(421av) Exploration of Significant Cellulase and Hemicellulase Activities Required for the Deconstruction of Ionic-Liquid Pretreated Biomass

Lignocellulosic biomass, comprised of polysaccharides derived from cellulose and hemicellulose, is a second generation renewable feedstock for carbon-based chemicals and fuels.  The recalcitrance of biomass to deconstruction is one of the main obstacles in the conversion of lignocellulosic biomass to monomeric sugars and the production of sugar platform fuels and chemicals.  Ionic Liquid (IL) pretreatment creates unique biomass substrates consisting of amorphous cellulose with largely intact hemicellulose and lignin. These substrates differ from many other pretreatments which remove hemicellulose and retain cellulose crystallinity. The unique substrate resulting from IL-pretreatment poses an interesting challenge for enzymatic deconstruction using traditional commercial mixtures.

Mono-component cellulase and hemicellulase enzymes from Aspergillus nidulans have been expressed in Pichia pastoris, purified and characterized for activity, substrate specificity, and thermal stability. Enzyme combinations were used in series of hydrolysis experiments to determine key enzyme activities and/or interactions that significantly contribute to the conversion of a variety of lignocellulosic biomass substrates to monomeric sugars.  Accessory hemicellulases appear to be an essential addition to the mono-component enzyme mixtures.  Exploration of these accessory enzymes also yields insight into an understanding of the differences in hemicellulose structure between feedstocks.  Some accessory hemicellulases include acetyl xylan esterases (AXE), α-glucuronidases, and arabinofuranosidases.  In addition to mono-component enzyme mixtures, next generation commercially-available enzyme mixtures were characterized and formulated using key activities to optimize hydrolysis at low enzyme loadings.  These simplified mixtures will be dependent on the substrate.  As part of these studies, an analysis of variance (ANOVA) was used to statistically model the main and interaction effects of key enzyme activities.