Rezex rpm monosaccharide pb2 column

For quantification of free sugars, high pressure liquid chromatography (HPLC) (Dionex Ultimate 3000, Thermo Fisher Scientific, USA) coupled to refractive index detection (ERC Refractomax 521, Thermo Fisher Scientific, USA) was used. Sugar separations were performed on a Rezex RPM-Monosaccharide Pb²⁺ column (Phenomenex Ltd., Germany) using water as mobile phase and a flow rate of 0.6 mL/min at 85 °C. The sugars were identified and quantified using sugar standards and subsequently generated calibration curves and the Chromeleon™ software (Version 6.8, Dionex, Germany).

To test the ability of DT-Bgl to degrade various substrates (Table 3), 100 μL 1 % (w/v) of these compounds was reacted with 10 μL 1 U of purified DT-Bgl. All reactions were carried out in glycine-NaOH buffer (100 mM, pH 8.5), at 60 °C for 15 min, unless specified. Reactions were stopped by boiling for 5 min, and the samples were analyzed on the HPLC 1260 system (Agilent Technologies, Santa Clara, USA), using a 7.8 × 300 mm Rezex RPM-Monosaccharide Pb⁺² column (Phenomenex Inc, Torrance, USA) fitted with a 7.8 × 50 mm LC guard column filled with the same stationary phase. An Agilent 385-Evaporative Light Scattering Detector was used, and the nebulizer and evaporator were kept at 30 °C with a nitrogen flow rate of 1.6 slm (standard liters per min). Nanopure water, filtered through a nylon membrane filter (0.45 µm pore size, Millipore, Darmstadt, Germany) and degassed, was used as the mobile phase. All the insoluble particles were filtered through a polytetrafluoroethylene syringe filter membrane (0.45 µm pore size) prior to injection into the column. All substrates and standards used were of high purity grade.