Beechwood xylan

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.

Conidia were inoculated at a concentration equal to 10⁶ conidia per ml in 3 ml Vogel's media (Vogel, 1956 ) with 2% wt/vol powdered Miscanthus giganteus (Energy Bioscience Institute, UC-Berkeley), Avicel PH 101 (Sigma-Aldrich, St. Louis, MO), beechwood xylan (Sigma-Aldrich), or pectin (Sigma-Aldrich) in a 24-well deep-well plate. The plate was sealed with Corning breathable sealing tape and incubated at 25°C in constant light and with shaking (200 rpm). Images were taken at 48 hr. Culture supernatants were diluted 200 times with 0.1 M NaOH before Dionex high-performance anion exchange chromatographic (HPAEC) analysis, as described below. N. crassa growth on xylan was also determined by measuring N. crassa biomass accumulation. N. crassa grown on xylan for 3 days was harvested by filtration over a Whatman glass microfiber filter (GF/F) on a Büchner funnel and washed with 50 ml water. Biomass was then collected from the filter, dried in a 70°C oven, and weighed.

Strain A. fumigatus Af293 was obtained from the Fungal Genetics Stock Center, which genome has been published in 2005. Escherichia coli Trans1-T1 (TransGen Biotech, Beijing, China) was cultivated in Luria-Bertani (LB) medium at 37°C for gene cloning and sequencing. P. pastoris GS115 (Invitrogen, Carlsbad, CA) cultivated in yeast peptone dextrose (YPD) medium at 30°C was used for gene expression. The plasmids pGEM-T Easy (Promega, Madison, WI) and pPIC9 (Invitrogen) were used as cloning and expression vectors, respectively. Beechwood xylan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and locust bean gum were purchased from Sigma-Aldrich (St. Louis, MO). Soluble wheat arabinoxylan was obtained from Megazyme (Wicklow, Ireland). The DNA purification kit, LA Taq DNA polymerase and restriction endonucleases were purchased from TaKaRa (Otsu, Japan). SV Total RNA Isolation System was purchased from Promega.T4 DNA ligase was from New England Biolabs (Hitchin, UK). All chemicals were of analytical grade and commercially available.

The xylanase activity was assayed by measuring the amount of reducing sugars produced from beechwood xylan (Sigma) by the ferricyanide method (Friedemann et al. 1962 (link)) using xylose as the standard. The reaction mixture consisted of 1% xylan in 75 mM of universal buffer, pH 7.0. Typically, the reaction mixture consisted of 20 μl of the enzyme and 300 μl of the xylan solution. The mixture was incubated at 40 °C for 10 min. One unit of xylanase activity was defined as the amount of enzyme that forms reducing groups corresponding to 1 μmol of xylose in 1 min under the above conditions. Reactions with other substrates were done in the same conditions, but with replacement of xylan by 1% CM-cellolose or microcrystalline cellulose or barley β-glucan. The protein concentration was determined using the molar absorption coefficient at 280 nm calculated from the protein sequence.
Characterization of properties of xylanases was performed as previously described (Lisov et al. 2014 (link)).
The molecular weight of purified proteins was determined by SDS-PAGE using 12% gel according to Laemmly (1970 (link)). The standard proteins were as follows: beta-galactosidase (116 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35 kDa), REase Bsp98I (25 kDa), beta-lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa).

Phosphoric acid swollen cellulose (PASC) was prepared as follows. 5 g of Avicel^® PH-101 was moistened with water and treated with 150 mL ice cold 85% phosphoric acid, stirred on an ice bath for 1 h. Then 500 mL cold acetone was added while stirring. The swollen cellulose was filtered on a glass-filter funnel and washed 3 times with 100 mL ice cold acetone and subsequently twice with 500 mL water. PASC was then suspended in 500 mL water and blended to homogeneity.
High-purity pachyman (β-d-1,3-glucan), barley β-glucan (β-d-1,3-1,4-glucan), lichenan (from Icelandic moss, β-d-1,3-1,4-glucan), mannan (borohydride reduced), konjac glucomannan (β-d-1,4), carob galactomannan, larch arabinogalactan, wheat arabinoxylan, cellotriose, cellotetraose, cellopentaose, cellohexaose, mannobiose, and xylobiose were purchased from Megazyme. Locust bean gum, carboxymethyl cellulose (CMC), beechwood xylan, and cellobiose were purchased from Sigma. 5-bromo-4-chloro-3-indolyl-β-d-cellobioside was purchased from Santa Cruz Biotechnology.

The substrate specificity of the purified recombinant enzymes was investigated at the optimal temperatures at a pH of 5.0 in the following substrates (1%, w/v): oat spelt xylan (Sigma, USA), birchwood xylan (Sigma, USA), beechwood xylan (Sigma, USA), sodium carboxymethyl cellulose (CMC-Na, Sigma, USA), locust bean gum (Aladdin, Shanghai, China), konjac mannan (Aladdin, Shanghai, China), and guar gum (Aladdin, Shanghai, China). The enzyme activity was quantified using the DNS method. The specific activities against four artificial substrates (10 mM) containing p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenyl-α-L-arabinofuranoside (pNPA), p-nitrophenyl-β-D-xylopyranoside (pNPX), and p-nitrophenyl β-D-cellobioside (pNPC) were determined using the method of Parry et al.46 (link). The kinetic constants of the four recombinant enzymes were determined after measuring the initial rates at various concentrations of various xylans (oat spelt xylan, birchwood xylan, and beechwood xylan, 1 to 20 mg/mL) at 50 °C at an optimal pH for 10 min. The Michaelis-Menten constant (K_m) and the maximum velocity (V_max) were calculated using Lineweaver-Burk plots.

Caldicellulosiruptor owensensis was purchased as free-dried culture from the German Collection of Microorganisms and Cell Cultures (DSMZ), DSM number is 13100. Escherichia coli Top10 and BL21(DE3) were respectively used for gene cloning and expression. The plasmid, pET-28b(+) (Novagen, USA), was used for recombinant plasmid construction and gene expression. Beechwood xylan and 4-nitrophenyl β-D-xylopyranoside (pNPX) were purchased from Sigma-Aldrich (St louis, MO, USA). All of the other chemicals were of analytical grade and purchased from Sinopharm Chemical Regent Co., Ltd. C.owensensis cells were resuspended and subcultured in anaerobic modified DSMZ medium 640 (cellobiose was replaced by xylose), and grown at 75°C with orbital shaking (75 rpm). E.coli Top10 and BL21(DE3) were cultured in aerobic Luria–Bertani (LB) broth at 37°C with orbital shaking (220 rpm).