Characterization and molecular cloning of a heterodimeric β-galactosidase from the probiotic strain Lactobacillus acidophilus R22

• Published in: FEMS microbiology letters 2007-04, Vol.269 (1), p.136-144
• Main Authors: Nguyen, Thu-Ha, Splechtna, Barbara, Krasteva, Stanimira, Kneifel, Wolfgang, Kulbe, Klaus D, Divne, Christina, Haltrich, Dietmar
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Ammonium; Ammonium sulfate; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; beta-galactosidase; beta-Galactosidase - chemistry; beta-Galactosidase - genetics; beta-Galactosidase - metabolism; Biological and medical sciences; Cations - metabolism; Cloning; Cloning, Molecular; Dimerization; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Enzymes; fermentation; foods; Fractionation; Fundamental and applied biological sciences. Psychology; galacto-oligosaccharides; Galactooligosaccharides; Galactosidase; Gel electrophoresis; Glucose; health; Hydrogen-Ion Concentration; Hydrophobicity; Kinetics; lactic-acid bacteria; Lactobacilli; Lactobacillus acidophilus; Lactobacillus acidophilus - classification; Lactobacillus acidophilus - enzymology; Lactobacillus acidophilus - genetics; Lactose; Magnesium; Metabolism. Enzymes; Metal ions; Microbiology; milk; Molecular Sequence Data; Nitrophenol; o-Nitrophenol; Oligosaccharides; Oligosaccharides - metabolism; prebiotics; Probiotics; Proteins; Sequence Analysis, Protein; Sugar; Temperature; transgalactosylation; Yogurt; β-Galactosidase
• ISSN: 0378-1097; 1574-6968; 1574-6968
• DOI: 10.1111/j.1574-6968.2006.00614.x

β-Galactosidase from the probiotic strain Lactobacillus acidophilus R22 was purified to apparent homogeneity by ammonium sulphate fractionation, hydrophobic interaction, and affinity chromatography. The enzyme is a heterodimer consisting of two subunits of 35 and 72 kDa, as determined by gel electrophoresis. The optimum temperature of β-galactosidase activity was 55°C (10-min assay) and the range of pH 6.5-8, respectively, for both o-nitrophenyl-β- d-galactopyranoside (oNPG) and lactose hydrolysis. The Km and Vmax values for lactose and oNPG were 4.04±0.26 mM, 28.8±0.2 μmol d-glucose released per min per mg protein, and 0.73±0.07 mM, 361±12 μmol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with Ki,s=31.7±3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg²⁺, which enhanced both activity and stability. The genes encoding this heterodimeric enzyme, lacL and lacM, were cloned, and compared with other β-galactosidases from lactobacilli. β-Galactosidase from L. acidophilus was used for the synthesis of prebiotic galacto-oligosaccharides (GOS) from lactose, with the maximum GOS yield of 38.5% of total sugars at about 75% lactose conversion.