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Title: Engineering oxidase activity in flavocytochrome b2
Author: Østergaard, Lars H.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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An attempt was made to convert the L-lactate dehydrogenase, flavocytochrome b2, into an oxidase. One approach was based on protein engineering by introduction of site-specific mutations. The mutations were concerned with the problem of generating access to and space around the bound FMN. A number of modified enzymes with substitution of amino acid residues in close proximity to the co-factor were constructed, purified and characterised. Most mutations had negative effects on the dehydrogenase activity. However, their oxidase activity did not improve either, and suggests that a single mutation may not be sufficient to swing the activity Another concern in the design of mutant enzymes is the extremely high degree of similarity between the two enzyme structures. However, a loop region exhibits no amino acid sequence similarity between members of this family of enzymes. This loop is located close to the flavin, but is disordered in the electron density map of both flavocytochrome b2 and glycolate oxidase. The function of this loop was studied by construction of various alterations in this region. The loop clearly interacted with the active site and is bound co-factor, as demonstrated by changes in absorption spectra and kinetic parameters. Unfortunately, none of these mutant enzymes exhibited the desired oxidase activity either. The approach of forced evolution seems ideal, when considering the above described difficulties. The method includes random mutagenesis of the gene coupled with selection. A selection system based on expression of mutant enzymes in E. coli has been established. The mutagenesis has been performed by error-prone PCR, which offers control over level of mutagenesis and may be targeted to specific areas. The technique has been combined with DNA shuffling to mimic the process of natural evolution, and thus exploiting the benefits of homologous recombination. This technique may also be applied to mix the two genes of flavocytochrome b2, and glycolate oxidase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available