Identifizierung aktiver und enantioselektiver Hydrolasen für den industriellen Einsatz Of all the types of enzyme catalyzed reactions in organic synthesis hydrolytic transformations are the easiest to perform. Hydrolases accept a broad range of substrates exhibit high stability and cofactors are not required. Since the number of commercially available biocatalysts is raising steadily in this thesis model substrates (secondary alcohols) are used to develop and evaluate assay systems for the fast and reliable identification of active and enantioselective Hydrolases. Under technical conditions the characterization of selected enzymes often shows that their properties are not satisfying for the generation of profitable processes. By substrate and medium engineering respectively immobilization and the improvement of an enzyme by directed evolution it is possible to increase the effectiveness of a biocatalytic process especially with respect to the enantioselectivity. The generation of a mutant library is performed by the classical method of directed evolution by error prone PCR. For the identification of more stereoselective enzyme variants a new high throughput assay system is used to screen the mutant library. The assay format is based on the Hydrolase catalyzed cleavage of acetates with subsequent determination of the released acetic acid by a cascade of enzymatic reactions. Hydrolases secondary alcohols difficult to resolve substrates error prone PCR Immobilization substrate engineering medium engineering thermophilic microorganisms Esterases Lipases Proteases enantioselectivity high throughput screening HTS pH assay acetate assay Hydrolasen Identifikation Industrie