Sugar beet arabinan

Linear arabinan, debranched arabinan, sugar beet arabinan, 1,4-β-D-mannan and galactan were purchased from Megazyme (Wicklow, Ireland). pNPX and pNPAF were purchased from Sigma Chemical Co., Ltd. (St. louis, USA).

Arabinose, pNP and 4-nitrophenyl-α-l-arabinofuranoside (pNP-Araf) were purchased from Sigma-Aldrich, St. Louis, MO. Wheat flour arabinoxylan, sugar beet arabinan and AXOS were purchased from Megazyme, Co. Wicklow, IE.
For the thermotolerance assay, XacAbf51 was incubated at 50 °C for up to 72 h and samples were collected to measure activity against pNP-Araf in McIlvaine buffer (pH 5.5) at 50 °C, for 10 min, using 0.5 µg mL⁻¹ (9 nM) enzyme and 10 mM substrate. The generation of p-nitrophenolate from pNP-conjugated monosaccharides was monitored at A_400nm (ε_{400nm, pH 12} = 17,500 mol⁻¹ L cm⁻¹). Activity against arabinoxylan at 10 mg mL⁻¹ was measured in McIlvaine buffer (pH 5.5) at 50 °C, for 10 min, using 15 µg mL⁻¹XacAbf51 (263 nM) or 16 µg mL⁻¹ (278 nM) TxAbfD3 and the generation of Arabinose from polysaccharides was determined by the 3,5-dinitrosalicylic acid (DNS) method [45 (link)]. To determine the kinetic properties of XacAbf51 and TxAbfD3, the reactions were performed in McIlvaine buffer (pH 5.5) at 50 °C, for 10 min, in the range from 7 µM to 14 mM of pNP-Araf using 0.5 µg mL⁻¹ (9 nM) XacAbf51 or 0.1 µg mL⁻¹ (2 nM) TxAbfD3 and from 0.3 to 140 mg mL⁻¹ of arabinan using 263 nM XacAbf51 or 278 nM TxAbfD3. The kinetic parameters were calculated by non-linear regression analysis of the Michaelis–Menten plot using the program OriginPro 8.1.

Alginate (A7003), medium viscosity alginate (A2033), low viscosity alginate (A2158), fucoidan (F5631), laminaran (L9634), gum Arabic (G9752), oat spelt xylan, and polygalacturonic acid (P3889) were obtained from Sigma-Aldrich. A sodium alginate from Laminaria hyperborea stipes was kindly provided by DuPont (Landerneau, France). High viscosity alginate (02154723, MP Biochemicals) and very low viscosity alginate (A18565, Alfa Aesar) were kindly provided by Dr Richard Blackburn (University of Leeds). Tamarind xyloglucan, potato galactan, guar galactomannan, sugar beet arabinan and citrus pectin were obtained from Megazyme International (Bray, Ireland).
The MM- and GG-blocks of alginates were prepared using the DuPont sodium alginate according to Heyraud et al. (1996) (link). A 0.5% alginate solution was hydrolyzed for 5 h at 100 °C in 0.3 M HCl. After selective precipitation and centrifugation, the blocks were dialyzed, freeze-dried, and re-suspended in distilled water, and respective M/G compositions monitored by NMR. The MM- and GG-blocks were further fragmented using alginate M-lyase (Lundqvist et al., 2012 (link)) and alginate G-lyase (Thomas et al., 2013 (link)), respectively. The enzymatic degradation and the purification of the released oligosaccharides was performed as described previously (Thomas et al., 2013 (link)).

Unless otherwise stated, all chemicals were of analytical grade and purchased from Sigma–Aldrich (St. Louis, MO, USA). Restriction enzymes, Taq DNA polymerase and their corresponding buffers were obtained from New England Biolabs (Ipswich, MA, USA). Oligonucleotides were synthesized by Eurogentec (Angers, France). Avicel PH-101 (Ref. 11365) and birchwood xylan (BWX) (Ref. X0502) were purchased from Sigma Aldrich. Cellohexaose, sugar beet arabinan, barley mixed-linkage glucan and wheat arabinoxylan were purchased from Megazyme (Wicklow, Ireland). Wheat straw (Apache variety), harvested in 2007 in southern France, was obtained from ARD (Pomacle, France) and milled to 0.5 mm as previously described [29 (link)] and cellulose nanocrystals from cotton linters (in a 2% w/w aqueous suspension) were kindly prepared by Laurent Heux (CERMAV, Grenoble, France) using an established method [30 (link)]. The resulting nanoparticles displayed lath form (200 ± 60 nm, length, 20 ± 10 nm, width and 7 nm height).