Enzymes for selective decoupling of woody biomass: From fundamentals to industrial potential
Sammanfattning: The need for an economy based on renewable materials has resulted in growing interest in the use of woody biomass in a wider field of application. However, the chemical complexity of lignocellulose and the dense structure of wood pose challenges in its processing. The aim in a materials biorefinery is to extract the individual wood-components in as native and intact form as possible; and highly specific enzymes could be used for this. In this work, lignocellulosic enzymes with the potential of selectively decoupling and decomposing wood polymers were investigated and characterised. One of the studies was devoted to assessing enzyme accessibility in wood structures and evaluating the importance of pre-treatment to open up the dense wood structure, in order to improve enzyme performance. It was shown that the physical contribution during steam explosion is crucial for enhanced enzymatic hydrolysis of wood. Moreover, the performance of two endo-mannanases produced by the bacterium Cellvibrio japonicus(CjMan5A and CjMan26A) were compared on mannan polymers with dissimilar backbone structures and decorations, including the industrially relevant spruce galactoglucomannan. The enzymes were shown to be differently affected by the backbone heterogeneity and the presence of side groups on the substrates, demonstrating the variation in substrate preferences among mannanases. It was further shown that chemical acetylation of mannans reduced substrate hydrolysis significantly. Acetylation was therefore suggested as a tool to limit the biodegradation of mannan-based material. The major part of the work described in this thesis was dedicated to investigating the role and function of glucuronoyl esterases (GEs), which are enzymes that hydrolyse the ester linkages between lignin and hemicelluloses that contribute to the recalcitrance of woody biomass. Extensive structure-function studies of bacterial GE candidates contributed to our understanding of the diversity of this relatively unexplored enzyme family. Both similarities and differences in substrate preferences among the GEs studied revealed enzymes more promiscuous than their characterised fungal counterparts. GE activity was further assayed on lignin-carbohydrate complexes isolated from woody biomass, and GE-mediated ester cleavage was demonstrated with advanced and complementary tools, including size-exclusion chromatography, 31P NMR and 2D NMR. These findings not only confirm the suggested biological role of GEs, but also demonstrate the potential of these enzymes in decoupling lignin from hemicelluloses in industrial settings.
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