Beech xylan

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.

Assays to evaluate the ability of Bo_M to bind to insoluble polysaccharides were performed on the insoluble fractions of ivory nut mannan (Carbosynth), cellulose (Merck), birch xylan (Merck), beech xylan (Merck), mixed-linkage barley glucan (Megazyme) and potato starch (Merck). 2.5% w/v solutions of insoluble polysaccharides, suspended in 50 mM tris(hydroxymethyl)aminomethane pH 8.0, 250 mM NaCl, 5% glycerol, were incubated with Bo_M (0.1 g/L) at 37 °C for 30 min (incubation step sample). The insoluble polysaccharides were collected by centrifugation (14,000 rpm, 5 min) before being washed with buffer and incubated for an additional 10 min (wash sample). The insoluble polysaccharides were collected by centrifugation once more and washed with 8 M urea to solubilize any bound protein (elution sample). SDS-PAGE and ImageLab (Bio-Rad) were used to visualize Bo_M and quantify its concentration. BSA was used as control and, similar to previous reports [21 (link)], did not bind to any of the tested substrates.