Untersuchungen zur Erweiterung der Substratspezifität von Carboxylester Hydrolasen Hydrolases Enantiopreference Directed evolution Error prone PCR Molecular modeling DNA shuffling High throughput screening Rational protein design Upases Esterases Amidases Tertiary alcohols Enantioselectivity Carboxylester hydrolases are among the enzymes used most frequently in biocatalysis. They show good solvent and storage stability and accept a broad range of substrates. While they hydrolyze esters of primary and secondary alcohols most of them are not able to convert esters of tertiary alcohols. In this thesis methods are examined to generate esterase variants by directed evolution and rational protein design which are able to hydrolyze those substrates with high enantioselectivity. Large enzyme libraries of random mutants are created and screened on new activities with a microtiterplate based assay system. Utilizing molecular modeling techniques positions were predicted to enhance enantioselectivity by directed mutagenesis.