Since the initial development of the glycosynthase methodology, progress has been mainly focused on beta-glycosidic bond formation with neutral hexosyl units to produce simple oligo- and polysaccharides. We propose to give a step further by i) enriching the range of catalysts available towards specific psychrophilic activities, and ii) extending the methodology towards in vitro synthesis of anionic polysaccharides for the development of novel biomaterials.
Glycosynthases are mutant glycosidases with a non-nucleophilic amino acid replacing the catalytic nucleophile. They are hydrolytically inactive, but, when presented with an activated donor sugar, condense the donor and an acceptor sugar in high yield. The usual Â-glycosyl fluoride donor is labile at high temperature and is readily hydrolysed above 50ºC. Therefore, the temperature used for the glycosynthase reaction is a compromise between donor stability and enzyme activity that renders conditions far from optimal. Thus, being able to perform the glycosynthase reaction at low temperature will clearly enhance product yields. Screening for cold glycosidase activities in psychrophilic bacteria and cloning of the corresponding genes will provide the necessary enzymatic toolbox.
Another challenge is to extend the possibilities for the synthesis of original oligosaccharides. In the frame of this project, we plan to address the synthesis of glycosaminoglycans (GAGs), current targets in biomedical applications. GAGs are a large family of anionic polysaccharides with disaccharide repeating units of uronic acid and hexosamine. Engineering and directed evolution will be performed on existing beta-glucanases or beta-glucosidases to accept charged donors. The proposed project will combine an existing repertoire of characterized glycosynthases and expertise in directed evolution and molecular biology with the overall goal of extending the knowledge on rational engineering of glycosynthases and oligosaccharide synthesis.
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