Part:BBa_K2286003

EchA: Epichlorohydrin epoxide hydrolase

EchA is an epoxide hydrolase derived from Agrobacterium radiobacterAD1, which can undergo the ring-opening reaction of various epoxides. It is reported that EchA Shows better activity for Epoxohydrin, Epibromohydrin and Epifluorohydri in various epoxides[1]。 Since this enzyme is derived from prokaryotic cells, the original sequence is suitable for expression in prokaryotic cells, and this sequence is codon optimized for petunia system 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
  COMPATIBLE WITH RFC[1000]

Characterization

Molecular weight

This epoxide hydrolase gene codes for a protein of 294 amino acids with a molecular mass of 35 kDa[2].

Effect of pH on enzyme activity

It is reported that the activity of the oxidative hydrolase derived from Pseudomonas sp. Strain AD1 was greatly affected by the pH[2]. Therefore, we tested the activity of EchA expressed by Escherichia coli MC1061 at different pH levels. The results showed that the best activity of EchA located about pH 8.5 in the 50 mM Tris / sulphate buffer at 37℃. This is consistent with the results reported in the literature[2].

Fig 1.Effect of pH on enzyme activity.

EchA in tobacco chassis

For the plant expression system, the sequence was optimized and transferred to tobacco to explore its potential for stable work in plants. After obtaining positive plants, we first carried out reverse transcription test. The results showed that EchA was stably transcribed in tobacco. Furthermore, we used tobacco leaves to extract crude enzyme solution for activity detection, and the results showed that EchA was able to work effectively in tobacco.

Fig 2.RT-PCR of positive plants.

Fig 3.The amount of CPD generated within seven hours Enzymatic reaction in 200mM Trs-SO4 at pH 8.5 and 37℃. The data were measured by Gas chromatography.

References

1.Rink, R., et al., Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1. J Biol Chem, 1997. 272(23): p. 14650-7.

2.Jacobs, M.H., et al., Characterization of the epoxide hydrolase from an epichlorohydrin-degrading Pseudomonas sp. Eur J Biochem, 1991. 202(3): p. 1217-22.