Isolation and characterization of unhydrolyzed oligosaccharides from switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with commercial enzyme preparations

• Structural characterization of abundant unhydrolyzed linkages of Switchgrass xylan.
• LC-MSn analysis of xylan end-point digestion oligosaccharide products.
• SG xylan structures limit α-arabinofuranosidase activity of commercial enzymes.
• A GH 62 α-arabinofuranosidase hydrolyzes the most abundant oligosaccharide products.

Switchgrass (Panicum virgatum, L.) is a potential renewable source of carbohydrates for use in microbial conversion to biofuels. Xylan comprises approximately 30% of the switchgrass cell wall. To understand the limitations of commercial enzyme mixtures, alkali-extracted, isolated switchgrass xylan was hydrolyzed by the action of two commercial enzyme cocktails, in the presence and absence of an additional α-arabinofuranosidase enzyme. The two 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 a GH62 α-arabinofuranosidase enzyme; further characterization confirmed the presence of (1→3)-α-arabinose linkages. These results demonstrate the lack of the required selectivity for arabinose-containing substrates in the commercial enzyme preparations tested. One product 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.

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