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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Random and rational evolution of tautomerase superfamily members : analysis and implications

Darty, Joseph Edward 10 April 2014 (has links)
P[Kappa]a is not responsible for the improved activity. Hence, stabilization of an enediolate intermediate may be important for catalysis. In the second part of this work, the Chloroflexus aurantiacus J-10-fl heterohexameric 4-OT tautomerase was employed in random and rational directed evolution studies to introduce a CaaD activity. Genetic selection and a high throughput screening assay were used to identify mutants. Genetic selection was unsuccessful due to plasmid instability in the host strain. A small mutant library in the screening assay precluded the identification of any mutants with CaaD activity. Finally, rational design using structure-function relationships was investigated and a single mutant was discovered for hh4-OT that incorporated CaaD activity into the enzyme, the [alpha]L9R hh4-OT, this mutant has been characterized kinetically and the evolutionary implications for the tautomerase superfamily are described. / text

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