Xylooligosaccharides (XOS) have gained increased interest as prebiotics during the last years. XOS and arabinoxylooligosaccharides (AXOS) can be produced from major fractions of biomass including agricultural by-products and other low cost raw materials. Endo-xylanases are key enzymes for the production of (A)XOS from xylan. As the xylan structure is broadly diverse due to different substi-tutions, diverse endo-xylanases have evolved for its degradation. In this review structural and functional aspects are discussed, focusing on the potential applications of endo-xylanases in the production of dif-ferently substituted (A)XOS as emerging prebiotics, as well as their implication in the processing of the raw materials. Endo-xylanases are found in at least eight different glycoside hydrolase families (GH), and can either have a retaining or an inverting catalytic mechanism. To date, it is mainly retaining endo-xylanases that are used in applications to produce (A)XOS. Enzymes from these GH-families (mainly GH10 and GH11, and the more recently investigated GH30) are taken as prototypes to discuss substrate preferences and main products obtained. Finally, the need of new and accessory enzymes (new specifici-ties from new families or sources) to increase the yield of different types of (A)XOS is discussed, along with in vitro tests of produced oligosaccharides and production of enzymes in GRAS organisms to fa-cilitate use in functional food manufacturing.

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Xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) were produced from the insoluble arabinoxylan fraction of pretreated wheat bran by endoxylanases. The glycoside hydrolase (GH) family 10 xylanases GsXyn10A from Geobacillus stearothermophilus and RmXyn10A-CM from Rhodothermus marinus produced the AXOS AX, AXX and AXX in addition to XOS. RmXyn10A-CM also produced XAXX due to its non-conserved aglycone region accommodating additional arabinose substitutions in subsite +2. The GH11 enzymes, Pentopan from Thermomyces lanuginosus and NpXyn11A from Neocallimastix patriciarum had minor structural differences affecting hydrogen bonds in subsites -3 and +3, with similar hydrolysis profiles producing XAXX as major AXOS and minor amounts of XAXX but different ratios of X/X. In vitro analysis of the prebiotic properties of (A)XOS produced by Pentopan revealed nearly complete uptake of X and X by the probiotic bacteria Lactobacillus brevis and Bifidobacterium adolescentis. In contrast to previous reports, the GH43 arabinofuranosidase BaAXHd-3 from B. adolescentis cleaved α-1,3-linked arabinose on some single substituted AXOS.

Hydrolysis of arabinoxylan (AX) by glycoside hydrolase family 10 (GH10) xylanases produces xylo- and arabinoxylo-oligosaccharides ((A)XOS) which have shown prebiotic effects. The thermostable GH10 xylanase RmXyn10A has shown great potential to produce (A)XOS. In this study, the structure of RmXyn10A was investigated, the catalytic module by homology modelling and site-directed mutagenesis and the arrangement of its five domains by small-angle X-ray scattering (SAXS). Substrate specificity was explored in silico by manual docking and molecular dynamic simulations. It has been shown in the literature that the glycone subsites of GH10 xylanases are well conserved and our results suggest that RmXyn10A is no exception. The aglycone subsites are less investigated, and the modelled structure of RmXyn10A suggests that loop βα in the aglycone part of the active site contains a non-conserved α-helix, which blocks the otherwise conserved space of subsite +2. This structural feature has only been observed for one other GH10 xylanase. In RmXyn10A, docking revealed two alternative binding regions, one on either side of the α-helix. However, only one was able to accommodate arabinose-substitutions and the mutation study suggests that the same region is responsible for binding XOS. Several non-conserved structural features are most likely to be responsible for providing affinity for arabinose-substitutions in subsites +1 and +2. The SAXS rigid model of the modular arrangement of RmXyn10A displays the catalytic module close to the cell-anchoring domain while the carbohydrate binding modules are further away, likely explaining the observed lack of contribution of the CBMs to activity.

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