Difference between revisions of "Part:BBa K1199044"

(→‎Activity toward other substrates)
(→‎Catalytic activity towards to CPD)
 
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==Improvement from iGEM17-UESTC-China==
 
==Improvement from iGEM17-UESTC-China==
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As a mutant with high activity toward 1,3-DCP, HheC-W249P has few changes in the activity of 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] (P84-W249P) with improved 2,3-DCP activity by molecular simulation and single-site saturation mutagenesis.
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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.
  
 
===Catalytic activity towards to 2，3-DCP===
 
===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.
 
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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[[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℃.]]
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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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===Catalytic 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 3'''.The activity of mutants on CPD.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃]]
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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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[[File:T--UESTC-China--W249P-1,3-DCP.png|500px|thumb|center|'''Fig 4'''.The activity of mutants on 1,3-DCP.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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The above experimental results show that 84 amino acid of HheC has an important effect on its catalytic activity toward the 2,3-DCP, 1,3-DCP and 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 1,3-DCP and CPD.  
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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.