Towards directed evolution of Fluoroacetate dehalogenases

Beers, Pauline van (2024) Towards directed evolution of Fluoroacetate dehalogenases. Bachelor's Thesis, Chemistry.

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Abstract

The enzymatic activity of RPA1163, dehH1, and FAc-DEX towards four fluorinated substrates was assessed by a colorimetric assay by Holloway et al. and by 19F NMR. RPA1163 showed activity towards 2-fluoropropionic acid but requires further research for fluoroacetic acid, difluoroacetic acid, and 2-fluoropropionic acid. dehH1 and FAc-DEX converted fluoroacetic acid and 2-fluoropropionic acid, with an indication for activity for difluoroacetic acid in the colorimetric assay. Neither enzyme converted 2,2-difluoropropionic acid. Next, the activity was investigated in vivo, by measuring the growth curve after the four substrates were added. No activity was seen for the difluorinated substrates. Furthermore, it was shown that the toxicity of fluoroacetic acid can be used as a selection marker. In addition, 2-fluoropropionic acid can be used as a sole carbon source with dehH1 and FAc-DEX, highlighting the possibility to do a growth-based selection based on a combination of detoxification and additional carbon source. A pH indicator agar plate selection did not display a difference between the wild type and the null variant; thus, it is not usable as a selection method. However, the growth-based selection showed that the wild type of dehH1 and FAc-DEX can take over in two rounds of increased selection pressure, which was not observed with RPA1163. This shows that this method is usable for a selection of a gene library of dehH1 and FAc-DEX.

Item Type:	Thesis (Bachelor's Thesis)
Supervisor name:	Mayer, C.
Degree programme:	Chemistry
Thesis type:	Bachelor's Thesis
Language:	English
Date Deposited:	05 Jul 2024 08:51
Last Modified:	05 Jul 2024 08:51
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/33044

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