Wheat arabinoxylan

Phosphoric acid swollen cellulose (PASC) was prepared as follows. 5 g of Avicel^® PH-101 was moistened with water and treated with 150 mL ice cold 85% phosphoric acid, stirred on an ice bath for 1 h. Then 500 mL cold acetone was added while stirring. The swollen cellulose was filtered on a glass-filter funnel and washed 3 times with 100 mL ice cold acetone and subsequently twice with 500 mL water. PASC was then suspended in 500 mL water and blended to homogeneity.
High-purity pachyman (β-d-1,3-glucan), barley β-glucan (β-d-1,3-1,4-glucan), lichenan (from Icelandic moss, β-d-1,3-1,4-glucan), mannan (borohydride reduced), konjac glucomannan (β-d-1,4), carob galactomannan, larch arabinogalactan, wheat arabinoxylan, cellotriose, cellotetraose, cellopentaose, cellohexaose, mannobiose, and xylobiose were purchased from Megazyme. Locust bean gum, carboxymethyl cellulose (CMC), beechwood xylan, and cellobiose were purchased from Sigma. 5-bromo-4-chloro-3-indolyl-β-d-cellobioside was purchased from Santa Cruz Biotechnology.

Penicillium oxalicum sp. 68 with the collection number CGMCC 7.328 in China General Microbiological Culture Collection Center was isolated from Chang bai mountain soil (Jilin Province, China). DH5α, E. coli BL 21 (DE3), P. pastoris GS115, pET-32a (+) and pPIC9K (Novagen, Madison, WI, USA) were used as host and expression vectors, respectively. pNP-α-l-arabinofuranoside (pNPαAraf), pNP-α-l-arabinopyranoside (pNPαArap) pNP-β-galactopyranoside (pNPβGal), pNP-α-galactopyranoside (pNPαGal), pNP-β-xylopyranoside (pNPβXyl), p-nitrophenyl-β-glucopyranoside (pNPβGlc), pNP-α-glucopyranoside (pNPαGlc) were purchased from Sigma (St. Louis, MO, USA). Sugar beet l-arabinan, linear-1,5-α-l-arabinan, and wheat arabinoxylan (low viscosity) were obtained from Megazyme International Ireland Ltd. (Wicklow, Ireland). All of other chemicals and reagents were analytical grade.

Binding studies to assess the ability of DmCE1A_CBM48 and DmCE1B_CBM48 to bind insoluble polysaccharides were performed using pull-down studies as described in Kmezik et al. (32 (link)), except 50 mM sodium phosphate buffer (pH 6.5) was used as the buffer, on the insoluble polysaccharides: ivory nut mannan (Carbosynth), cellulose (Merck), birch xylan (Merck), beech xylan (Merck), mixed-linkage barley glucan (Megazyme), and potato starch (Merck). All polysaccharides were washed three times in the aforementioned buffer before being used in the assay. Binding studies using soluble polysaccharides by affinity gel electrophoresis were performed as previously described (67 , 68 (link)) using carboxymethylcellulose, galactomannan (Megazyme), glucomannan (Megazyme), wheat arabinoxylan (Megazyme), and xyloglucan (Megazyme). All polysaccharides were used at a concentration of 0.5% w/v in the polyacrylamide gels.

Strains of B. pseudocatenulatum were cultured until they reached the exponential phase, centrifuged, and then, the resulting pellets were suspended to an OD₆₀₀ of 0.2 in modified peptone yeast extract (PY) medium (100 mM PIPES, pH 6.7, 2 g/L peptone, 2 g/L BBL trypticase peptone, 2 g/L bacto-yeast extract, 8 mg/L CaCl₂, 19.2 mg/L MgSO₄ ∙ 7H₂O, 80 mg/L NaCl, 4.9 mg/L hemin, 0.5 g/L L-cysteine hydrochloride and 100 ng/L vitamin K1). These suspension cultures were inoculated (1% vol/vol) into modified PY medium supplemented with 0.5% (wt/vol) XOS (Xylo-Oligo95P, B Food Science, Aichi, Japan) (PY-XOS), wheat arabinoxylan (Megazyme, Bray, Ireland) (PY-AX) or beechwood xylan (Sigma-Aldrich, Darmstadt, Germany) (PY-XY) and covered with sterile mineral oil (50 μL) to prevent evaporation. Growth was monitored anaerobically by measuring the OD₆₀₀ using a PowerWave 340 plate reader (BioTek, Winooski, VT, USA) every 30 min in an anaerobic chamber for 48 h. The organic acids produced in PY-XY were analysed using high-pressure liquid chromatography as described [8 (link)].