Molecular Cloning and Expression of the β-Galactosidase Gene from Bifidobacterium longum LL04 in Escherichia coli

A Bifidobacterium strain with β-galactosidase (β-gal) activity was isolated from infant feces on transgalactooligosaccharide (tGOS) propionate plates and de Man, Rogosa, and Sharpe (MRS) plates containing 5-bromo-4-chloro-3-indolyl-β-d-galacto-pyranoside (X-gal). The strain was identified as a Bifidobacterium longum and was named B. longum LL04. The gene encoding the β-galactosidase enzyme β-gal from B. longum LL04 was revealed by nucleotide sequence analysis as a 3.2-kb DNA fragment. B. longum β-gal (3,069 bp) is a 1,023-amino acid-long polypeptide with a predicted molecular mass of 114.5 kDa. The amino acid sequence of β-gal was homologous to those found in the LacZ families. The acid-base, nucleophilic, and substrate-recognition sites conserved in the LacZ family were found in β-gal. The coding region of the β-gal gene was then cloned downstream of a T7 promoter for overexpression in Escherichia coli.