Xylo oligosaccharides

Glucose, xylose, oat spelt xylan (OSX), and beech wood xylan were purchased from Sigma (St. Louis, USA) while cellobiose and maltose were purchased from BioShop Inc. (Ontario, Canada). Cello-oligosaccharides and xylo-oligosaccharides (XOS) were purchased from Megazyme (Wicklow, Ireland), and mixed XOS (DP-2–7, 95% pure) were obtained from Cascade Analytical Reagents and Biochemicals (Oregon, USA). Wheat bran hemicellulose and propoxylated wheat bran hemicellulose were kindly provided by Prof. Yaman Boluk (University of Alberta, Canada). Gluconic acid was obtained from Thermo Fisher Scientific (Massachusetts, USA). Aspergillus niger Glucose oxidase (Cat. no. G0543, with ≤0.1 units/mg protein catalase) and bovine liver catalase were purchased from Sigma (St. Louis, USA).
To prepare insoluble OSX, 2 g OSX was suspended in 200 mL of 50 mM Tris-HCl pH 8.0 for 48 h at room temperature and then washed three times. The washed OSX was then filtered through a 0.45-μm nylon membrane, and the dry weight of the retentate was measured.

For monitoring xylan degradation in vitro, 1 mL sterile-filtered cultivation supernatant from 72 h growth in MA medium containing 1% xylan (Lenzing AG) was mixed with 9 mL sterile MA medium containing 1% xylan (Lenzing AG) and incubated in a 50 mL sterile reaction tube on a rotary shaker (180 rpm) at 30°C. 200 µL samples were drawn at given time points and the enzymatic reactions immediately stopped by addition of 200 µL 100 mM NaOH. The remaining, undissolved xylan was removed by centrifugation at 4°C and 20,000g for 10 min. Samples were diluted 1:10 with 10 mM NaOH for HPAEC-PAD analysis. HPAEC-PAD analysis was performed with a Thermo Scientific Dionex ICS-5000 system (Thermo Scientific). Samples were separated on a Dionex CarboPac PA1 (2 × 250 mm) carbohydrate column (Thermo Scientific). The column was preconditioned at 0.26 mL/min in 150 mM NaOH/250 mM NaOAc for 5 min, followed by 150 mM NaOH for 15 min. Samples were then injected and oligosaccharides separated by a linear gradient to 150 mM NaOH/250 mM NaOAc in 20 min. Quantification was done by calibration of peak areas with authentic standards of xylooligosaccharides (Megazyme International, Ireland).

The hydrolysis of oat spelt xylan, birchwood xylan, beechwood xylan (1%, w/v) and xylo-oligosaccharides (1 mg/mL) was performed at the optimal pH of each enzyme.
The reaction mixture was incubated for 12 h at 45 °C in a water bath, and subsequently, the enzyme reaction was terminated after boiling for 10 min and freezing. All products were freeze-dried and redissolved in a methanol (50%). A total of 6 μL of each aliquot was spotted onto a TLC plate (Qingdao Haiyang Chemical Plant, Qingdao, China), and the plates were developed with a solvent system consisting of chloroform-acetic acid-water (3:6:1, v/v). The plates were sprayed with a staining solution containing a 9:1 (v/v) mixture of methanol and sulfuric acid with 0.2% orcinol and heated at 85 °C for 5–10 min. Xylose (X1) and xylo-oligosaccharides (Megazyme, Ireland) (xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6)) were used as standards.

Rice straw was ground with a coffee grinder. Rice straw powder at 10% (w/v) was suspended in 2% NaOH and heated at 121 °C for 20 min. Aliquots of the suspension were adjusted at different pH (7.0, 9.0 and 10.5) with HCl.
Xylanases activity was assayed using in tubes containing 500 μL of rice straw, at different pH, to which 50 μL of enzyme solution (0.15 units) were added. One unit is defined as 1 μmol of reducing sugars min⁻¹ mg⁻¹ from oat-spelt xylan, at pH 7.0 and 65 °C. Enzyme treatments were carried out at two temperatures: 65 and 90 °C, during 24 h. After this time, the tubes were centrifuged and 20 μL of 1:10 dilution of the supernatant were used to determine reducing sugars as described above. Analysis of soluble sugars released by the enzymatic treatment was carried out by ion exchange chromatography using a Dionex (Thermo Fisher Scientific) instrument equipped with CarbonPac PA100 column and a pulsed amperometric detector (Dionex Thermo Fisher Scientific). Xylose (Sigma-Aldrich) and xylooligosaccharides, from two to six units (Megazyme) were used as chromatographic standards (Additional file 5: Figure S2).

Wheat AX (medium viscosity, 31 centistokes) was obtained from Megazyme, CMC from Nacalai Tesque and soluble starch from Sigma-Aldrich. Monosaccharides d-xylose and d-glucose were purchased from Nacalai Tesque. Xylulose and l-arabinose were purchased from Sigma-Aldrich. Xylo-oligosaccharides (xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose; X1–X6) as standards for TLC were purchased from Megazyme and Silica Gel 60 F254 TLC plates (5 cm × 10 cm) were obtained from Merck. Colorimetric detection reagent, orcinol monohydrate, was purchased from Sigma-Aldrich. α-Cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) were purchased from Shimadzu GLC. Angiotensin II, 3-aminoquinoline (3-AQ), and N-acetyl-renin substrate were purchased from Sigma-Aldrich. Ammonium dihydrogen phosphate was purchased from Merck Millipore. Trifluoroacetic acid (TFA) was purchased from Wako Pure Chemical Industries.

Sarocladium strictum type strain CBS 346.70 was obtained from the American Type Culture Collection no. 34717. Glucose, xylose, cellobiose, oat spelt xylan, and beechwood xylan were purchased from Sigma (St. Louis, USA), while other cello-oligosaccharides as well as xylo-oligosaccharides were purchased from Megazyme (Wicklow, Ireland). Propoxylated wheat bran hemicellulose and Thermobifida fusca xylanase 11A [27] (link) were kindly provided by Prof. Yaman Boluk (University of Alberta, Canada) and Prof. David Wilson (Cornell University, USA), respectively.

B. intestinalis DSM 1739323 was obtained from the Deutsche Sammlung von Mikrooganismen und Zellkulturen or German Collection of Microorganisms and cell Cultures (DSMZ, Braunschweig, Germany). The NEB Turbo chemical competent Escherichia coli cells and Q5 High-Fidelity 2X Master Mix (used for PCR) were purchased from New England Biolabs (Ipswich, MA). The E. coli BL21-CodonPlus (DE3)-RIL competent cells were obtained from Agilent (Santa Clara, CA). The pET-46 Ek/LIC vector was purchased from Novagen (San Diego, CA). The QIAprep Spin Miniprep kit was obtained from Qiagen, Inc. (Valencia, CA), and the Talon Metal Affinity resin was obtained from Clontech Laboratories, Inc. (Mountain View, CA). Amicon Ultra-15 centrifugal filter units with 10 kDa, 30 kDa, and 50 kDa molecular mass cutoffs (MMCOs) were obtained from Millipore (Billerica, MA). Soluble wheat arabinoxylan with medium viscosity (WAX), rye
arabinoxylan (RAX) and xylo-oligosaccharides were obtained from Megazyme (Bray, Ireland), and oat spelt xylan (OSX) was purchased from Sigma-Aldrich (St. Louis, MO).