Intra- and extracellular β-galactosidases from Bifidobacterium bifidum and B-infantis:: Molecular cloning, heterologous expression, and comparative characterization

Three beta -galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta -galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta -galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta -galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta -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 a-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 beta -galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta -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 beta -galactosidases were highly specific for hydrolysis of beta -D-galatiosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.