Abstract
Pyranose dehydrogenases (PDHs; EC 1.1.99.29; AA3_2) demonstrate ability to oxidize diverse carbohydrates. Previous studies of these enzymes have also uncovered substrate-dependent regioselectivity, along with potential to introduce more than one carbonyl into carbohydrate substrates. Enzymatic oxidation of carbohydrates facilitates their further derivatization or polymerization into bio-based chemicals and materials with higher value; accordingly, PDHs that show activity on xylooligosaccharides could offer a viable approach to extract higher value from hemicelluloses that are typically fragmented during biomass processing. In this study, AbPDH1 from Agaricus bisporus and AmPDH1 from Leucoagaricus meleagris were tested using linear xylooligosaccharides, along with xylooligosaccharides substituted with either arabinofuranosyl or 4-O-(methyl)glucopyranosyluronic acid residues with degree of polymerization of two to five. Reaction products were characterized by HPAEC-PAD to follow substrate depletion, UPLC-MS-ELSD to quantify the multiple oxidation products, and ESI-MSn to reveal oxidized positions. A versatile method based on product reduction using sodium borodeuteride, and applicable to carbohydrate oxidoreductases in general, was established to facilitate the identification and quantification of oxidized products. AbPDH1 activity toward the tested xylooligosaccharides was generally higher than that measured for AmPDH1. In both cases, activity values decreased with increasing length of the xylooligosaccharide and when using acidic rather than neutral substrates; however, AbPDH1 fully oxidized all linear xylooligosaccharides, and 60–100% of all substituted xylooligosaccharides, after 24 h under the tested reaction conditions. Oxidation of linear xylooligosaccharides mostly led to double oxidized products, whereas single oxidized products dominated in reactions containing substituted xylooligosaccharides. Notably, oxidation of specific secondary hydroxyls vs. the reducing end C-1 depended on both the enzyme and the substrate. For all substrates, however, oxidation by both AbPDH1 and AmPDH1 was clearly restricted to the reducing and non-reducing xylopyranosyl residues, where increasing the length of the xylooligosaccharide did not lead to detectable oxidation of internal xylopyranosyl substituents. This detailed analysis of AbPDH1 and AmPDH1 action on diverse xylooligosaccharides reveals an opportunity to synthesize bifunctional molecules directly from hemicellulose fragments, and to enrich for specific products through appropriate PDH selection.
Highlights
Hemicelluloses comprise a diverse group of polysaccharides present in plant cell walls, and are thought to strengthen cell wall structures through interactions with both cellulose and lignin (Ebringerová, 2006; Scheller and Ulvskov, 2010)
The yield of recombinantly produced and purified AbPDH1 and AmPDH1 was 1.65 and 11.5 mg/l, respectively, where over 90% purity was reached for both enzymes (Figure S2)
Activity on xylose and xylooligosaccharides was measured using Fc+ ion as the electron acceptor, given the particular sensitivity of the corresponding assay (Sygmund et al, 2008). Both AbPDH1 and AmPDH1 were active on 10 mM X-X4 and A3X; AbPDH1 was distinguished by its additional activity on A2XX and XA3XX under the conditions used (Figure 1)
Summary
Hemicelluloses comprise a diverse group of polysaccharides present in plant cell walls, and are thought to strengthen cell wall structures through interactions with both cellulose and lignin (Ebringerová, 2006; Scheller and Ulvskov, 2010). In addition to Araf, arabinoglucuronoxylans found in cell walls of both grasses and coniferous trees, contain glucuronic acid and MeGlcpA substitutions (Scheller and Ulvskov, 2010). Biocatalysts that transform hemicelluloses have mainly been studied and developed to deconstruct corresponding polysaccharides to sugars that can be fermented to fuels and chemicals (Chundawat et al, 2011). These enzymes include glycoside hydrolases (GHs), carbohydrate esterases (CEs), and auxiliary activities (AAs) that are classified by the Carbohydrate-active enzyme (CAZy) database Enzymatic functionalization of hemicelluloses, including oligosaccharides liberated during lignocellulose processing, opens new possibilities to make high value bioproducts that retain the carbon and energy stored in starting carbohydrate structures
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