Characterization of novel GH115 Îą-glucuronidases for enzymatic tailoring of xylans

Xylans represent abundant renewable resources for developing applications ranging from prebiotics to plastic-replacing packaging materials. Accessory hemicellulases can be used to tailor the side-group chemistry of xylans to thereby control properties such as nutrient value, rheology, solubility, adsorption behavior, as well as barrier characteristics of xylan-based films and coatings. Glucopyranosyluronic acid (GlcA) and (methyl)-glucopyranosyluronic acid (MeGlcA) are common substituents of the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. α-Glucuronidases from glycoside hydrolase family GH115 have the potential to tailor high-molecular-weight xylans, because they are the only known enzymes that can target GlcA/MeGlcA from both internally and terminally substituted regions of xylans. Two family GH115 enzymes (SdeAgu115A from the marine bacterium Saccharophagus degradans 2-40T and AxyAgu115A from alkaliphilic bacterium Amphilbacillus xylanus) were selected for characterization. Both enzymes demonstrated activity towards glucuronoxylans and oligomers thereof with preference towards internally substituted residues. The first five-domain crystal structure of a GH115 enzyme was also reported, which comprised an additional insertion C+ domain likely to participate in substrate binding. Consistent with the natural habitats of the source organisms, AxyAgu115A demonstrated better performance in alkaline condition compared to other reported GH115 α-glucuronidases, whereas salt activation was only observed for SdeAgu115A. Unlike SdeAgu115A, AxyAgu115A displayed similar activity towards beechwood glucuronoxylan and the more highly branched spruce arabinoglucuronoxylan. The ability of AxyAgu115A to accommodate comparatively complex xylans was consistent with conclusions that could be drawn through docking analysis. Synergistic action between SdeAu115A and AxyAgu115A, and two α-arabinofuranosidases from families GH51 (E-AFASE) and GH62 (SthAbf62A) towards spruce arabinoglucuronoxylan was identified, suggesting that MeGlcA and Araf could sterically hinder α-arabinofuranosidase and/or α-glucuronidase activity. Finally, AxyAgu115A was applied herein to recover xylan-derived oligosaccharides extracted from mixed hardwood chips through liquid hot water extraction. As part of this study, an LC-MS/MS method was developed to facilitate label-free characterization of diverse xylooligosaccharides.Ph.D.2019-12-19 00:00:0