Difference between revisions of "Part:BBa K2286000"
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HheC-W249P is a single site mutant of the Halohydrin dehalogenase (haloalcohol dehalogenase), whose mutant locates at the 249 (W-> P) | 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 | 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. | + | 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 <em>Agrobacterium radiobacterAD1</em>, 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. |
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===Enzyme activity on 1,3-DCP=== | ===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]. | + | 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 <em>E.coli</em> 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]. |
| − | [[File:T--UESTC-China--DhaA31-WT.png|500px|thumb|center|'''Fig | + | [[File:T--UESTC-China--DhaA31-WT.png|500px|thumb|center|'''Fig. 1''' Activity on 1，3-DCP.Data were measured in Tris-H2SO4 buffer at pH8.5 and 37℃.]] |
===Enzyme activity on CPD=== | ===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. | |
| − | References | + | [[File:T--UESTC-China--DhaA31-CPD.png|500px|thumb|center|'''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. | 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. | ||
Latest revision as of 02:38, 2 November 2017
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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].
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.
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.