Novel biocatalysts for technical application by evolution and design [Elektronische Ressource] / von Thorsten Eggert

Novel biocatalysts for technical applications by evolution and design Habilitationsschrift zur Erlangung der Venia legendi für das Fachgebiet Molekulare Mikrobiologie an der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf von Dr. rer. nat. Thorsten Eggert Dezember 2006 Für Barbara und Laila Th. Eggert Table of contents Table of contents 1. Zusammenfassung 1 2. General introduction 5 3. Scope and objectives 7 4. Biocatalyst identification by high-throughput screening and 9 selection systems 4.1 Introduction 9 4.2 High-throughput screening of chiral alcohols 12 4.3 Spectrophotometric screening assay for hydroxynitrile lyase activity 14 4.4 Screening and selection systems for C-C-bond forming enzymes 16 4.5 Discussion 19 5. Biocatalyst production in Bacillus subtilis 21 5.1 Introduction 21 5.2 Optimization of heterologous protein secretion by signal sequence 24 screening 5.3 Optimization of heterologous protein secretion by directed evolution 28 5.4 Discussion 29 6. Novel biocatalysts by evolution and design 33 6.1 Introduction 33 6.2 First generation of directed evolution: complete saturation mutagenesis 39 6.