Biogel p2 extrafine column

Five hundred mg of low viscosity wheat flour AX (P-WAXYL, Ara : Xyl 38:62) were added to 24.5 ml of deionized water at 60°C and dissolved by stirring on a magnetic stirrer until complete dissolution. Then, the solution was equilibrated to 40°C and 0.5 ml of 0.5 M sodium phosphate buffer, pH 6, were added. This solution was placed in a water bath at 40°C and 97.5 U of endo-1,4-β-D-xylanase from Neocallimastix patriciarum (Megazyme #E-XYLNP) were added and incubated at 40°C for 16 h. Reactions were terminated by incubating the solutions at 95°C for 5 min. Solutions were centrifuged at 9,400 g for 10 min to remove insoluble materials. Digestion products were freeze-dried, desalted and pre-purified using a Sephadex G-10 column (90 cm³ bed-volume in a 1.5 cm diameter column; Merck) and size-fractionated using a Biogel P2 Extrafine column (140 cm³ bed-volume in a 1.6 cm diameter column; BioRad). Columns were connected to a Biologic-LP instrument, distilled water was used as mobile phase and the flow rates were 0.24 ml/min. The entire process was repeated three times.

The following saccharides were used as standards: glucose, fructose, sucrose (Sigma Aldrich, USA); GFn DP3–10 inulin-type FOS OligoTech® (Elicityl, France); Oligofructose Orafti®P95 (Beneo, Germany), containing GFn and Fn DP 2–7 inulin-type FOS; 6-kestose and neokestose (kindly donated by Professor M. Iiusuka). The acceptor substrate1-kestose was purchased from Wako Pure Chemical Industries (Japan); levanbiose was obtained as recently reported^{38 (link)}; and blastose, 6-kestose, neo-kestose and 6-neo-nystose were isolated (5–10 mg) from SacB reactions and purified by size exclusion chromatography (SEC) followed by phase-reverse chromatography (rpHPLC). SEC fractionation was performed in a Bio-Gel P2 Extra Fine column (2.5 × 100 cm, Bio-Rad) using MQ water as eluent at 0.2 mL/min. rpHPLC was carried out in a Waters 1525 HPLC system equipped with a Spherisorb S5 ODS2 Semi-Prep column (20 × 250 mm, Waters), employing MQ water as mobile phase at 7 mL/min.