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A7 Structural biology of glycosyltransferases for the optimisation of biotechnical processes

(Heinz/Seibel)

Aim of the subproject A7 is the x-ray analysis of a dextransucrase (including mutants) and a fructosyltransferase to gain a detailed knowledge of the active centres of these enzymes in sugar-free and sugar-bound form. The structures will allow for a rational mutagenesis of the genes to evocate changes in substrate specificities (subproject A3, Buchholz-Hofer). An equally important aim is to design changes of the overall surface charge of the proteins. In cooperation with subproject C2 (Seidel-Morgenstern) an improvement of separation processes by means of mathematical simulation studies as well as the improved immobilisation of enzymes (subproject C3 Buchholz-Jördening) should be accomplished.
The program envisages the isolation, purification and crystallization of a native dextransucrase followed by the x-ray structural analysis. For that purpose, glycosyltransferases (GTF) from various organisms will be used. Using expression in E. coli, GTF from S. oralis, strep-tag fusioned gtfD from S. mutans as well as fructosyltransferase from S. oralis will be produced.
Using expression in B. megaterium, DsrS of L. mesenteroides will be produced. The subsequent crystallization of the cleaned and functionally active enzymes will be employed by using commercially available sparse-matrix screens.

In parallel mechanism-based inhibitors such as 6-deoxysaccharose are being synthesized to support crystallization. The structural data of these enzym-substrate complexes will allow for the identification of amino acids vital for substrate recognition and catalysis. The modelling of downstream processes for chromatography based on interaction of protein surfaces and column materials is supposed to be acquired in close cooperation with Teilprojekt C2 (Seidel-Morgenstern). In summary an optimization of biocatalysis as well as the processing of enzymes for biocatalytic processes will be reached.

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