Using isolated cell wall xylan to identify recalcitrant oligosaccharides

Herbaceous biomass is a renewable source of carbohydrates with potential for use in microbial conversion to biofuels.  Xylan comprises 20-40% of herbaceous biomass cell wall material and its full depolymerization benefits the economics of bioconversion.  To understand the limitations of commercial enzyme mixtures, alkali-extracted, isolated xylans from select biomass sources were hydrolyzed, independently, by the action of two commercial enzyme cocktails.  Alkali-extracted xylans were used to eliminate potential effects of: cellulose; lignin; and esterification on the accessibility and/or recognition of enzymes to their target glycosidic bonds.  Additionally, digestions were performed at low solids loadings to limit the effects of product inhibition.  The most abundant enzymatic digestion products from each commercial enzyme treatment were separated and characterized by LC-MSⁿ, linkage analysis, and NMR.  The most abundant oligosaccharide from each commercial cocktail was susceptible to hydrolysis when supplemented with α-arabinofuranosidase enzymes from certain, but not all, GH families; further characterization confirmed the presence of (1→3)-α-arabinose linkages in these oligosaccharides.  These results demonstrate the lack of the required selectivity for arabinose-containing substrates in the commercial enzyme preparations tested.  Additionally, one oligosaccharide product, characterized by LC-MSⁿ, linkage analysis, and NMR, from each condition remained intact and was found to contain (1→2)-β-xylose-(1→3)-α-arabinose side chains; this linkage acts as a source of oligosaccharide recalcitrance.  The identification and purification of these oligosaccharides permits enzyme screening for either better activity on the most relevant substrates and the discovery of auxiliary enzymes  to improve hydrolysis of specific bonds.