Lactose

The production of GOSs was measured using 5% (wt/vol) lactose as a substrate in the assay buffer (20 mM sodium phosphate buffer, pH 7.0, containing 150 mM NaCl) at 25 °C. The reactions were initiated by mixing 125 μL of substrate solution containing 20% (wt/vol) lactose and 375 μL of protein solution containing 0.4 μM BgaD-D and/or an appropriate concentration of monobody (133–266 μM). Samples were withdrawn periodically and boiled for 10 min to terminate the reaction. The amounts of monosaccharides, disaccharides and GOSs were determined using a CK04S column (Mitsubishi Chemical) on an HPLC (LC-30AD, Shimadzu) equipped with an evaporative light scattering detector (ELSD-LTII, Shimadzu). The assay samples were eluted from the column using H₂O at a flow rate of 0.4 mL/min at 80 °C. For separation and determination of lactose and other disaccharides (DP2), an Asahipak NH2P-40 3E column (Shodex) was used with a gradient of H₂O (solvent A) and acetonitrile (solvent B) at a flow rate of 0.3 mL/min at 30 °C. Sugar concentrations were determined from peak areas. Glucose, galactose, lactose and 4′-galactosyllactose purchased from Wako Chemicals, and tetrasaccharides and larger oligosaccharides prepared as described above, were used as reference compounds for producing standard curves for these assays. Experiments were performed in triplicate.