Enzyme evolution : exploring substrate scope and catalytic promiscuity Enzyme katalytische Promiskuität Hydrolasen Schweineleber Esterase Epoxid Hydrolase Haloalkan Dehalogenase Protein Engineering catalytic promiscuity pig liver esterase hydrolases protein engineering enzyme evolution Enzymatic evolution and the corresponding relationship to substrate scope and catalytic promiscuity were targeted in this thesis. As enzyme examples pig liver esterase (PLE) oleate hydratases and linoleate isomerases as well as epoxide hydrolases (EH) and haloalkane dehalogenases (HLD) were used. The substrate scope and the enantiopreference of PLE was analyzed by molecular modeling and substrate docking since different enantiomeric excesses were detected for the conversion of malonate diethyl esters depending on the PLE isoenzyme. Additionally fatty acid converting enzymes with high identity were found and analyzed to comprehend the switch of both activities. Furthermore the evolutionary connection between EH and HLD was investigated by interconversion studies to implement an HLD acitivity in an EH. By directed evolution and rational design both possibilities of protein engineering were realized. Finally a new methodology for targeted continuous in vivo evolution was established by a temperature dependent mutagenesis frequency.