From the Departments of Pathology, ¶ Food
Microbiology, and
Environmental Sanitation, Osaka Prefectural
Institute of Public Health, 1-3-69, Nakamichi, Higashinari-ku, Osaka
537-0025, Japan
Received for publication, September 5, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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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.
Azo dyes are synthetic organic colorants that are characterized by
great structural variety. Synthetic azo dyes are extensively used in
the textile, food, and cosmetics industries. More than 7 × 105 tons of these dyes are produced annually worldwide (1).
Most azo dyes are released into the environment as waste from
the textile, food, cosmetic, and dyestuff manufacturing industries (2). They are frequently found in a chemically unchanged form even after
waste-water treatment (3-5), so they are regarded as pollutants. The
treatment system of colored waste-water, based on physical or chemical
procedures, is effective but suffers from such shortcomings as high
cost, formation of hazardous byproducts, and intensive energy
requirements. In contrast, biological degradation of these dyes does
not have similar problems. An uncharged azo dye can be transformed into
colorless compounds by reductive cleavage of the azo bond in anoxic
sediment environments (6-8). To establish biological waste-water
treatment of azo dye, it is essential to discover the microorganisms
that carry the azo dye-degrading enzymes.
To accomplish this, we have isolated three bacterial strains that
reduce azo dyes from soil and sewage samples. These strains were
identified as Bacillus sp. OY1-2, Xanthomonas sp.
NR25-2 and Pseudomonas sp. PR41-1. The enzymes produced by
these bacteria catalyze the reduction of Methyl Red and produce
dimethyl p-phenylenediamine and o-aminobenzoic
acid (Fig. 1 and Ref. 9). Among them, an enzyme from Bacillus sp. OY1-2, which was able to reduce azo
dyes such as Methyl Red, Rocceline, and Sumifix Red B in the presence of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-NADPH, was purified from bacterial cell extracts by means of
column chromatography and subsequently
characterized.1
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Fig. 1.
Reduction of Methyl Red by azoreductase.
Methyl Red was treated with crude azoreductase solution in the presence
of -NADPH. Products were analyzed by gas chromatography and
identified as dimethyl p-phenylenediamine and
o-aminobenzoic acid (9).
Molecular cloning of the gene encoding this enzyme is essential for
further characterization as well as for technological applications of
this enzyme. In this report, we show the molecular cloning and
characterization of the gene encoding the azoreductase from
Bacillus sp. OY1-2 and present the characteristics of
recombinant azoreductase expressed in E. coli.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains, Plasmids, and Culture Conditions--
Azo
dye-degrading bacteria isolated from soil near a waste-water plant from
a textile factory were identified as Bacillus sp. OY1-2
based on biological characterization (9). This strain can grow in
brain-heart infusion broth (Difco). E. coli strains C600hfl
and XLI-Blue were cultured in Luria broth consisting of 10 g of
bactotryptone (Difco), 5 g of yeast extract (Difco), and 10 g
of NaCl per liter. The E. coli strains GI724 and GI618 were cultured in RMG medium consisting of 40 g of cazamino acids
(Difco), 5 g of glycerol, 1 mM MgCl2,
6 g of Na2HPO4, 3 g of
KH2PO4, 0.5 g of NaCl, 1 g of
NH4Cl per liter. Recombinant proteins were expressed by
E. coli strains GI724 or GI618 in ID medium consisting of
0.4 g of cazamino acid, 5 g of glucose, 1 mM
MgCl2, 6 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, 1 g of
NH4Cl per liter. A phage vector gt10 was used for the
construction of the genomic library. The plasmid pT7Blue(R)T (Novagen,
Inc.) and pUC18 were used for the subcloning of genes. The plasmids
pTrx-Fus (Invitrogen Co.) and pTrcNd (reconstructed from pQE30 (Qiagen
Inc.) were used for expression of recombinant azoreductase.
N-terminal Amino Acid Sequence of Native and Recombinant Azoreductase-- Samples of native and recombinant azoreductase were separated on SDS-PAGE2 and transferred to polyvinylidene difluoride membranes (Bio-Rad). The blotted protein strips were used for amino acid sequencing on a PE Biosystems 470/120A protein sequencer.
Construction of Bacillus sp. OY1-2 Genomic DNA
Library--
Bacillus sp. OY1-2 genomic DNA was prepared by
mechanical disruption as described previously (11). Briefly, bacterial
pellet from 5 ml of liquid culture was suspended in 0.5 ml of lysis
buffer consisting of 0.3 M Tris-HCl, pH 8.0, 0.1 M NaCl, and 6 mM EDTA. The cell suspension was
transferred into a conical 2-ml screw-cap vial, which is one-fourth
filled with 0.17-mm acid-washed sterile glass beads. Cells were
disrupted by vigorous shaking with 0.5 ml of chloroform on a Mini-Bead
Beater cell disrupter (Biospec Products, Bartlesville, UK) for 5 min.
DNA in the upper layer after centrifugation was further purified by
phenol/chloroform extraction, concentrated by ethanol precipitation,
and dissolved in 300 µl of TE buffer consisting of 10 mM
Tris-HCl, pH 8.0 and 1 mM EDTA. The purified genomic DNA
was completely digested with EcoRI and ligated into the
EcoRI site of gt10. Genomic library constructs were
introduced into E. coli strain C600hfl by means of in
vitro packaging using gigapack plus (Stratagene).
Generation of Probe for Screening--
The N-terminal amino acid
sequence was used to design oligonucleotide primers for amplifying the
DNA fragment encoding the N-terminal of azoreductase (Fig.
