Library design and screening strategies for efficient enzyme evolution

Enzymes are the catalysts of nature and they facilitate thousands of chemical conversions in living organisms. Enzymes work in an aqueous environment at neutral pH and ambient temperature. Such mild reaction conditions are also highly advantageous for synthetic organic synthesis because this can reduce energy use and prevent the formation of undesired side-products. Enzymes have been successfully applied as catalyst in synthetic organic synthesis for more than a century but their biocatalytic use also has serious limitations. It is often possible to evolve enzyme variants with improved properties in the laboratory but this requires time-consuming and expensive massive screening of mutant enzyme collections. In this thesis a number of tools and strategies are explored that facilitate the knowledge driven laboratory evolution of enzymes. A number of these approaches and principles are experimentally evaluated and are focused on enhancing the biocatalytic potential of haloalkane dehalogenases. This unique class of enzymes catalyzes the hydrolytic cleavage of carbon-halogen bonds. In one of the studies a haloalkane dehalogenase was engineered for the conversion of a toxic waste compound into valuable chiral building blocks that can be used for the synthesis of pharmaceuticals such as propranolol and viloxazine