Reaktionstechnische Untersuchungen zur enzymatischen $\textit{de novo}$ Synthese von GDP-$\beta$-L-Fucose und der $\textit{in situ }$Fucosylierung von Oligosaccharide

Many inter- and intracellular recognition processes base on the interaction of oligosaccharides with other (macro-)molecules. Well-known examples are viral or bacterial infections, inflammation or fertilization. In mammals, the monosaccharide and deoxy sugar l-fucose is crucial for a multitude of biological processes. $\textit{In vivo}$, enzymatic synthesis of fucosylated oligosaccharides is catalyzed by the $\textit{Leloir}$-fucosyltransferases (FucTs). These enzymes require GDP-$\beta$-L-fucose (GDP-Fuc) as the donor. GDP-Fuc is one of the most expensive and unavailable nucleotide sugars. In consequence, large-scale $\textit{in vitro}$ syntheses of fucosylated compounds still remain difficult due to the low availability of GDP-Fuc. $\textbf{Objective}$ The objective of this work was the development of processes for enzymatic synthesis of GDP-Fuc by using the enzymes of the de novo pathway: GDP-α-D-mannose-4,6-dehydratase (GMD) and GDP-$\beta$-L-fucose synthetase (GFS). These enzymes catalyze the synthesis of GDP-Fuc starting from GDP-α-d-mannose (GDP-Man) via the intermediate GDP-4-keto-6- deoxy-α-d-mannose (GKDM). Since GDP-Fuc strongly decreases GMD activity by a feedback inhibition, the design of processes that omit this kinetic drawback is imperative. By coupling the reaction system for GDP-Fuc synthesis with a α1,2-fucosyltransferase (α1,2-FucT) reaction, the concept of a one-pot fucosylation of oligosaccharides was investigated within this framework. $\textbf{Analytical methods & enzyme production}$ Robust means for the quantification of enzyme activities were provided. An enzyme assay based on capillary electrophoresis and suitable for GMD activity measurement was developed by using a genetic algorithm approach. Recombinant GMD and GFS as well as a recombinant α1,2-FucT from $\textit{Helicobacter pylori}$ were produced by fermentation of the respective rec. $\textit{E. coli}$ strains. Further downstream processing of GMD was performed by the means of ion exchange chromatography, GFS and the α1,2-FucT were purified by immobilized metal affinity chromatography (IMAC). [...