| Popis: |
Development of a novel biosensor for high throughput screening of enantioselective alcohol oxidases Enantioselective alcohol oxidases are of interest as biocatalytic tools because of their potential. for use in the production of enantiopure compounds. The initial aim of this project was to identify hitherto unexploited alcohol oXidases; however the results obtained from the screening of potential source organisms were not consistent under the conditions applied and yield was found to be low in all cases studied. During these investigations a need for high throughput screening methods was identified and beeause of this a new aim was initiated, that of developing a biosensor designed for the high throughput screening of libraries of enzymes on the basis of the H202 produced as a by-product of oxidase activity. A biosensor was designed and created in Saccharomyces cerevisiae that indicated the presence of H202 by increased levels of GFP. Aryl-alcohol oxidase (AAO) from Pleurotus eryngii was expressed for the first time in S. cerevisiae in order to provide a positive control for these experiments and the biosensor was found capable of detecting the H20 2 produced as a by-product of the action of this enzyme on the substrate veratryl alcohol. Mutant libraries of AAO were produced, transformed into the biosensor yeast and screened by FACS analysis, this approach proved unsuccessful in identifying any mutants of interest. Further optimisation is required before the biosensor can be a useful tool in screening libraries of oxidase enzymes on the basis of activity. |
