β glucan

D-Cellooligosaccharides [D-cellobiose (C₂) to D-cellohexaose (C₆)], β-glucan from barley (low viscosity), curdlan from Alcaligenes faecalis, xyloglucan, and glucomannan were purchased from Megazyme International Ireland Ltd. (Wicklow, Ireland). All other materials including D-glucose (C₁), sodium carboxymethylcellulose (CMC), Avicel PH-101, chitosan, beechwood xylan, locust bean gum, para-nitrophenyl (pNP)-glucopyranoside, and pNP-cellobioside were provided by Sigma-Aldrich (St. Louis, MO, United States).

Polysaccharide-binding assays were performed as described by Bleackley et al. [31 (link)]. All experiments were performed in triplicate. In brief, the test defensins were added to increasing amounts of insoluble polysaccharide yeast β-glucan (Megazyme, Sydney, Australia) or chitin from shrimp cells (Sigma Aldrich, St. Louis, MI, USA), and the mixture was incubated on a rotator wheel at room temperature for 30 min before centrifugation at 17,000× g to pellet the insoluble polysaccharide. The supernatant was then removed, and 25 µL of this supernatant was analysed using SDS-PAGE. The test defensins (unbound) were visualised with RAPIDstain (Calbiochem, Victoria, Australia) as per the manufacturer’s protocol and imaged with Gel Doc (BioRad, Hercules, CA, USA).

Cellulose nanofibers (CNF) were prepared from never-dried bleached kraft birch pulp by fluidization in deionized water six times (six passes) using a Voith LR40 homogenizer, as described by Österberg et al. (53 (link)). The resulting CNF samples with a solid content of 2.8% (wt/vol) were stored at 4°C. Avicel PH-101 (~50-μm particle size; catalog no. 11365), xylan from birch wood (catalog no. 95588), Whatman qualitative filter paper grade 1 (catalog no. WHA1001185), Whatman qualitative filter paper grade 3 (catalog no. WHA1003185), and peptidoglycan from Micrococcus luteus (catalog no. 53243) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chitin from shrimp shells (catalog no. P-CHITN), glucomannan (Konjac; low viscosity; catalog no. P-GLCML), carboxymethyl cellulose 4M (CMC; catalog no. P-CMC4M), xyloglucan (tamarind; catalog no. P-XYGLN), hexaacetyl-chitohexaose (catalog no. O-CHI6), cellohexaose (catalog no. O-CHE), and β-glucan (yeast; alkali soluble; catalog no. P-BGYST) were obtained from Megazyme (Bray, Ireland). Thermo Fisher Scientific (Waltham, MA, USA) was the supplier of the Pierce bicinchoninic acid (BCA) protein assay kit (catalog no. 23225). All other chemicals were reagent grade.

To prepare protein samples, each metagenomic clone was cultivated overnight at 37 °C in 40 mL LB-Cm and then centrifuged at 5,000 × g for 15 min at 4 °C, in order to obtain 50-fold concentrated fractions. Supernatants were transferred under sterile conditions in a Spin-X UF 10 K concentrator (manufacturer’s protocol, Corning B.V., Amsterdam) and pellets were resuspended in 800 μL of sterile 50 mM NaPi buffer. Mechanical cell lysis was performed on pellets, after adding glass beads (Sigma Aldrich, Saint-Quentin-Fallavier, France), at 40 s at 5 m.sec⁻¹ with a FastPrep^®−24 (MP-Biomedicals, NY, USA).
Agar-well plate enzymatic assays were performed to detect endoglucanase, xylanase, β-glucanase, lichenase and xyloglucanase activities of 50-fold concentrated extracellular or cell-associated proteins. Samples were loaded (80 μL/well) in Omnitray plates containing 45 mL of LB-NaPi supplemented with 0.5% CMC, OS xylan, β-glucan, lichenan or xyloglucan (from Megazyme, Wicklow, Irlande), 12.5 μg/μL Cm, 0.5 mM MgCl₂ and 0.5 mM CaCl₂. The plates were incubated for 24 hours at 37 °C and stained with Congo red as described for the functional screening. Clear halos revealed substrate degradation around wells containing polysaccharidic enzymes. Here again, intensity of the halo comparable to those of the EC100 E. coli/pEpiFOS-5 control is considered as negative.