Open AccessCharacterization and molecular cloning of a heterodimeric β‐galactosidase from the probiotic strain Lactobacillus acidophilus R22
Author(s) -
Nguyen ThuHa,
Splechtna Barbara,
Krasteva Stanimira,
Kneifel Wolfgang,
Kulbe Klaus D.,
Divne Christina,
Haltrich Dietmar
Publication year - 2007
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2006.00614.x
Subject(s) - lactose , lactobacillus acidophilus , chemistry , enzyme , biochemistry , enzyme assay , hydrolysis , affinity chromatography , probiotic , biology , bacteria , genetics
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 ( o NPG) and lactose hydrolysis. The K m and V max values for lactose and o NPG 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 o NPG with K i,s =31.7±3.5 mM. The enzyme showed no specific requirements for metal ions, with the exception of Mg 2+ , 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.