Turning Bacillus circulans xylanase into a glycosynthase

Oligosaccharide syntheses have become increasingly important for the pharmaceutical industry since many of these biomolecules are considered potent therapeutic agents. Due to the complex structure of oligosaccharides, classical chemical synthesis is demanding and intricate and hence strategies to precisely derivatize carbohydrate moieties and thus optimize their potential use, are still limited. The most promising alternative to tedious and laborious synthetic chemistry is to employ enzymes in large-scale productions of oligosaccharides. This approach has recently been advanced in pioneering studies by Withers and colleagues, who engineered glycosidases for this purpose. Next steps are now to modify the activity of widely known enzymes, and in particular to screen and select for mutants that exhibit variant substrate-binding properties and/or enzymatic activities. Since Bacillus circulans xylanase (Bcx) is one of the best characterized glycosidase, both at the structural and at the functional level, and due to its small size (20 kDa), this enzyme meets all required standards for directed evolution in combination with phage display technology in order to effectively alter its catalytic property. The goal of this project is to engineer Bacillus circulans xylanase by bringing forth mutant enzymes that bind modified (donor) sugar substrates and specifically transglycosylate these carbohydrate moieties to acceptors that are otherwise eschewed by the wild-type enzyme. Screening and selection of evolved enzymes is performed by leveraging mechanism-based sugar inhibitors as part of a modified phage display technology. Structural analyses of mutant enzymes that exhibit novel properties will be undertaken applying computational modeling using available three-dimensional structural information of wild-type Bcx. Further details particularly concerning molecular interactions will be determined by entertaining collaborations with laboratories specialized in x-ray and NMR protein structure determination technologies. Conducting this project I hope to gain insights that lead to a better understanding of the molecular mechanisms of glycosidases and glycosynthases, which may be also applied to promote advancements in carbohydrate chemistry and to further biomedical and pharmaceutical research.