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Table of contents; List of figures; List of schemes; List of abbreviations; 1 Introduction; 1.1 Value of biocatalysis in chemical synthesis; 1.2 Natural halogenated compounds; 1.3 Halogenating enzymes; 1.4 The FAD-dependent halogenase family; 1.5 Biocatalytic application of tryptophan halogenases; 1.6 Directed enzyme evolution; 1.7 Halogenated marine metabolites; 1.8 FAD-dependent halogenase Bmp5; 2 Motivation; 3 Material and Methods; 3.1 Analytics; 3.2 Laboratory Equipment; 3.3 Chemicals and solvents; 3.4 Molecular biological methods; 3.5 Biochemical methods; 3.6 Directed evolution.
6.1.2 Expression of Bmp5 in E. coli BL21 (DE3) pGro76.1.3 Protein purification via HisTALON affinity chromatography; 6.1.4 Bmp5 expression in E. coli BL21 (DE3); 6.2 Identification of Bmp5; 6.2.1 Detection of His6-tagged Bmp5 by Western blot analysis; 6.2.2 Identification of Bmp5 by peptide mass fingerprint; 6.3 Enzymatic bromination of 4-HBA after purification of His6-Bmp5; 6.4 Generation of GST-tagged Bmp5; 6.4.1 Subcloning of bmp5 into pETM30 vector; 6.4.1.1 Identification of positive pETM30-GST-bmp5 clones by colony-PCR; 6.4.2 Expression and purification of GST-Bmp5.
6.4.3 Attempts for enzymatic bromination of 4-HBA after purification of GST-Bmp56.5 Establishment of cofactor regeneration for Bmp5 in E. coli lysate; 6.5.1 Investigation of glucose dehydrogenase; 6.5.2 Optimization of cofactor regeneration in Bmp5-catalyzed bromination employing alcohol dehydrogenase; 6.5.3 Identification of reaction components by GC-MS analysis; 6.5.4 Study of Bmp5's bromination ability in presence of chloride; 6.5.5 Examination of iodination ability; 6.6 Investigation of Bmp5's substrate scope; 6.7 Preparative synthesis of 2,4-dibromophenol using Bmp5-ADHcombiCLEAs.
7 Establishment of a marine brominase7.1 Establishment of the brominase Bmp5 for heterologous expression; 7.2 Expression of GST-Bmp5 fusion protein; 7.3 His6-Bmp5 revealed sufficient enzymatic activity after purification; 7.4 Establishment of cofactor regeneration system for Bmp5-catalyzed reactions; 7.5 Evidence of Bmp5 expression by Western blot analysis and peptide mass fingerprint; 7.6 Detected reaction compounds confirmed proposed Bmp5-catalyzed reaction mechanism; 8 Outlook; 9 Summary; Literature.
6.1.2 Expression of Bmp5 in E. coli BL21 (DE3) pGro76.1.3 Protein purification via HisTALON affinity chromatography; 6.1.4 Bmp5 expression in E. coli BL21 (DE3); 6.2 Identification of Bmp5; 6.2.1 Detection of His6-tagged Bmp5 by Western blot analysis; 6.2.2 Identification of Bmp5 by peptide mass fingerprint; 6.3 Enzymatic bromination of 4-HBA after purification of His6-Bmp5; 6.4 Generation of GST-tagged Bmp5; 6.4.1 Subcloning of bmp5 into pETM30 vector; 6.4.1.1 Identification of positive pETM30-GST-bmp5 clones by colony-PCR; 6.4.2 Expression and purification of GST-Bmp5.
6.4.3 Attempts for enzymatic bromination of 4-HBA after purification of GST-Bmp56.5 Establishment of cofactor regeneration for Bmp5 in E. coli lysate; 6.5.1 Investigation of glucose dehydrogenase; 6.5.2 Optimization of cofactor regeneration in Bmp5-catalyzed bromination employing alcohol dehydrogenase; 6.5.3 Identification of reaction components by GC-MS analysis; 6.5.4 Study of Bmp5's bromination ability in presence of chloride; 6.5.5 Examination of iodination ability; 6.6 Investigation of Bmp5's substrate scope; 6.7 Preparative synthesis of 2,4-dibromophenol using Bmp5-ADHcombiCLEAs.
7 Establishment of a marine brominase7.1 Establishment of the brominase Bmp5 for heterologous expression; 7.2 Expression of GST-Bmp5 fusion protein; 7.3 His6-Bmp5 revealed sufficient enzymatic activity after purification; 7.4 Establishment of cofactor regeneration system for Bmp5-catalyzed reactions; 7.5 Evidence of Bmp5 expression by Western blot analysis and peptide mass fingerprint; 7.6 Detected reaction compounds confirmed proposed Bmp5-catalyzed reaction mechanism; 8 Outlook; 9 Summary; Literature.