Lactobacillus plantarum is a useful microorganism that metabolizes galactose-containing polysaccharides. Genome analysis has shown that L. plantarum contains four beta-galactosidase-related genes. Here, we cloned the beta-galactosidase gene that encodes the glycoside hydrolase family 2 (GH2) enzyme LpGal2. Recombinant LpGal2 (rLpGal2, 72 kDa) is a homodimer with maximal enzymatic activity at pH 7.0 and 50 degrees C. Under these conditions, rLpGal2 hydrolyzes p-nitrophenyl-beta-D-galactopyranoside (pNP beta Gal) with a specific activity of 2.16 x 10(-3) U/mg and substrate specificity for 8-1,6-galactobiose to produce D-Galactose. In addition, rLpGal2 can also hydrolyze beta-1,6-galactan to D-Galactose, whereas other galactose-containing oligosaccharides and polysaccharides tested could not be hydrolyzed. This finding demonstrates that LpGal2 functions as an exo-beta-1,6-galactosidase with narrow substrate specificity. To our knowledge, this is the first report of a beta-galactosidase derived from L. plantarum with exo-beta-1,6-galactosidase activity that has potential application for structure analysis of polysaccharides.