Novel Retaining Glycoside Hydrolases : Potential candidates for transglycosylation and hydrolysis

Sammanfattning: Our society is moving towards renewable resources, where biomass, rich incarbohydrates, is producing chemicals and fuel. However, there are severallimitations when it comes to valorisation of the carbohydrates from renewablebiomass. One major hurdle is the over-functional nature of carbohydrates, making them difficult to process by conventional chemistry. Carbohydrate activeenzymes can help overcome this limitation by providing excellent tools to utiliserenewable feedstocks, supplying competitive alternatives to the traditionalchemical process.The enzymatic toolbox is the green alternative when it comes to synthesis ofglycoconjugates and to make the transition towards bioeconomy, use of thesetools is an essential step. In nature, glycosylation is executed mostly byglycosyltransferases. However, they are not ideal for industrial applications dueto their need to use expensive activated donors. Whereas, transglycosylases(classified under glycoside hydrolase families: GHs) do not need any activateddonor, making them perfect candidates. The only limitation with them is thatthere are not many that have been characterised. Transglycosylases are classifiedin the same families as their hydrolysing counterparts, and are closely related insequence and structure, making it difficult to select them based on sequencesimilarities. A typical exception from this is the cyclodextrin glucanotransferases(CGTases) which belongs to GH13.This thesis investigates the transglycosylation activity of cyclodextringlucanotransferases, for expanding the utilisation of transglycosylases. The focusof the work was on the elongation of the carbohydrate part of alkyl glycosides. Anovel cyclodextrin glucanotransferase (CspCGT13) from Carboxydocella sp.was characterised and compared with available commercial enzymes to evaluatethe applicability in alkyl glycoside modification. The novel enzyme showedsignificant coupling activity with γ-cyclodextrin as the donor, however it was notas efficient as the commercial CGTases. Later, the coupling activity wasimproved by protein engineering and bioinformatic analysis, making it acompetitive candidate for alkyl glycosides modification.The majority of the enzymes in the GH-families are hydrolases and are widelyavailable. Using glycoside hydrolases in synthesis requires reduced hydrolyticactivity. In this thesis, oligosaccharide synthesis was studied by using glycosideshydrolases. Significantly reduced hydrolysis was achieved for an endo-xylanasefrom the thermophilic bacterium Rhodothermus marinus DSM 4252T throughprotein engineering. The enzyme variants displayed enhanced transglycosylationactivity.In addition, novel candidates from the enzymatic toolbox from another strain ofthis marine thermophilic bacterium were also investigated in this thesis, aimingto gain more insight into the hydrolytic mechanism used for saccharificationprocesses. Six novel exo-hydrolases from a single GH family (GH3) originatingfrom R. marinus DSM 4253 were characterised. The study showed these enzymesto have broad substrate specificities and activities at moderately high temperature. Also, more information was obtained regarding their structuralfeatures and genomic distributions, providing more knowledge to tailor theenzymes for industrial applications.