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 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 beta-D-glucosidase from Bifidobacterium breve clb had 46% identity with that of beta-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 beta-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.
Nunoura
N
N
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Ohdan
K
K
Tanaka
K
K
Tamaki
H
H
Yano
T
T
Inui
M
M
Yukawa
H
H
Yamamoto
K
K
Kumagai
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics*; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D015183:Restriction Mapping; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics*
D004800:Enzymes, Immobilized; D009844:Oligosaccharides; D001617:beta-Glucosidase