Arabinoxylan

Preparation of reagents: 10 g of 3,5-dinitrosalicylic acid plus 2 g of phenol, 0.5 g of sodium sulphite and 10 g of sodium hydroxide were dissolved in 800 mL of water. The volume was adjusted to 1 L and the solution stored in a sealed Duran bottle at room temperature (stable for >2 months). Rochelle’s salt solution was prepared by dissolving 80 g of potassium sodium tartrate in 120 mL of water and then adjusting the volume to 200 mL with water. The solution was stored in a sealed Duran bottle at room temperature and is stable for several years.

Preparation of substrate solutions: 1.0 g of beechwood xylan, birchwood xylan or wheat flour arabinoxylan was added to 90 mL of 100 mM sodium acetate buffer (pH 4.5) and dissolved by stirring at approximately 50 °C for 10 min on a magnetic stirrer hotplate. The volume was adjusted to 100 mL with 100 mM sodium acetate buffer (pH 4.5) and the solution stored in a well-sealed Duran bottle at room temperature. Substrate was also prepared in 100 mM sodium phosphate buffer (pH 6.0) using the same procedure. Two drops of toluene was added to each bottle to prevent microbial contamination.

Preparation of endo-xylanase preparations: Pure suspensions of endo-xylanase in ammonium sulphate (3.2 M) as supplied by Megazyme (see “Materials” section) were centrifuged in a microfuge at 13,000 rpm for 6 min and the supernatant solution removed with a micropipettor and discarded. The enzyme pellet was dissolved in 1 mL of either 100 mM sodium acetate buffer (pH 4.5) containing bovine serum albumin (BSA), (0.5 mg/mL) or 100 mM sodium phosphate buffer (pH 6.0) containing BSA (0.5 mg/mL), depending on the pH optima of the enzyme. This solution was then added to 9 mL of the same buffer and then further diluted in the same buffer to an enzyme concentration suitable for assay and stored on ice between use. A. niger and T. viride endo-xylanases were dissolved in acetate buffer at pH 4.5 whereas N. patriciarum and C. mixtus xylanases were dissolved in phosphate buffer at pH 6.0.

Assay procedure: Multiple aliquots of 1.8 mL of substrate solution in 16 × 120 mm glass test tubes were pre-equilibrated for 5 min at 40 °C. The reaction was initiated by adding 0.2 mL of pre-equilibrated, suitably diluted endo-xylanase solution and incubating the tubes at 40 °C. The reaction was terminated after various time intervals by adding 3 mL of DNSA reagent solution with vigorous stirring. Reagent blanks were prepared by adding 3 mL of DNSA reagent to 1.8 mL of substrate solution plus 0.2 mL of the buffer solution as used in the assay, and the tube contents were mixed immediately. Enzyme blanks were prepared by adding 3 mL of DNSA reagent to 1.8 mL of substrate solution plus 0.2 mL of the enzyme solution as used in the assay and the tube contents mixed immediately. The xylose/xylo-oligosaccharide standards were prepared by adding 3 mL of DNSA solution to 1.8 mL substrate solution plus 0.2 mL of xylose or xylo-oligosaccharide standard (0–2 μmoles/0.2 mL). All tubes (reaction, reagent blanks, enzyme blanks and xylose and xylo-oligosaccharide standards) were placed in a boiling water bath and incubated for 15 min. The tubes were removed from the boiling water bath, and 1 mL of 40 % Rochelles salt solution was added immediately and the tube contents mixed immediately on a vortex mixer. The tubes were cooled at room temperature over approximately 15 min, and the contents were then re-mixed. The absorbance of the xylose and xylo-oligosaccharide standards was measured against the reagent blank at 540 nm. Concurrently, the absorbance of the reaction solutions was measured against the enzyme blank at 540 nm. The rate of hydrolysis was calculated as micromoles of xylose reducing sugar equivalent released per minute. One unit of A. niger endo-xylanase activity is defined as the amount of enzyme required to release 1 μmole of xylose reducing sugar equivalents per minute from the xylan or arabinoxylan substrate at pH 4.5 and at 40 °C.