Part:BBa_K2043006
xylE-FBD10 from Pseudomonas putida codon optimized for E. coli
Fabric Binding Domain 10 (BBa_K2043017) in the N-terminal cloned by the Paris Bettencourt team in 2016 in the context of the Frank&Stain project. This enzymes originally comes from Pseudomonas putida, which we codon optimised for E. coli.
In order to facilitate working with this enzyme, we added a His-tag at the C-terminal. This tag allows for purification in an easier way.
The Fabric Binding Domain has affinity for cellulose. It is positively charged (+1) .
We chose to work with this enzyme because it seemed to be a good candidate for degrading Anthocyanins. Anthocyanins, the key pigments present in wine, are polyphenolic molecules that are naturally found in many plants. Our project consisted in the degradation of wine strains, and therefore enzymes with the ability to degrade polyphenolic molecules were of interest to us.
In particular, Catechol-dioxygenases are good candidates because they degrade Catechol, which is structurally similar to Anthocyanins.
Testing the part
We tested the activity of XylE using cell extract of cells expressing our protein.
We tested our cell extract for XylE activity in Potassium Phosphate 100mM at pH 7.5, with 30mM of Catechol as substrate, as recommended in the literature. Measurements were taken after 12 min, timepoint after which all the substrate had been consumed.
Control corresponds to cells that do not express our proteins. In all cases, values measured correspond to reaction product.
As the image indicates, there is a clear difference between our native enzyme and the control. We measured the reaction product at 475nm, which results from the oxidation of Catechol. Since much more reaction product is produced with cells expressing XylE than in the control, we can affirm that the enzyme was functional.
he binding of the FBD10 had a negative effect on the enzymatic activity, but nonetheless the activity of the enzyme was still observed.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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Cerdan, P., Rekik, M., & Harayama, S. (1995). Substrate Specificity Differences Between Two Catechol 2, 3‐Dioxygenases Encoded by the TOL and NAH Plasmids from Pseudomonas putida. European journal of biochemistry, 229(1), 113-118.
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NCBI Reference Sequence: NP_542866.1
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