Ics 3000 hplc system

The HPIC method is based on the method outlined in Dionex application note 143 with modifications based on information provided in the IonPac AS11-HC product manual (Thermo Fisher Scientific, 031333-09).
The HPIC was conducted using an ICS-3000 HPLC system (Thermo Fisher Scientific) fitted with a quaternary pump, equipped with an eluent generator (EGC-KOH), an IonPac AG11-HC guard column (2 mm × 50 mm, 052963) coupled to a IonPac AS11-HC analytical column (2 mm × 250 mm, 052961) and suppressed conductivity detection (ASRS 300, 2 mm suppressor) in external water mode (1 mL min^–1).
For each analysis, 10 μL of sample was injected and separated using a mixture of KOH and methanol as per the separation method outlined in Supplementary Table S2. The flow rate used was 0.38 mL min^–1 and the column and detection compartments were maintained at 30°C.

Monosaccharide composition of cellulose, arabinoxylan, β-glucan and glucomannan including glucose, galactose, mannose, arabinose and xylose were measured according to the previous method [20 (link)]. Briefly, the biomass of four polysaccharides was extracted with water and ethanol at 100 °C, then about 300 mg of dried biomass was weighted in a 100 mL Pyrex glass bottle. Throughout the process of the hydrolysis reaction, 72% sulfuric acid was incubated, and after 60 min deionized water was added. The mixture was autoclaved. Meanwhile, a sugar recovery standard was prepared. Released monosaccharides were analyzed using an ICS-3000 HPLC system (Thermo Fisher Scientific, Sunnyvale, CA, USA) equipped with a pulsed-amperometric detector. Samples were injected onto a 150 mm × 3 mm CarboPac PA20 column (Thermo Fisher Scientific, Sunnyvale, CA, USA) with a 50 mm × 3 mm guard column of the same material. Elution was performed at 30 °C with 2 mM potassium hydroxide at a flow rate of 0.4 mL/min.

Reaction products were analysed on a Dionex ICS-3000 HPLC system operated by Chromeleon software version 6.80 (Dionex) using a Dionex Carbopac PA1 column. Solvent A was water, solvent B was 1 M sodium hydroxide and solvent C was 200 mM NaOH with 170 mM Na acetate. The following programme was employed: pre-wash and column calibration -14–-7 min 60% B, 40% C (1 mL min⁻¹); -6–0 min 100% A (1 mL min⁻¹); sample injection 0–5 min 100% A (0.5 mL min⁻¹); gradient elution 5–20 min 0–30% B (0.5 mL min⁻¹).
To analyse the products of polysaccharide degradation by secretomes, 1 mL assays were prepared using substrate at 1 mg mL⁻¹, which was incubated with ~400 μg mL⁻¹ protein for up to 4 days at 30°C in 50 mM sodium citrate buffer, pH7, prior to analysis by HPAEC-PAD. Samples (200 μl) were also taken from cultures during growth on a range of carbohydrates. These samples were boiled to stop all enzyme reactions, concentrated by lyophilisation, resuspended in 50 μl of water, and analysed by HPAEC-PAD to observe oligosaccharide production and degradation during growth. Finally, pure protein (100 nM) was incubated with polysaccharide at 1 mg mL⁻¹, for 16 hours at 30°C in 50 mM sodium citrate buffer, pH 5.0, prior to analysis by HPAEC-PAD.