Amine oxidases in the deracemisation of racemic chiral amines

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Project objective

i. Characterisation of known amine oxidases: in order to improve the activity of the A. niger amine oxidase, the gene has been subcloned resulting in approximately 3 fold greater levels of expression. The enzyme has been partially purified and Turner and coworkers are trying to characterize this amine oxidize with respect to substate specificity and stereo selectivity using different substrates. We shall begin by completing this characterization in order to asses the properties of the enzyme and provide a basis for the in vitro evolution/ selection methods detailed below.ii. Directed evolution of amino acid/ amine oxidizes coupled with high-throughput screening: a major part of the project will be directed towards the generation of libraries of mutant amino acid and amine oxidizes followed by high throughput screening to identify enantioselective amine oxidizes for use in the deracemisation reactions. The similarity in apparent mechanism of action between amino acid oxidizes and type II amine oxidizes suggest that it should be possible to evolve one activity from the other.iii. Use of amine oxidizes to the deracemisation of racemic chiral amines: moreover the screening/ directed evolution work it will be necessary to determine substate specificity and stereo selectivity of the new amine oxidizes that could be discovered. New enzymes will be characterized against a defined set of substrates in order to generate a range of amine oxidizes with complementary specificities.

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Participants

• UNIVERSITY OF EDINBURGH · EDINBURGHCoordinatorUnited Kingdom

Links

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