Open access publications in the SLU publication database

Abstract

Presently, plant biomass is considered as one of the major future renewable sources for the production of second-generation biofuels. While the first generation biofuels essentially are based on starch and sucrose rich feed stocks and which production may compete with food production, the second-generation biofuels may be based on lignocellulose as feedstock, which is less problematic from an ethical point of view. The degradation of carbohydrates in plant biomass to fermentable sugars requires the concerted action of several diverse classes of carbohydrate active enzymes (CAZymes) for a total and efficient conversion of the plant biomass. Through a carefully balanced synergism mechanistically different CAZymes are able to degrade the stable and recalcitrant pol- ymers in the plant cell walls, such as cellulose, to soluble and fermentable monosaccharides. It is crucial to study the properties and function of these enzymes if we want to strive for a sustainable production of chemicals and biofuels, as they serve as a reservoir of environmentally friendly molecular tools. The main focus of the research work presented in this thesis is biochemical and structure-function characterizations of two classes of CAZymes: fungal glycoside hydrolase family 3 (GH3) β-glucosidases, and bacterial lytic polysaccharide monooxygenases, often referred to as LPMOs. GH3 β- glucosidases catalyse the conversion of disaccharides, produced by other CAZymes e.g. cellulases, to glucose. H. jecorina Cel3A, R. emersonii Cel3A and N. crassa NcGH3-3 are three industrially relevant fungal GH3 β-glucosidases for which the structures have been determined using X-ray crystallographic methods. The H. jecorina Cel3A, R. em- ersonii Cel3A enzymes has also been characterized biochemically. The LPMOs act in the very initial stage of plant cell wall degradation and cleave glycosidic bonds in crys- talline polysaccharides via an oxidative mechanism, which facilitates access to new chain ends for other CAZymes. To elucidate the structural and biochemical properties of LPMOs with bacterial origin, the structure of an AA10 LPMO the LPMO10A from Enterococcus faecalis was determined using X-ray crystallography. Furthermore, structural changes of the active site metal configuration by so-called X-ray induced photoreduction, were determined. During this reduction process, which mimics the active enzyme, the bound active site copper atom is reduced from Cu(I) to Cu(II), which causes changes in the ligation configuration.