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-beta-D-xylanase) and two different alpha-L-arabinofuranosidases (AXH-m and AXH-d3) were used stepwise to manufacture arabinoxylo-oligosaccharides (AXOS) with alpha-L-Araf (1-->2)-monosubstituted beta-D-Xylp residues or alpha-L-Araf (1-->2)- and (1-->3) doubly substituted beta-D-Xylp 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: alpha-L-Araf-(1-->2)-[alpha-L-Araf-(1-->3)]-beta-D-Xylp-(1-->4)-beta-D-Xylp-(1-->4)-D-Xylp, d-II: alpha-L-Araf-(1-->2)-[alpha-L-Araf-(1-->3)]-beta-D-Xylp-(1-->4)-D-Xylp, m-I: alpha-L-Araf-(1-->2)-beta-D-Xylp-(1-->4)-beta-D-Xylp-(1-->4)-D-Xylp and m-II: alpha-L-Araf-(1-->2)-beta-D-Xylp-(1-->4)-D-Xylp. To our knowledge, this is the first report on structural (1)H and (13)C 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 alpha-L-Araf (1-->2)-substituted AXOS could also be prepared in similar yields by treating the doubly substituted AXOS further with AXH-d3.