Substrate specificity of mussel and snail β-galactosidases

The substrate specificity of the mussel (C. gigas) and snail (A. vulgaris) β-galactosidases towards 2-amino benzoic acid labeled di- and trisaccharides (lactose-AA: Galβ1,4Glc-AA; galacto-N-biose-AA: Galβ1,3GalNAc-AA; 2-fucosyllactose-AA: Fucα1,2Galβ1,4Glc-AA; 3-fucosyllactose-AA: Fucα1,3[Galβ1,4]Glc-AA; N-acetyllactosamine-AA: Galβ1,4GlcNAc-AA and Galβ1,6GlcNAc-AA) were analyzed on reverse-phase HPLC (ODS HypersilTM, 250 × 4 mm, ThermoFisher Scientific–Bonn, Germany) with solvent A: 0.2% (v/v) 1-butylamin, 0.5% (v/v) orthophosphoric acid, 1% (v/v) tetrahydrofuran in H₂O and solvent B: solvent A/acetonitrile = 50/50 (v/v). The elution was performed by a linear gradient of solvent B from 5–100% in 23 min, at a flow rate of 1 mL/min. Quantification was done by peak integration after fluorescence detection at ex/em 360 nm/425 nm [33 (link)].
Separation of pNP-labeled sugars (pNP-lactose: pNP-Glcβ1,4Gal, pNP-galacto-N-biose: pNP-GalNAcβ1,3Gal) was done on reverse-phase HPLC (ODS HypersilTM, 250 × 4.6 mm, ThermoFisher Scientific–Bonn, Germany) with solvent A composing of 0.1 M ammonium acetate, pH 6.0 and solvent B containing 50% (v/v) acetonitrile in H₂O. Elution was achieved by a linear gradient of solvent B from 5–50% in 30 min, at a flow rate of 1 mL/min. Quantitative values were obtained by peak integration after UV detection at 280 nm.