Transglycosylation by Glycoside Hydrolases - Production and modification of alkyl glycosides

Abstract: To enable the transition to a green, bio-based economy, an extensive enzymatic toolbox competitive to traditional chemical procedures is needed. One strong area for enzymes is carbohydrate chemistry, due to the over-functionalized nature of carbohydrates, difficult to handle in traditional chemistry. Glycosylation can be catalyzed by four main classes of enzymes, glycosyltransferases, glycoside phosphorylases, transglycosylases and glycoside hydrolases. For industrial implementation, transglycosylases are ideal catalysts that do not need the expensive activated donors associated with glycoside phosphorylases and glycosyltransferases. In addition, they completely lack the hydrolytic activity intrinsic in the closely related glycoside hydrolases. Unfortunately, very few transglycosylases with limited substrate specificities exist in nature, while a wide abundance of glycoside hydrolases are available. To expand the enzymatic toolbox for synthetic chemists it would be favorable to convert glycoside hydrolases into transglycosylases, by limiting their hydrolytic activity. This dissertation investigates the transglycosylation activity of glycoside hydrolases with synthesis and modification of alkyl glycosides, a widely applicable type of surfactants, as model reactions. Reduced hydrolysis for β-glycosidases from the thermophilic Thermotoga neapolitana was achieved through protein engineering, limiting water content and increasing pH. Complete elimination of the hydrolytic activity with maintained transglycosylation was achieved for synthesis of hexyl-β-D-glucoside and the factors resulting in the success are discussed. In addition, extension of the glycosidic part of alkyl glycosides through the coupling activity of cyclodextrin glucanotransferases is explored. An enzyme kinetics study was undertaken to deduce the optimal reaction conditions to promote coupling for a commercial enzyme. Moreover, a novel cyclodextrin glucanotransferase from Carboxydocella species was characterized, shown to have good coupling activity with γ-cyclodextrins as donor. This previously poorly studied donor can be used to extend the range of alkyl glycosides that can be produced and thereby the number of applications available.