Enhancing the Activity and Regioselectivity of the Abg Glycosynthase through Site Directed Mutagenesis and Directed Evolution

Carbohydrates have important functions in many biological processes, such as cell recogniton, cell migration and signaling events. Chemical synthesis of these complex molecules is still a very challenging task and can hardly be automated. Thus, enzymatic derivatisation is not only a considered but essentially the only efficient alternative methodology to synthetic chemistry. In this context, glycosyltranferases and glycosidases have been studied to some extent. In addition to these, a new breed of enzymes, namely glycosynthases have recently been introduced. I attempt to work on this novel group of enzymes and plan to further improve the usability of the Abg glycosynthase. This class of enzymes is derived from glycosidases, but, since altered in one key amino acid within the acitve site, they do not hydrolyse substrates but, due to the still working transglycosylation activity, can now efficiently polymerize modified sugar oligomers. Yet, only a few examples of glycosynthases are published by now. My intention is to increase the useful repertoire of glycosynthases by enhancing the activity of the well characterized glucosynthase of Agrobacterium sp. by means of site directed mutagenesis as well as directed evolution. Furthermore, I plan to shift the substrate specificity of this enzyme to xyloside and change the regioselectivity of this transfer. Eventually, identified mutant enzymes will be analysed for there usability to synthesise modified carbohydrate compounds. I hope to gain insights that lead to a better understanding of the molecular mechanism of how glycosynthases work which in due course may be helpful to broaden their usability for carbohydrate chemistry.