A heterodimeric beta-galactosidase was discovered in the novel strain Lactobacillus curieae M2011381. The gene encoding the enzyme was expressed in Escherichia coli BL21 (DE3). The specific enzyme activities of the recombinant holoenzyme (LacLM) and large subunit (LacL) measured 11.4 U/mg and 3.8 U/mg, respectively. The kcat/Km values of LacLM and LacL were 740 mM(-1) s(-1) and 1.40 mM(-1) s(-1), respectively. LacLM showed maximum activity at pH 8.0 and 55 degrees C, and it could maintain its activity at a neutral pH and below 45 degrees C. LacLM displayed both hydrolysis and transgalactosylation activity on 200 g/L lactose. When LacLM was added to milk, the lactose was hydrolyzed after 6 h without galactooligosaccharide generation. The sequence alignment and homology modeling of the structures of the holoenzyme and subunits revealed that LacL has a catalytic domain with a catalytic dyad, Glu470 and Glu538, and small subunit LacM is a beta-sheet domain with a conserved Trp294. The molecular docking of LacLM helped to illustrate the roles of both subunits in the reaction with lactose.