Crystal Structure of α-Galactosidase from Lactobacillus acidophilus NCFM: Insight into Tetramer Formation and Substrate Binding

Efficient synthesis of α‐galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α‐galactosidase (BtGH97b)

“…13 15 The transfer products were identical among all reactions with homologous substrates and 16 acceptors. The products from reactions using α-GalF in the presence of formate ion were isolated 17 by HPLC and the main products were subjected to structural analyses.…”

“…The major trisaccharide was subjected to 1 H-NMR analysis. Three 14 doublet signals with similar integrated values were detected at δ=5.40 (J = 3.4 Hz), δ=5.27 (J = 15 3.4 Hz), and δ=4.65 ppm (J = 8.2 Hz), respectively, and no other notable doublet signals were 16 detected in the anomeric region (Fig. 4D).…”

Enzymology and Structure of the GH13_31 Glucan 1,6-α-Glucosidase That Confers Isomaltooligosaccharide Utilization in the Probiotic Lactobacillus acidophilus NCFM

“…LaGH13_31 was produced in a 5-liter bioreactor (Biostat B; B. Braun Biotech International, Melsungen, Germany) according to a fed-batch protocol developed previously for production of other L. acidophilus NCFM recombinant enzymes (14), with the exception that the induction (OD 600 of 8.3) was carried out at 16°C with 40 M isopropyl-␤-D-thiogalactopyranoside (IPTG). The fermentation was terminated after 23 h of induction, and 61 g of cell pellet (harvested by centrifugation at 12,200 ϫ g for 10 min at 4°C) was resuspended in 60 ml of a buffer containing 10 mM HEPES, 10 mM imidazole, 10% glycerol, 0.5 M NaCl, and 2 mM CaCl 2 (pH 7.5) (buffer A) and disrupted by passage through a French press at 600 ϫ 10 5 Pa. After Benzonase nuclease (Novagen) treatment, the suspension was centrifuged (at 43,000 ϫ g for 65 min) and sterile filtered (0.22-m pore size).…”

“…The fermentation was terminated after 23 h of induction, and 61 g of cell pellet (harvested by centrifugation at 12,200 ϫ g for 10 min at 4°C) was resuspended in 60 ml of a buffer containing 10 mM HEPES, 10 mM imidazole, 10% glycerol, 0.5 M NaCl, and 2 mM CaCl 2 (pH 7.5) (buffer A) and disrupted by passage through a French press at 600 ϫ 10 5 Pa. After Benzonase nuclease (Novagen) treatment, the suspension was centrifuged (at 43,000 ϫ g for 65 min) and sterile filtered (0.22-m pore size). LaGH13_31 was purified by immobilized metal ion affinity chromatography using a 5-ml HisTrap HP column (GE Healthcare, Uppsala, Sweden) as described elsewhere (14). This purification step was followed by anion exchange chromatography using an 8-ml Mono Q 10/100 GL column (GE Healthcare) equilibrated in 10 mM HEPES, pH 7.0, and 2 mM CaCl 2 (GE Healthcare) and installed on an Ä KTAexplorer chromatograph (GE Healthcare).…”

α-Galactosidase/Sucrose Kinase (AgaSK), a Novel Bifunctional Enzyme from the Human Microbiome Coupling Galactosidase and Kinase Activities

“…On the basis of the high level of sequence homology between members of family GH27 and GH36, which reflects intrinsically structural homology, Comfort et al (19) were able to infer the catalytic residues of Thermotoga maritima TmGalA, as well as to confirm their identity by elegant biochemical studies. For family GH36, only the crystal structures of T. maritima TmGalA (Protein Data Bank (PDB) 1zy9) and Lactobacillus brevis LbR11 (PDB 3mi6) have been deposited within the Protein Data Bank by Structural Genomics Consortia, and the crystal structure of Lactobacillus acidophilus NCFM LaMel36A (PDB 2xn0) has been solved very recently (20).…”