Part:BBa_K2043001

catA from Acinetobacter pittii, codon optimized for E. coli 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]

This part encodes Catechol-1,2-dioxygenase from Acinetobacter pittii, codon optimized for E. coli. This enzyme is an intradiol dioxygenase that catalyses oxidative ring cleavage of catechol. Anthocyanins, the key pigments of wine, are polyphenolic molecules naturally found in many plants. These compounds are structurally similar to catechol, making Catechol-1,2-dioxygenase a good candidate for anthocyanin degradation. Catechol-1,2-dioxygenase is also found in many species of soil bacteria. In order to facilitate working with this enzyme, we added a C-terminal His-tag for easier purification.

Figure 1
Figure 1 shows the action of Catechol-1,2-dioxygenase in degrading the phenolic ring of catechol, resulting in the opening of this molecule. By similarly degrading the phenol rings of anthocyanins, this enzyme should remove reduce anthocyanin pigmentation.

Testing the part

We tested the expression and activity of CatA using cell extract of cells expressing our protein.
First, we performed an SDS-PAGE to check whether the protein was being expressed.


Figure 2
Although we did not observe expression of the enzyme (Figure 2), we tested the cell extract for CatA activity in 50 mM sodium phosphate at pH 7 using 30 mM catechol as a substrate, following the protocol from the literature (Lin, 2015) (Figure 3).
The control represents cell extract from E. coli not producing the enzyme. In all cases, values measured correspond to the oxidized catechol reaction product, measured at 260 nm.

Figure 3
Based on this result, we can conclude that our codon-optimized enzymes degrades catechol to its reaction product.

References
Lin, J., & Milase, R. N. (2015). Purification and Characterization of Catechol 1, 2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants. The protein journal, 34(6), 421-433.

Image of Catecholase degradation reaction taken from wikipedia commons, created by user Ehoates, CC BY-SA 3.0.

NCBI Reference Sequence: YP_004995593.1