Discovery and investigation of glycoside hydrolase family 5 enzymes with potential use in biomass conversion
Abstract: Glycoside hydrolases (GHs) cleave glycosidic bonds in glycoconjugates, oligosaccharides and polysaccharides such as cellulose and various hemicelluloses. Mannan is a major group of hemicelluloses. In higher plants, they usually serve as storage carbohydrates in seeds and tubers or as structural polysaccharides cross-linking with cellulose/lignin in cell walls. In industrial fields, this renewable biomass component can be used in various areas such as production of biofuels and health-benefit manno-oligosaccharides; and mannan degrading enzymes, especially mannanases, are important molecular tools for controlling mannan polysaccharides properties in biomass conversion. In this thesis, the evolution, substrate specificity and subfamily classification of the most important GH family, i.e., glycoside hydrolase family 5 (GH5), are presented providing a powerful tool for exploring GH5 enzymes in search for enzymes with interesting properties for sustainable biomass conversion. Additionally, three GH5_7 mannanases from Arabidopsis thaliana (AtMan5-1, AtMan5-2 and AtMan5-6) were investigated in the present study. Bioinformatics tools, heterologous expression, and enzymology were applied in order to reveal the catalytic properties of the target enzymes, increase understanding of plant mannanase evolution, and evaluate their potential use in biomass conversion. This approach revealed: (1) AtMan5-1 exhibits mannan hydrolase/transglycosylase activity (MHT), (2) AtMan5-2 preferably degrades mannans with a glucomannan backbone, and (3) AtMan5-6 is a relatively thermotolerant enzyme showing high catalytic efficiency for conversion of glucomannan and galactomannan making this plant mannanase an interesting candidate for biotechnological applications of digesting various mannans. Moreover, these studies suggest an evolutionary diversification of plant mannanase enzymatic function.
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