The binding abilities of CBMs for soluble polysaccharides were investigated through affinity electrophoresis according to an established method [46 (link)]. In brief, five micrograms of the recombinant CBMs as well as bovine albumin (BSA) were respectively mixed with loading buffer and then used for non-denaturing polyacrylamide gel electrophoresis (PAGE) in a 10% gel. Soluble polysaccharides (arabinoxylan, glucuronoxylan, carboxymethyl xylan, arabinan or arabinogalactan) were added to a final concentration of 0.1% (w/v) when making separating gels. The electrophoresis was carried out in ice bath for 2 h with a voltage of 150 V, followed by Coomassie blue staining. The relative mobility (r) of CBM (distance migrated by CBM band divided by the distance migrated by dye) was calculated to show the CBM affinity for soluble polysaccharides. To determine the Kd of CrCBM13 and CrCBM2, the affinity electrophoresis was carried out using the gels containing arabinoxylan or glucuronoxylan with a gradient concentration (0/0.1/0.2/0.4/0.8 g/L for arabinoxylan and 0/0.75/1.25/2.5/5 g/L for glucuronoxylan). The plots of 1/r versus ligand concentration were then used to determine of Kd by regression analysis (Additional file 1 ), and the unit of Kd was finally converted from g/L to μM according to the average molecular weights of arabinoxylan and glucuronoxylan.
To investigate the affinity of CBMs for insoluble substrates, the microcrystalline cellulose, corncob xylan, delignified corncob or Carolina poplar were ground to the powders with particle sizes below 75# mesh. The CBMs and substrates were then dispersed in 1 mL of Na2HPO4-NaH2PO4 buffer (100 mM, pH 7.0) to a final concentration of 0.5 mg/mL and 10 mg/mL respectively in a 2 mL low-binding tube (Eppendorf, Germany). Subsequently, the tube was incubated in a shaker at 37 °C with a rotational speed of 200 rpm for 1 h. The supernatant was then collected through centrifugation, followed by the determination of protein concentration using Bradford assay. The concentration of the protein solution without any substrate is defined as 100%.
To investigate the affinity of CBMs for insoluble substrates, the microcrystalline cellulose, corncob xylan, delignified corncob or Carolina poplar were ground to the powders with particle sizes below 75# mesh. The CBMs and substrates were then dispersed in 1 mL of Na2HPO4-NaH2PO4 buffer (100 mM, pH 7.0) to a final concentration of 0.5 mg/mL and 10 mg/mL respectively in a 2 mL low-binding tube (Eppendorf, Germany). Subsequently, the tube was incubated in a shaker at 37 °C with a rotational speed of 200 rpm for 1 h. The supernatant was then collected through centrifugation, followed by the determination of protein concentration using Bradford assay. The concentration of the protein solution without any substrate is defined as 100%.
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