Intra- and Extracellular β-Galactosidases from Bifidobacterium bifidum and B. infantis: Molecular Cloning, Heterologous Expression, and Comparative Characterization

Three β-galactosidase genes from Bifidobacterium bifidum DSM20215 and one β-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum β-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published β-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis β-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 β-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal β-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum β-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four β-galactosidases were highly specific for hydrolysis of β-d-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.