Bovine liver catalase

PAH produced after transfection was collected by lysing the cells with RIPA lysis buffer (ThermoFisher) and incubating for 30 s at room temperature. Fresh lysates were placed on ice until use. For enzyme analysis, 200 μl of cell lysate were incubated for 5 min at room temperature with 80 μl of HEPES buffer (30 mM), 35 μl bovine liver catalase (20 mg/ml; Sigma-Aldrich, St. Louis, MO, United States) and 35 μl L-phenylalanine (10 mM; Sigma-Aldrich). Then, 70 μl ammonium iron (II) sulfate hexahydrate (100 μM; Sigma-Aldrich) and 250 μl HEPES (30 mM) were added and incubated for 1 min at room temperature. Finally, 26.4 μl of tetrahydro-L-Biopterin (BH₄) (5 mg/ml; Cayman Chemical Company, Ann Arbor, MI, United States) dissolved in dithiothreitol (2 mM; Applichem Inc., Council Bluffs, IA, United States) were added to the reaction mixture, and the mixture was incubated for 3 h at 30°C with agitation (300 rpm). Samples were collected and stored at −80°C for further analysis. Final concentrations of reagents in the reaction mix were: 1 mg/ml catalase, 0.5 mM L-Phe, 10 μM ammonium iron (II) sulfate hexahydrate and 600 μM BH₄.

Exponential-phase bacterial cells were centrifuged (6,000 × g, 15 min), resuspended in MRS medium containing 0, 5, 10, 15, and 30 mM H₂O₂, and incubated at 37°C. After 1 h, H₂O₂ was eliminated using bovine liver catalase (10 U/ml; Sigma-Aldrich, MO, USA), and viable cells were counted by plating dilutions on MRS medium.

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.