Coding

Part:BBa_K2286000

Designed by: Demin Xu   Group: iGEM17_UESTC-China   (2017-10-20)
Revision as of 02:38, 2 November 2017 by Tedemin (Talk | contribs) (→‎Enzyme activity on CPD)

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HheC-W249P: Agrobacterium radiobacterAD1 halohydrin dehalogenase mutant

HheC-W249P is a single site mutant of the Halohydrin dehalogenase (haloalcohol dehalogenase), whose mutant locates at the 249 (W-> P) site of Halohydrin dehalogenase. HheC catalyzes the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Its substrate is mainly 1,3-dichloro-2-propanol (1,3-DCP) and 2,3-dichloropropane-1-ol (2,3-DCP). For 1,3-DCP, HheC-W249P activity is 4.32 times than the wild type. For 2,3-DCP, The activity of HheC-W249P was 16.9 times than wild type[1]. This enzyme is derived from Agrobacterium radiobacterAD1, so the original sequence is suitable for expression in prokaryotic cells. However this sequence is codon optimized for petunia and is more suitable for plant system expression.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 619

Characterization

Molecular weight

This halohydrin dehalogenase gene codes for a protein of 762 amino acids with a molecular mass of 29kDa[2].

Enzyme activity on 1,3-DCP

It is reported that W249P is a highly reactive mutant for 1,3-DCP. In order to confirm the characteristics of its activity, we used crude enzyme solution expressed by E.coli MC1061 to detect its activity. The results showed that the activity of W249P to 2,3-DCP was 3.87 times that of wild type. This is similar to the results reported in the article[1].

Fig. 1 Activity on 1，3-DCP.Data were measured in Tris-H2SO4 buffer at pH8.5 and 37℃.

Enzyme activity on CPD

In addition, we also tested the activity of W249P on CPD. The results showed that the activity of W249P was about 2.5 times that of wild type.

Fig. 2 Activity on CPD.Data were measured in Tris-H2SO4 buffer at pH8.5 and 37℃.

References

1.Wang, X., et al., Improvement of the thermostability and activity of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by engineering C-terminal amino acids. J Biotechnol, 2015. 212: p. 92-8.

2.Dvorak, P., et al., Immobilized synthetic pathway for biodegradation of toxic recalcitrant pollutant 1,2,3-trichloropropane. Environ Sci Technol, 2014. 48(12): p. 6859-66.



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