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J. Biol. Chem., Vol. 276, Issue 12, 9059-9065, March 23, 2001
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From the Departments of Azo dyes are regarded as pollutants because they
are not readily reduced under aerobic conditions. Bacillus
sp. OY1-2 transforms azo dyes into colorless compounds, and this
reduction is mediated by a reductase activity for the azo group in the
presence of NADPH. A 1.2-kbp EcoRI fragment containing the
gene that encodes azoreductase was cloned by screening the genomic
library of Bacillus sp. OY1-2 with digoxigenin-labeled
probe designed from the N-terminal amino acid sequence of the purified
enzyme. An open reading frame encoding the azoreductase, consisting of
178 amino acids, was predicted from the nucleotide sequence. In
addition, because only a Bacillus subtillis hypothetical
protein was discovered in the public databases (with an amino acid
identity of 52.8%), the gene encoding the azoreductase cloned in this
study was predicted to be a member of a novel family of reductases.
Southern blot analysis revealed that the azoreductase gene exists as a
single copy gene on a chromosome. Escherichia
coli-expressing recombinant azoreductase gave a ten times greater
reducing activity toward azo dyes than the original Bacillus sp. OY1-2. In addition, the expressed azoreductase
purified from the recombinant E. coli lysate by
Red-Sepharose affinity chromatography showed a similar activity and
specificity as the native enzyme. This is the first report
describing the sequencing and characterization of a gene encoding the
azo dye-reducing enzyme, azoreductase, from aerobic bacteria and its
expression in E. coli.
The nucleotide sequence(s) reported in this paper has been
submitted to the DDBJ GenBankTM/EBI Data Bank With accession number(s) AB002631.
Molecular Cloning and Characterization of the Gene Coding for
Azoreductase from Bacillus sp. OY1-2 Isolated from
Soil*
§,, and
Pathology, ¶ Food
Microbiology, and Environmental Sanitation, Osaka Prefectural
Institute of Public Health, 1-3-69, Nakamichi, Higashinari-ku, Osaka
537-0025, Japan
*
This work was supported by a cooperative research project of
Osaka Prefectural Government on Advanced Technology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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