Cloning and Nucleotide Sequence of theβ-d-Glucosidase Gene fromBifidobacterium breveclb, and Expression ofβ-d-Glucosidase Activity inEscherichia coli

Publisher Website
Google Scholar
Abstract
Genomic DNA encoding a β-d-glucosidase (EC 3.2.1.21), which has β-d-fucosidase activity, was cloned from Bifidobacterium breve clb. We sequenced a 1.9-kbp cloned DNA fragment that contained a single open reading frame encoding 460 amino acids with a calculated molecular mass of 51,513 Da. A putative ribosome binding site was found 5 bp upstream of the initiation codon. The amino acid sequence of this β-d-glucosidase from Bifidobacterium breve clb had 46% identity with that of β-glucosidase from Microbispore bispore. The enzyme of Bifidobacterium breve clb was expressed in Escherichia coli. A cell-free extract prepared from the recombinant strain showed 80 to 90-fold more β-d-glucosidase activity than that from Bifidobacterium breve clb. 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 clb. The enzymatic properties were similar to those of Bifidobacterium breve clb. 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.

This publication has 0 references indexed in Scilit: