Sugar pak column

To detect glucose by HPLC, the ascal lysate was filtered by a 0.22-μm filtration membrane (Millipore) and analyzed by HPLC (Waters) equipped with Waters Sugar-Pak column (300 mm by 6.5 mm). EDTA calcium salt (500 mg/L) was used as mobile phase at a flow rate of 0.5 mL/min. The column temperature was 80°C, and a refractive index detector was used. To quantify glucose in ascal lysates, glucose standard solutions (0.5, 1, 2, 5, 10, and 20 mg/mL) were prepared. These standards were subjected to HPLC, and a calibration curve was generated by plotting the peak area (Fig. S11a). The concentration of glucose in the samples was determined based on this calibration curve.
To detect glucose by a chemical method, a glucose test kit (Rongsheng) was used according to the manufacturer’s instructions.

One gram of freeze‐dried sample and 30ml of 80% methanol aqueous solution were used to extract these samples with sonication for 1 h. The extract was filtered in a 0.2 μm membrane filter. The separations and analyses were performed with an HPLC system (Dionex ultimate 3000, Thermo Dionex), consisting of a Shodex Refractive Index (RI)‐101 detector (Shodex), a column heater set at 70°C, and a sugar‐pak column (300 × 6.5mm, Waters); the isocratic mobile phase was deionized‐distilled H2O delivered at 0.5 ml/min. The calibration curve with four different points was used to obtain the free sugar standard. The results are in mg/100 g of dry weight.

The substrate specificity of the purified pTsbg was determined at 85 °C using 4 mg mL⁻¹ solutions of the following chromogenic substrates in Z buffer (pH 7): ONPG, p-Nitrophenyl-β-d-fucopyranoside, p-Nitrophenyl-β-d-mannoside, p-Nitrophenyl-α-d-mannoside, p-Nitrophenyl-β-d-glucoside, p-Nitrophenyl-α-d-glucoside, p-Nitrophenyl-β-d-xyloside, and p-Nitrophenyl-α-d-xyloside.
GOS and lactose concentrations were determined by HPLC (HPLC Waters Breeze I), using a Waters Sugar-Pak column eluted at 90 °C with 0.1 M EDTA disodium salt in Milli-Q water at a flow rate of 0.5 mL min⁻¹, and a Waters 2414 refractive-index detector. Purified protein was incubated at 70 °C and 650 rpm in phosphate buffer 0.1 M (pH 6.8), supplemented with 40% lactose. Samples were taken at 0, 0.5, 1, 2, 4, 6, and 24 h and immediately transferred to 99 °C for 5 min to inactivate the enzyme and stored at − 20 °C for subsequent analysis. Carbohydrates were quantified by external calibration, using standard solutions of galactose, glucose, lactose, raffinose, and stachyose.