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Abstract

The subject of this thesis is the development of novel enantioselective esterolytic functionalities with the aim to expand the biocatalytic toolbox. For that purpose, two basic strategies are known: the evolution of existing activities and the quest for undiscovered functionalities. Both concepts are addressed in this work.
Although directed evolution is a well-established method, its application is still limited in many cases, because many systems do not allow for the fast and efficient isolation of improved variants from large libraries. Therefore, the main goal of this thesis is the development of new approaches giving very high throughputs in directed evolution of enantioselective esterases and lipases.
Since FACS analyses and sorts cells at a very high speed, this instrument provides an ideal basis for the establishment of a high-throughput screening method. Hence, the first aim is to set-up a chemical and biological system, which labels the cells with fluorescent dyes according to the enantioselectivity of their enzymes. In a second step, the variants of the library could then be fractionated. Such a system could be used to efficiently evolve enantioselective enzymes.
Selection is another strategy allowing at least in theory extreme numbers of mutants to be evaluated. Therefore, the attempt is made to apply this technique for the isolation of enantioselective lipases and esterases. So far, this challenging problem is not solved. Thus, another important aim of this thesis is the development of a novel concept to establish the complex biochemical link between enantioselectivity and the growth-rate of the cells.
A further objective of the thesis is the establishment of a platform for the quick characterisation of novel esterolytic activities. In order to evaluate the potential of enzymes for biocatalytic processes, a variety of representative test substrates is required. This compound library could, for instance, be applied for testing enzymes obtained from metagenomic screening projects.
In the course of investigations concerning hydrolytic activities of proteins, an esterolytic activity of HisF from Thermotoga maritima was discovered. Due to the potentially interesting evolutionary insights, the nature of this promiscuous activity was investigated as a spin-off project.