Difference between revisions of "Part:BBa K2043004"

Revision as of 17:34, 21 October 2016

xylE from Pseudomonas putida codon optimized for E. coli

This part corresponds to Catechol-2,3-dioxygenase 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.

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.
First, we performed an SDS-PAGE to check whether the protein was being expressed.

The enzyme was successfully expressed, and therefore we continued to the next step, which was testing our protein's activity.
We tested our cell extract for CatA 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 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.

Sequence and Features

Kobayashi, T., Ishida, T., Horiike, K., Takahara, Y., Numao, N., Nakazawa, A., ... & Nozaki, M. (1995). Overexpression of Pseudomonas putida catechol 2, 3-dioxygenase with high specific activity by genetically engineered Escherichia coli. Journal of biochemistry, 117(3), 614-622.

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.

NCBI Reference Sequence: NP_542866.1