Engineering and analysis of the enantioselectivity of esterases The focus of this thesis is the engineering and analysis of the enantioselectivity of esterases using 3 phenylbutyric acid (3 PBA) as model substrate. An ultra high throughput assay for identification of enantioselective esterases has been developed based on the combination of in vivo selection and flow cytometry. The in vivo selection medium consists of a couple of pseudo enantiomers of 3 PBA; one enantiomer is coupled to glycerol (GE) and hydrolysis of this substrate will enable cell survival. The other enantiomer is coupled to the toxin 2 3 dibromopropanol (BE) the hydrolysis of this substrate will cause cell death. Thus cell survival is a function of the enantioselectivity of the enzyme expressed. The pseudo enantiomeric substrates are structurally similar to allow selection for enantioselectivity instead of selection for enzyme substrate affinity. Next esterase BS2 was chosen as negative control to establish the selection system since it hydrolyses both pseudo enantiomers with low enantioselectivity (E~3 and 1 respectively). High enantioselective esterases towards 3 PBA: esterases PestE and CL1 (E > 100 both (R) selective) were identified in a screening and used as positive controls. Further the hyperthermophilic esterase PestE was crystallized. After elucidation of the enzyme structure the high enantioselectivity of the enzyme towards 3 PBA could be explained by molecular modelling ... Durchflusscytometrie Esterasen Gerichtete Evolution Biotechnologie High throughput screening Proteindesign Protein engineering in vivo selection