Step-wise enzymatic preparation and structural characterization of singly and doubly substituted arabinoxylo-oligosaccharides with non-reducing end terminal branches

Shearzyme (GH10 endo-1,4-β- d-xylanase) and two different α- l-arabinofuranosidases (AXH-m and AXH-d3) were used stepwise to manufacture arabinoxylo-oligosaccharides (AXOS) with α- l-Ara f (1→2)-monosubstituted β- d-Xyl p residues or α- l-Ara f (1→2)- and (1→3) doubly substituted β- d-Xyl p residues from wheat arabinoxylan (AX) in a rather straightforward way. Four major AXOS (d-I, d-II, m-I and m-II) were formed in two separate hydrolyses. The AXOS were purified and the structures were confirmed using TLC, HPAEC-PAD, MALDI-TOF-MS and 1D and 2D NMR spectroscopy. The samples were identified as d-I: α- l-Ara f-(1→2)-[α- l-Ara f-(1→3)]-β- d-Xyl p-(1→4)-β- d-Xyl p-(1→4)- d-Xyl p, d-II: α- l-Ara f-(1→2)-[α- l-Ara f-(1→3)]-β- d-Xyl p-(1→4)- d-Xyl p, m-I: α- l-Ara f-(1→2)-β- d-Xyl p-(1→4)-β- d-Xyl p-(1→4)- d-Xyl p and m-II: α- l-Ara f-(1→2)-β- d-Xyl p-(1→4)- d-Xyl p. To our knowledge, this is the first report on structural 1H and 13C NMR analysis of xylobiose-derived AXOS d-II and m-II. The latter compound has not been reported previously. The doubly substituted AXOS were produced for the first time in good yields, as d-I and d-II corresponded to 11.8 and 5.6 wt% of AX, respectively. Singly α- l-Ara f (1→2)-substituted AXOS could also be prepared in similar yields by treating the doubly substituted AXOS further with AXH-d3.