Final Report Summary - WALLESTER (Lignin and carbohydrate acylation in nonwoody plant cell-walls: Structural role, enzymatic cleavage and biotechnological relevance) The scientific aim of WALLESTER was to investigate lignin versus hemicellulose acylation in different non-woody angiosperms in connection with plant cell-wall structure the development of biotechnological applications based on the use of esterases and other cell-wall degrading enzymes. This would be a more complete and environmentally-friendly utilisation of plant carbohydrates and lignin, leading to improved exploitation of lignocellulosic biomass. The scientific aim was addressed through four specific scientific objectives: (a) To establish if acetylated and coumaroylated lignins are substrates for acetyl and feruloyl esterases? (b) To ascertain if feruloyl esterases act at the lignin-polysaccharide interface? (c) To elucidate the relationship between lignin and carbohydrate depolymerisation: how does a micro-organism enzymatically deconstruct lignocellulosic biomass? (d) To enhance the added-value potential of lignocellulosic biomass through the development of enzymatic biotools to facilitate the separation and utilisation of each component. It was ascertained that feruloyl and acetylxylan esterases are key enzyme components for improved biomass solubilisation, but structural acetylation of plant polymers itself was not a barrier to enzymatic degradation. 2D-NMR was sufficiently developed to follow enzymatic effects on lignin and hemicellulose components of plant biomass in 'real-time'. The esterases act mainly on the carbohydrate portion of the biomass but one particular feruloyl esterase also interacts with monolignols removing a significant portion of acetate groups from Abaca lignin. An isolated lignin-carbohydrate complex was split by the action of the enzymes demonstrating that the esterases can act at the lignin-carbohydrate interface but in whole fibres, the complex interaction of the polymeric matrix restricts enzyme access to this linkage as well as the acetyl groups on the lignin. Lignin is insoluble in aqueous environments and enzyme activity can be severely reduced if not abolished in organic solvents. Feruloyl esterases were robust in DMSO concentrations up to 20 % and at low organic solvent concentrations, hydrolysis activity could be enhanced. While DMSO is also used to solubilise isolated hemicellulose, and theoretically could change the plant cell wall matrix sufficiently to enable better access of enzymes their substrate interaction sites, the presence of DMSO in hydrolysis reactions of whole lignocellulose fibres reduced overall solubilisation, although acetic acid release was enhanced. The use of enzymes to break open and degrade plant cell wall structures has many important applications within the sustainable and 'green' bio-economy platform. Efficient breakdown and modification of plant-based raw materials would lead to both more efficient energy savings in food and pulp / paper industries, reduction in the use of environmental-unfriendly chemicals, and knowledge on how to remove lignin and hemicellulose fractions without their necessary destruction would facilitate the development of new value chains for traditional industries as well as developing the biorefinery concepts. The improved knowledge on the stability of feruloyl esterases and wider application knowledge will result in a better exploitation of this type of enzyme, as well as the demonstrating that supplementation of commercial enzyme preparations with feruloyl esterase activity can aid in better plant biomass deconstruction. The results of WALLESTER will be of interest to academic researchers looking at the enzymatic treatment of plant-derived material for either total breakdown or modification / functionalisation of biopolymers, and to industries looking at using enzymes for modification of ingredients / biocomponents, energy saving and developing sustainable, environmental-friendly processes, such as the (health) food industry, pulp and paper, and biofuel sectors.