Glucomannan

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.

NaCl and tryptone were purchased from Biofroxx (Heidelberg, Germany). Yeast extract, glucose, and agar were supplied from Panreac AppliChem (Barcelona, Spain). M9 salts and carboxymethylcellulose were provided from Sigma-Aldrich (Darmstadt, Germany). Ortho-nitrophenyl-β-D-galactopyranoside (ONP-β-gal) and para-nitrophenyl-α-D-galactopyranoside (PNP-α-gal) used for testing the enzymatic activities were purchased from Biosynth (Staad, Switzerland). Cellic C-Tec2 (a commercial cocktail consisting of a blend of cellulases, β-glucosidases, and hemicellulases) was kindly provided from Novozymes A/S (Bagsværd, Denmark). The kit for L-lactic acid detection (L-lactic acid, L-lactate) assay kit (K-LATE) and xylan beechwood, wheat arabinoxylan, and glucomannan (konjac) were purchased from Megazyme (Bray, Irland). The citrus waste which is a by-product of fruit juice manufacturing was gifted from Ortogel SpA, Sicily, Italy.

The chemicals and reagents used in this study were analytical grades. Xyloglucan (tamarind seed), β-glucan (barley), glucomannan (konjac), lichenan (lichen of Iceland), arabinoxylan (wheat) were purchased from Megazyme (Bray, Ireland); laminarin (laminaria), xylan (birch), pectin, carboxymethylcellulose sodium (CMC-Na) and Avicel were purchased from Sigma Aldrich (Shanghai, China); soluble starch (potato) and chitin were obtained from Sangon (Shanghai, China); and phosphoric acid-swollen cellulose (PASC) was prepared from Avicel according to the protocol described by Zhang [61 (link)] et al. Whatman filter paper was provided from Maidstone, UK.
Corn bran was collected from Nanyang, Henan Province, China. Destarched corn bran was prepared by amylase and papain treatment according to the method used by Rose and Inglett [62 (link)] with modifications. Amylase and papain were purchased from Imperial Jade Bio-Technology Co., Ltd. (Ningxia, China). Apple pomace was purchased from Yuanzhi Biotechnology Co., Ltd. (Shaanxi, China).
Endoglucanase 1 (EG1) from Volvariella volvacea and GH10 xylanase from Eupenicillium parvum 4–14 were expressed in Pichia pastoris KM71H according to previous methods [51 (link),63 (link)], and cellobiohydrolase 1 (CBH1) from Hypocrea jecorina was purchased from Sigma-Aldrich (Shanghai, China).

Sodium acetate, hydrogen peroxide (H₂O₂), sodium hydroxide (NaOH), sodium borohydride (NaBH₄), iron(III) sulfate pentahydrate (Fe₂(SO₄)₃), and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, MO). Amylopectin was obtained from Carbosynth (Compton, UK). Amylose, xyloglucan, arabinoxylan, xylan, glucomannan, galactomannan, mannan, curdlan, galactan, β-glucan, arabinan, and lichenan were purchased from Megazyme (Bray, Ireland). Microcrystalline cellulose was purchased from ACROS Organics. Formic Acid (FA) was purchased from Fisher Scientific (Belgium, UK). Acetonitrile (HPLC grade) was purchased from Honeywell (Muskegon, MI). Nano-pure water was used for all experiments.

Mannobiose (M₂), mannotriose (M₃), mannotetraose (M₄), mannopentaose (M₅), mannohexaose (M₆), glucomannan, galactomannan, and galactose-free β-mannan (prepared from carob galactomannan with removal of all α-galactosyl residues by α-galactosidase) were purchased from Megazyme International (Bray, Ireland). Microcrystalline cellulose (MCC; Funakoshi, Tokyo, Japan), carboxymethylcellulose (CMC; Hercules Inc., Wilmington, DE), xylan from beech wood (Sigma), and chitin (Wako Pure Chemical Industries, Osaka, Japan) served as the carbon sources in cultures and in enzymatic assays.

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).

Bacterial stocks, previously stored at −80°C, were struck onto Brain-heart infusion (BHI; Becton Dickinson) agar plates supplemented with 10% (vol:vol) horse blood. Plates were incubated in an anaerobic growth chamber (Coy Laboratory Products; atmosphere 3% hydrogen, 20% CO₂, and 77% N₂). Single colonies were picked and grown overnight on a defined Bacteroides minimal medium (BMM) (McNulty et al., 2013 (link)) containing 5 mg/mL d-glucose. Bacteria were then diluted 1:500 (vol:vol) into BMM supplemented with a carbon source at a final concentration of 0.5% (wt:wt) and distributed into the wells of a 96-well half-area plate (Costar; Cat. No.: 3696). Plates were sealed with an optically clear membrane (Axygen; Cat. No.: UC500) and growth at 37°C was monitored by measuring optical density at 600 nm every 15 min (Biotek Eon instrument with a BioStack 4). Carbon sources tested include d-glucose, PFABN, SBABN, and glucomannan (Megazyme). All conditions were tested in quadruplicate. Readings obtained from control wells inoculated with bacteria but lacking a carbon source were averaged and subtracted from data obtained from carbon-supplemented cultures to generate background subtracted OD₆₀₀ growth curves.