| The goal of the proposed project is to engineering the halide-binding site of halohydrin dehalogenase in order to introduce activity in the reverse reaction (which is epoxide ring opening) with non-anionic nucleophiles. The current enzyme can form and open epoxides only with anionic leaving groups/nucleophiles. Activity with non-anionic nucleophiles, such as amines, will greatly expand the biocatalytic applicability of the enzyme. The approach is based on a combination of protein design, aimed at introducing the structural features required for nucleophile activation in the halide-binding loop region, and directed evolution, aimed at optimizing the stability and catalytic activity of the designed variants. Growth selection and screening will be used to obtain active enzymes, of which the biocatalytic properties and structures will be investigated. |
