Aspergillus niger glucose oxidase

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.

In order to investigate the capacity of the different mutants to produce or consume NO, cell cultures at an initial OD600 of about 0.25 were incubated microoxically with KNO₃ as the sole nitrogen source for 24 h, harvested by centrifugation, washed twice with 25 mM Na₂HPO₄/NaH₂PO4 buffer (pH 7.4), and resuspended in 1.5 ml of the same buffer. NO production and consumption activities were determined by using an ISONOP NO electrode APOLLO 4000^®(World Precision Instruments). The reaction chamber (2 ml) was temperature-controlled, magnetically stirred and contained: 1410 μl of 25 mM Na₂HPO₄/NaH₂PO₄ buffer (pH 7.4) and 250 μl of cell suspension (0.4–0.7 mg protein) for NO production or 760 μl of 25 mM Na₂HPO₄/NaH₂PO₄ buffer (pH 7.4) and 900 μl of cell suspension (1.5–2.5 mg protein) for NO consumption. To generate an anoxic atmosphere, 100 μl of an enzymatic mix containing Aspergillus niger glucose oxidase (40 units⋅ml⁻¹), bovine liver catalase (250 units⋅ml⁻¹) (Sigma-Aldrich), 90 μl of 1 M sodium succinate and 100 μl of 320 mM glucose were added to the chamber. Once a steady base line was obtained, 50 μl of 50 mM NaNO₂ (NO production) or 50 μl of 2 mM NO (NO consumption) was added to the chamber to start the reaction.