Xylotriose

Mannobiose, mannotriose, mannotetraose, mannopentaose and mannohexaose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose were from Megazyme (Ireland). Mannose, xylose, 2-picoline borane, 2-aminobenzamide, ethyl acetate, methanol and ammonium formate were purchased from Sigma-Aldrich (Germany).
Acetylated galactoglucomannan (AcGGM) from Norway spruce (Picea abies) was produced in house from dried wood chips^{79 (link)}. A simplified (lower DP range) version of this substrate, named GH26-AcGGM was produced by treating the AcGGM with a β-mannanase (R. intestinalis β-mannanase RiGH26).
Acetylated (arabino)glucuronoxylan (AcAGX) was produced in house from birch (Betula pubescens) chips^{80 (link)}. A simplified (lower DP range and deacetylated) version of this substrate, named GH10-AGX was produced by treating the AcAGX with sodium hydroxide to remove all acetylations followed by subsequent treatment with the commercial xylanase Shearzyme (Novozymes, Denmark).

Sugarcane bagasse (SCB) was provided from a sugar factory in Iran, Khuzestan Province (haft tapeh). It was first dried at room temperature and was then ground using a laboratory mill to pass a 0.5-mm sieve and stored under darkness at room temperature. All chemicals were supplied from Sigma-Aldrich unless otherwise specified. Guluronic acid was provided from Chemos (Germany). Sulfuric acid (72%) was purchased from Thermo Fisher (Germany). Sodium hydroxide (NaOH) pellets and acetic acid (glacial) 100% were supplied by Merck (Darmstadt, Germany). Ethanol (99%) was purchased from Solveco Group (Rosenberg, Sweden). Ultrapure water (18,2 MΩ/cm) was provided through a Milli-Q instrument (Millipore, Billerica, MA, USA). Oligosaccharide standards (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose) were provided from Megazyme (Ireland). Standards for FT-IR analysis, include Wheat Arabinoxylan (Megazyme), Avicel (Sigma-Aldrich) and microcrystalline cellulose; from Cellupharm (Sweden) AB.

Xylan from birchwood, beechwood, and Larchwood, were purchased from Sigma Aldrich (Saint Louis, Missouri). Xylan from quinoa (Chenopodium quinoa) stalks was extracted as described in our previous work.^{10 (link)} Analytical grade xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose were obtained from Megazyme (Wicklow, Ireland).

Kinetics of rBxTW1 was evaluated against several synthetic and natural substrates for xylanolytic enzymes. The enzyme was assayed by using pNPX, p-NP-α-l-arabinopyranoside and p-NP-α-l-arabinofuranoside (Sigma-Aldrich). The analysis was also carried out against xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose (Megazyme) together with beechwood xylan (Sigma-Aldrich).
Kinetics for each specific substrate were obtained by using increasing substrate concentrations from 0.078 to 20 mM. The activity data were fitted by least-squares to the Lineweaver–Burk linear equation of the Michaelis–Menten model. The effect of product inhibition from xylose was also determined by evaluating the pNPX hydrolysis in the presence of 2.5, 5 and 10 mM xylose and obtaining the corresponding K_i.

The composition of specific oligosaccharides contained in the HOS preparations (modified minimal salts media) was determined using a Dionex™ ICS-6000 high-performance anion exchange chromatography (HPAEC) system (Thermo Fisher Scientific, Madison, WI) fitted with a CarboPac™ PA200 analytical column (250 mm × 3 mm) and a corresponding microbore guard column (50 mm × 3 mm). The pulsed amperometric detection (PAD) system of ICS-6000 had a AgCl reference electrode and a gold working electrode. The mobile phases were composed of solvent A: 100 mM NaOH, and solvent B: 100 mM NaOH mixed with 320 mM sodium acetate. A gradient elution method was used as follows; hold 100% solvent A for 15 min, then ramp to 50% solvent B at a linear rate for 40 min, afterward increase solvent B to 100% in 1 min and hold constant for 4 min; finally, return the mobile phase to 100% solvent A in 1 min. The eluent flow rate was 0.5 ml/min, injection volume was 10 μl, and the column temperature was 35°C. Pure standards (>95%) of cellobiose, xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose, mannobiose, mannotriose, arabinobiose, and arabinotriose, purchased from Megazyme (Wicklow, Ireland), were used to calibrate the instrument.

Fast Blue BB (FBBB) [4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi-(zinc chloride) (Rebello et al., 2016 (link)), bile extract porcine, pancreatin from porcine pancreas, pepsin from porcine gastric mucosa, α-amylase from human saliva, 2,2′-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2′-diazobis-(2-aminodinopropane)-dihydrochloride (AAPH), fluorescein, 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich, Co. (St. Louis, MO, United States). Standards of chlorogenic acid, 2-coumaric acid, 2,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, vanillic acid, and vanillin were provided by Extrasynthese (Lyon, Genay Cedex, France). Standards of 6-hydroxy-2,5,7, 8-tetramethyl-2-carboxylic acid (Trolox), gallic acid, caffeic acid, ferulic acid, vitexin, D-(+)-glucose, D-(+)-galactose, D-(+)-xylose, D-(–)-arabinose, and stachyose were acquired from Sigma-Aldrich, Co. (St. Louis, MO, United States). Standards of 3³-α-L-plus 2³-α-L-arabinofuranosyl-xylotetraose (XA³XX/XA²XX), 2³,3³-di-α-L-arabinofuranosyl-xylotriose (A^{2, 3}XX), and xylotriose were obtained from Megazyme (Wicklow, Ireland). Raffinose and cellobiose standards were supplied by Merck (Darmstadt, Germany).

The oligosaccharide profile after enzymatic hydrolysis of different substrates was investigated applying HPAEC–PAD with Dionex ICS-5000 system (Thermo Fisher Scientific) using a Dionex CarboPac PA200 (250 × 3 mm, 5.5 μm) analytical and a guard (50 × 3 mm) columns (Thermo Fisher Scientific). The separation was performed using a constant mobile phase composition of 100 mM NaOH at 0.5 mL/min, and a linear gradient from 0 to 30 min of 0–120 mM sodium acetate and thereafter a constant concentration of 160 mM sodium acetate until 40 min. AXOS standards xylobiose (X²), xylotriose (X³), xylotetraose (X⁴), xylopentaose (X⁵), xylohexaose (X⁶), 3²-α-L-arabinofuranosylxylobiose (A³X), 2³-α-L-arabinofuranosyl-xylotriose (A²XX), 3³-α-L-arabinofuranosyl-xylotetraose (XA³XX), 2³-α-L-arabinofuranosyl-xylotetraose (XA²XX) and 2³,3³-di-α-L-arabinofuranosyl-xylotriose (A²⁺³XX) were purchased from Megazyme.