Dionex ics 3000 system

Degradation products from cellulose and xylan were analyzed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The cellooligosaccharides were separated using a Dionex ICS3000 system (Thermo Scientific, San Jose, CA, USA) equipped with a CarboPac PA1 2 × 250 mm analytical column. A stepwise gradient with an increasing amount of eluent B (eluent B is 0.1 M NaOH and 1 M NaOAc; eluent A is 0.1 M NaOH) was applied starting right after sample injection, as follows: 0–10% B over 10 min, 10–30% B over 25 min, 30–100% B over 5 min, 100–0% B over 1 min, 0% B over 9 min. Data analysis was performed using Chromeleon 7.0 software. Cellooligosaccharide standards with a degree of polymerization of one to five (DP1—DP5) and xylo-oligosaccharide standards with a degree of polymerization of one to six (DP1 –DP6) were purchased from Megazyme (Wicklow, Ireland) and used to identify the products.

The specificity of RmCE towards different monosaccharides was determined in MOPS buffer (pH 6.3) and with 200 mM of substrate (d-glucose, d-mannose, d-galactose, d-xylose, d-lyxose, l-arabinose) at an enzyme concentration of 0.3 mg/mL. All reactions were monitored at 70 °C and 5 time points were taken during 24 h. Enzyme inactivation was achieved by transferring the sample (5 μL) in 100 mM of NaOH (90 μL). Conversion of substrate to product was evaluated by high-performance anion exchange chromatography–pulsed amperometric detection (HPAEC–PAD) using the Dionex ICS-3000 system (Thermo Fischer Scientific) (CarboPac PA20 column-3 × 150 mm) as described by Verhaeghe et al. but with a 15-min isocratic method of 20 mM NaOH [40 (link)]. The monosaccharides were quantified by determining conversion based on the peak areas. Product concentration was then plotted in function of time and a linear correlation was fitted representing the activity of the enzyme (in μmol/min). Finally, specific activities were calculated by dividing the increase in product (units) by the enzyme concentration (mg). One unit (U) of RmCE activity was defined as the amount of enzyme required to produce 1 μmol of product from substrate per minute at 70 °C and pH 6.3.

The soluble cellooligosaccharides were separated and quantified by high-performance anion-exchange chromatography with a pulsed amperometric detection (HPAEC-PAD; Dionex ICS-3000 system, Thermo Fisher Scientific, Waltham, MA, USA), using a gradient method (0–11 min 100 mM NaOH, 11–20 min 90% 100 mM NaOH and 10% 100 mM NaOH/100 mM NaOAc, followed by 20–26 min 100% 100 mM NaOH for recalibration) and a flow rate of 0.5 ml/min. Analytical standards with different concentrations of αG1-P, glucose, cellobiose, cellotriose, cellotetraose and cellopentaose were used to detect and perform a quantitative analysis of cellodextrins. The results were visualised and plotted in SigmaPlot software (Systat Software Inc.).

A 2‐mg aliquot of starch, obtained as above, was used to determine the distribution of amylopectin chain length, based on HPAEC‐PAD analysis, modified from the method given by Sestili et al. (2016). After debranching, the supernatant was diluted to 0.4 mg/mL and a 10 μL aliquot was injected onto a CarboPac PA‐100 4 × 250 mm column, using a Dionex ICS 3000 system equipped with an autosampler (Thermo Fisher). The sample was eluted by imposing a linear gradient of 0–200 mm NaOAc in 50 mm NaOH over 10 min, followed by a convex gradient of 200–390 mm NaOAc over the subsequent 120 min, then by 1 m NaOAc for 5 min and finally by 0 mm NaOAc for 10 min. After baseline subtraction from water injections, peak areas were normalized to the total peak area of the injected sample. Starch crystallinity was determined from X‐ray powder diffraction patterns, as described by Sparla et al. (2014).

Oligosaccharide analysis was carried out by High Performance Anion Exchange Chromatography (HPAEC) on a Dionex ICS-3000 system (Thermo Scientific, USA) equipped with a 4×250 mm CarboPac PA-1 column. A pulsed amperometric detector with a gold electrode and an Ag/AgCl pH reference electrode were used. The system was run with a gradient of 30–600 mM NaAc in 100 mM NaOH run at 1 mL/min. Chromatograms were analyzed using Chromeleon 6.8 chromatography data system software (Thermo Scientific). A mixture of glucose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose (0.1 mg/mL of each component) was used as reference for qualitative determination of elution time of each component. The decay rate of detector signal from DP 2 to DP 7 calculated from reference sample is 4.44:2.76:2.02:1.45:1.36:1.00, which is used to correct the DP 2 to DP 7 of all samples and other components are without correction.