activity toward other substrates===
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===Catalytic activity towards to CPD===
  
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haloalcohol dehalogenase HheC can catalyze o-halide into epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanisms, which are involved in the catalytic degradation of many halogen compounds and have a wide range of catalytic substrates. Therefore, in order to clarify whether the high activity of the mutants are suitable for other substrates, under the same conditions, we select 1,3- dichloropropane-2-ol (DCP) and 3-chloropropane-1,2-diol (CPD) as the substrate for further detection. The results show that the activity of P84A-W249P toward both substrates is lower than W249P. And the activity of P84A toward CPD has a certain increase. For 1,3-DCP, its activity is lower than W249P, but still has a very high activity.
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haloalcohol dehalogenase HheC can catalyze o-halide into epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanisms, which are involved in the catalytic degradation of many halogen compounds and have a wide range of catalytic substrates. Therefore, in order to clarify whether the high activity of the mutants are suitable for other substrates, under the same conditions, we select 3-chloropropane-1,2-diol (CPD) as the substrate for further detection. The results show that the activity of P84A-W249P towards CPD is lower than W249P. And the activity of P84A towards to CPD increased a little.
  
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[[File:T--UESTC-China--W249P-CPD.png|500px|thumb|center|'''Fig. 2''' The activity of mutants on CPD.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃]]
  
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The above experimental results show that amino acid of HheC locating in 84 site has an important effect on its catalytic activity toward the 2,3-DCP,  CPD.Compared with W249P, the mutant P84A-W249P successfully improved its catalytic activity toward 2,3-DCP. While the mutant P84A not only improves its catalytic activity toward 2,3-DCP, but also maintains its high activity for CPD.
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<img src="https://static.igem.org/mediawiki/2017/7/7c/T--UESTC-China--W249P-CPD.png
 
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              <p>Figure 2. The activity of mutants on CPD
 
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              <p>Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃</p>
 
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              <img src="https://static.igem.org/mediawiki/2017/3/3d/T--UESTC-China--W249P-1%2C3-DCP.png
 
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              <p>Figure 3. The activity of mutants on 1,3-DCP
 
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              <p>Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃</p>
 
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 03:06, 2 November 2017

HheCW249P 2,3-DCP(2,3-dichloropropanol)->Glycerol

HheC displayed high regioselectivity and moderate to high enantioselectivity that can be applied for the kinetic resolution of chiral spiroepoxides.And HheC-W249P is mutant of HheC ,displayed higher activity than the wide type .

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 AgeI site found at 697
  • 1000
    COMPATIBLE WITH RFC[1000]


Improvement from iGEM17-UESTC-China

As a mutant with high activity toward 1,3-DCP, HheC-W249P has few changes towards to 2,3-DCP. This year we UESTC-China used this part as the substrate to successfully obtain two mutants BBa_K2286006 (P84A) and BBa_K2286008 (P84A-W249P) with improved 2,3-DCP activity by molecular simulation and single-site saturation mutagenesis.

Catalytic activity towards to 2，3-DCP

Halogen ion release occurs when the haloalcohol dehalogenase catalyzes the the o-halanol to the epoxide. The interaction of the halide with Hg2+ allows Hg2+ to be separated from SCN-, which forms a colored complex with Fe3 +. This complex can be determined by absorption at 460 nm. Therefore, we determined these two mutants activity by the halide ion method.

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

The results show that the catalytic activity of mutant P84A to 2,3-DCP is 2.42 times that of W249P, and the catalytic activity of P84A-W249P to 2,3-DCP is 1.15 times that of W249P at 37℃ and pH 8.5.

Catalytic activity towards to CPD

haloalcohol dehalogenase HheC can catalyze o-halide into epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanisms, which are involved in the catalytic degradation of many halogen compounds and have a wide range of catalytic substrates. Therefore, in order to clarify whether the high activity of the mutants are suitable for other substrates, under the same conditions, we select 3-chloropropane-1,2-diol (CPD) as the substrate for further detection. The results show that the activity of P84A-W249P towards CPD is lower than W249P. And the activity of P84A towards to CPD increased a little.

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

The above experimental results show that amino acid of HheC locating in 84 site has an important effect on its catalytic activity toward the 2,3-DCP, CPD.Compared with W249P, the mutant P84A-W249P successfully improved its catalytic activity toward 2,3-DCP. While the mutant P84A not only improves its catalytic activity toward 2,3-DCP, but also maintains its high activity for CPD.