Pyranose dehydrogenases (PDHs) are enzymes with ability to oxidize diverse carbohydrates in multiple positions. Oxidation of the anomeric carbon results in lactone which spontaneously hydrolyses to carboxylic acid in water. Secondary hydroxyls are oxidized to ketones which exist primary as hydrates in water complicating the analysis. In addition to monosaccharides, PDHs have shown activity on some di- and trisaccharides. Targeted enzymatic oxidation of carbohydrates facilitates their further derivatization or polymerization into bio-based chemicals and materials. We have compared activity and performance of two PDHs from Agaricus bisporus and Leucoagaricus meleagris on linear (XOS) and arabinofuranosyl substituted (AXOS) xylooligosaccharides with degree of polymerization of two to five. Reaction mixtures were analyzed by UPLC-MS-ELSD to quantify the different oxidation products and ESI-MSn to reveal oxidized positions along the oligosaccharide. A workable method was developed applying sodium borodeuteride reduction for labeling of formed carbonyls to facilitate the identification, quantification and structural analysis of oxidized products. Activity of both enzymes was found to decrease with increasing length of the (A)XOS. Oxidation of linear XOS mostly led to bifunctionalized double oxidized products, and the oxidation was clearly targeted to the reducing and non-reducing xylopyranosyl residues. Single oxidized products dominated in AXOS reactions. Selectivity between oxidation of specific secondary hydroxyls versus the reducing end C1 was found to depend on both the enzyme and the substrate. The developed MS methods and data analysis, with the obtained results will be discussed in detail in the current paper. The sensitive and fast MS methods will be shown to have broad applicability to study carbohydrate oxidoreductases in general.

Mass spectrometry analysis of pyranose dehydrogenase action on xylooligosaccharides

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.