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 bet a-D-fucosidase activity, was cloned from Bifidobacterium breve db. We sequenced a 19-kbp cloned DNA fragment that contained a single open re ading frame encoding 460 amino acids with a calculated molecular mass of 51,513Da, A putative ribosome binding site was found 5bp upstream o f the initiation codon, The amino acid sequence of this beta-D-glucosi dase 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 prepar ed from the recombinant strain showed 80 to 90-fold more beta-D-glucos idase activity than that from Bifidobacterium breve db. The recombinan t enzyme was purified to homogeneity from cell-free extracts of the re combinant strain using 4 column chromatographies. The recovery of enzy me from the recombinant strain was about 138-fold-higher than that of Bifidobacterium breve db. The enzymatic properties were similar to tho se of Bifidobacterium breve db. For application of this recombinant en zyme, we attempted to synthesize a disaccharide that seemed to be spec ifically assimilated by Bifidobacteria using the condensation activity of the enzyme.