Cloning and nucleotide sequence of the β-D-glucosidase gene from Bifidobacterium breve clb, and expression of β-D-glucosidase activity in Escherichia coli
In: Bioscience, biotechnology, and biochemistry, Jg. 60 (1996), Heft 12, S. 2011-2018
Online
academicJournal
- print, 44 ref
Zugriff:
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.
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Cloning and nucleotide sequence of the β-D-glucosidase gene from Bifidobacterium breve clb, and expression of β-D-glucosidase activity in Escherichia coli
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Autor/in / Beteiligte Person: | NUNOURA, N ; OHDAN, K ; TANAKA, K ; TAMAKI, H ; YANO, T ; INUI, M ; YUKAWA, H ; YAMAMOTO, K ; KUMAGAI, H |
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Zeitschrift: | Bioscience, biotechnology, and biochemistry, Jg. 60 (1996), Heft 12, S. 2011-2018 |
Veröffentlichung: | Tokyo: Japan Society for Bioscience Biotechnology and Agrochemistry, 1996 |
Medientyp: | academicJournal |
Umfang: | print, 44 ref |
ISSN: | 0916-8451 (print) |
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