Cloning and nucleotide sequence of the beta-D-glucosidase gene from Bifidobacterium breve clb, and expression of beta-D-glucosidase activity in Escherichia coli

Genomic DNA encoding a beta-D-glucosidase (EC 3.2.1.21), which has beta-D-fucosidase activity, was cloned from Bifidobacterium breve db. We sequenced a 19-kbp cloned DNA fragment that contained a single open reading frame encoding 460 amino acids with a calculated molecular mass of 51,513Da, A putative ribosome binding site was found 5bp upstream of the initiation codon, The amino acid sequence of this beta-D-glucosidase from Bifidobacterium breve db had 46% identity with that of beta-glucosidase from Microbispore bisipore. The enzyme of Bifidobacterium breve db was expressed in Escherichia coli, A cell-free extract prepared from the recombinant strain showed 80 to 90-fold more beta-D-glucosidase activity than that from Bifidobacterium breve db. The recombinant enzyme was purified to homogeneity from cell-free extracts of the recombinant strain using 4 column chromatographies. The recovery of enzyme from the recombinant strain was about 138-fold-higher than that of Bifidobacterium breve db. The enzymatic properties were similar to those of Bifidobacterium breve db. For application of this recombinant enzyme, we attempted to synthesize a disaccharide that seemed to be specifically assimilated by Bifidobacteria using the condensation activity of the enzyme.