Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B-infantis: Molecular cloning, heterologous expression, and comparativecharacterization

Three beta -galactosidase genes from Bifidobacterium bifidum DSM20215 and o ne beta -galactosidase gene from Bifidobacterium infantis DSM20088 were iso lated and characterized. The three B. bifidum beta -galactosidases exhibite d a low degree of amino acid sequence similarity to each other and to previ ously published beta -galactosidases classified as family 2 glycosyl hydrol ases. Likewise, the B. infantis beta -galactosidase was distantly related t o enzymes classified as family 42 glycosyl hydrolases. One of the enzymes f rom B. bifidum, termed BIF3, is most probably an extracellular enzyme, sinc e 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 en zyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta -galactosidase domain and a C-termina l galactose binding domain. The other two B. bifidum beta -galactosidases a nd the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrola ses, 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.