Aspergillus oryzae β galactosidase

Lacto-N-fucopentaose I (LNFP I: Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), p-nitrophenyl 6-O-sulfo-β-GlcNAc (GlcNAc6Sβ-pNP), and GlcNAcβ-O-Ser were purchased from Carbosynth. GlcNAcβ-pNP was obtained from Nacalai Tesque, and core 2-pNP [Galβ1-3(GlcNAcβ1-6)GalNAcα-pNP] was obtained from Toronto Research Chemicals. N,N′-Diacetylchitobiose (GlcNAcβ1-4GlcNAc), core 2-Thr [Galβ1-3(GlcNAcβ1-6)GalNAcα-O-Thr], core 3-Thr [GlcNAcβ1-3GalNAcα-O-Thr], and core 4-Thr [GlcNAcβ1-3(GlcNAcβ1-6)GalNAcα-O-Thr] were purchased from Tokyo Chemical Industry. GalNAcβ-pNP, GlcNAcβ1-2Man, and porcine gastric mucin (PGM, type III) were from Sigma-Aldrich. PGM was dialyzed against water and lyophilized prior to use. Lacto-N-triose II (GlcNAcβ1-3Galβ1-4Glc) was prepared by digesting lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc, LNnT) by Aspergillus oryzae β-galactosidase (Sigma-Aldrich) followed by separation with Toyopearl HW-40C gel chromatography (20 mm × 600 mm) (Tosoh). LNnT was a gift from Glycom A/S. The purity was confirmed by high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) as described later.

Aspergillus oryzae β-galactosidase (E. C. 3.2.1.23), lactulose, d-fructose, 6-galactobiose, and 50% glutaraldehyde were purchased from Sigma-Aldrich (Steinheim, Germany). Sepharose 4B was supplied by Pharmacia Biotechnology (Uppsala, Sweden). d-galactose, raffinose and o-nitrophenyl-β-d-galactopyranoside (ONPG) from Fluka (Steimheim, Germany). Fructooligosaccharides (Orafti P95) and inulin (Orafti HP) were kindly gifted by Orafti (Tienen, Belgium).