Einfluss von Mikrowelleneinstrahlung auf beta-Galaktosidase katalysierte Transglykosylierungsreaktionen

This dissertation contains extensive investigations in the area of microwave assisted biocatalysis. Beta-galactosidase catalyzed transglycosylation reactions have been in the focus of this work. Three beta-galactosidases were used, two commercially available and one recombinantly expressed enzyme of the mesophilic organism Bacillus circulans. The observed microwave effect on enzymatic activity was a time-dependent consecutive activation and inactivation of the commercial enzyme. It could be shown that an increased irradiation intensity at comparable temperature leads to a more rapid inactivation of the biocatalyst. This had a decisive effect on the synthesis of nucleotide oligosaccharides. It was found that microwave irradiation had a stabilizing effect on product concentrations and distribution. This could again be explained by the time-dependent inactivation of the enzyme. This effect was also found for the synthesis of galacto oligosaccharides, a class of nondigestible milk oligosaccharides with health promoting characteristics. During the synthesis of glycan structures with chemical linker, microwave irradiation influenced the balance of structural isomers evolving in this reaction. The time-dependent activation and inactivation of the biocatalyst was found to be the main reason for the advantageous course of synthetic reactions performed under microwave irradiation. In cooperation with Prof. Kanaya (Department of Material and Life Science, Graduate School of Engineering, Osaka University) the protein structure of the recombinant beta-galactosidase was investigated. This is a first step for the observation of influences of electromagnetic fields on the protein structure and thereby altered behavior of the biocatalyst during synthesis. By crystallization studies the protein structure of this recombinant beta-galactosidase could be solved and first CD-spectroscopy measurements were performed. This work is the basis for systematic search for protein structure elements, which allow a given biocatalyst to be highly efficient under microwave irradiation. With such information in the future criteria could be found to judge on natural ability or enable biocatalysts by mutation for their usage under microwave irradiation