2A). The reaction mixture (50 µl) consisted of long and accurate (LA) PCR buffer II
(Mg2+-free); 2.5 mM MgCl2; 200 mM each dATP, dCTP, dGTP, and dTTP; 10 ng of DNA from
Bacillus sp. OY2-1; 1.25 units of Takara LA Taq
DNA polymerase (Takara Shuzo Co., Ltd., Japan); and 0.5 mM each primer AZR-1 and AZR-2 (Fig. 2A). PCR was carried out
for 30 cycles in a Takara Thermal Cycler Personal (Takara Shuzo Co., Ltd.), with each cycle consisting of denaturation for 30 s at 94 °C, annealing for 30 s at 55 °C, and extension for 1 min
at 72 °C. The PCR product was extracted from the gel after
separation on a 1% agarose gel and was directly subcloned into the
pT7Blue(R)T vector. The subclones were sequenced by the dideoxy chain
termination method (12) with a Model 310 genetic analyzer (PE
Biochemicals Inc). A hybridization probe was synthesized by PCR
using a PCR DIG labeling kit (Roche Diagnostics Co., Germany).
The reaction mixture (50 µl) consisted of 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 200 mM each dATP, dCTP,
and dGTP and 130 mM dTTP; 70 mM
digoxigenin-11-dUTP; 1.25 units of Taq DNA polymerase; a 1 µM concentration of primers M13 M1 & M13 RV (Takara Shuzo
Co., Ltd.); and 10 ng of plasmid carrying the PCR product encoding the
N-terminal of azoreductase. PCR was performed under the same conditions
as described above.
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Cloning and DNA Sequencing of the Azoreductase Gene-- A phage library was screened essentially as previously described (13). Approximately 1 × 105 plaques from the genomic library were plated with E. coli C600hfl and incubated at 37 °C for 6 h. Nylon filters (Nytran 13N, Schleicher & Schuell Co.) were processed for hybridization. The filters were prehybridized in ExpressHyb hybridization solution (CLONTECH Laboratories, Inc.) at 68 °C for 30 min and then hybridized for 1 h at 48 °C with a 10 ng/ml concentration of digoxigenin-labeled probe described above in the same buffer as used for prehybridization. The filters were washed for 5 min in 2× SSC and 0.1% SDS at room temperature followed by washing for 15 min in 0.2× SSC and 0.1% SDS at 48 °C. The hybridized probe was detected after 30 min of incubation at room temperature with alkaline phosphatase-conjugated anti-digoxigenin antibody (Fab; Roche Diagnostics Co.) diluted 1:5000. The enzyme-catalyzed color reaction was carried out using a nitro blue tetrazolium salt (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) system (Wako Pure Chemical Industries, Japan) in Buffer 3 consisting of 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2. The DNA inserts in the positive clones were subcloned into the EcoRI site of pUC18 for further characterization. The EcoRI fragment in the subclone was digested by SphI, NlaIV, or HincII; further subcloned in pUC18; and sequenced as shown in Fig. 2 by the dideoxy chain termination method with a Model 310 genetic analyzer.
Database Search-- Protein and DNA sequences with homology to the deduced amino acid sequence of the azoreductase ORF were searched using TBLASTN from the National Center for Biological Information.
Southern Blot Hybridization-- Detection of the restriction DNA fragment carrying the azoreductase gene was performed according to Southern (14). One µg of genomic DNA was completely digested with restriction enzymes, separated on a 0.7% agarose gel, and vacuum-transferred to Nytran 13N nylon filters. The filters were prehybridized in ExpressHyb hybridization solution at 68 °C for 30 min followed by hybridization with the same solution containing a 10 ng/ml Dig-labeled 1.2-kbp EcoRI DNA fragment carrying the whole coding region of azoreductase. After hybridization, the filters were washed for 5 min with 2× SSC and 0.1% SDS at room temperature followed by washing twice with 2× SSC/0.1% SDS for 15 min at 68 °C. The hybridized Dig-labeled probe on the filters were detected by alkaline phosphatase-conjugated anti-digoxigenin antibody, followed by color development using NBT/BCIP as substrates in Buffer 3.
Expression of Azoreductase in E. coli--
The entire open
reading frame of azoreductase was amplified by PCR. Briefly, the
reaction mixture (50 µl) consisted of LA-PCR buffer II
(Mg2+-free); 2.5 mM MgCl2; 200 mM each dATP, dCTP, dGTP, and dTTP; 10 ng plasmid
pT7B-AZR5-8; 1.25 units of Takara LA Taq DNA polymerase, and 0.5 mM each of primers AZR-rec-S-Nde
(CATATGAAACTAGTCGTTATTAAC) and AZR-rec-E-Xba
(TCTAGAGCAGATAGACTATTGGCTCC). PCR was carried out for 30 cycles in a
Takara Thermal Cycler Personal, with each cycle consisting of
denaturation for 30 s at 94 °C, annealing for 30 s at
55 °C, and extension for 1 min at 72 °C. The PCR product was
extracted from the gel after separation on 1% agarose gel electrophoresis and subcloned into pT7Blue(R)T for confirmation of the
nucleotide sequence and then transferred into expression vectors
pTrx-Fus or pTrcNd after digestion by NdeI and
XbaI. Expression of reductase in pTrx-Fus system was
performed by adding tryptophan at a concentration of 0.1 mg/ml in ID
medium. Expression in pTrcNd was performed by adding
isopropyl--D-thio-galactopyranoside (IPTG) at a
concentration of 1 mM in Luria-Bertani medium. Cells from 10 ml of induced culture were suspended in 0.5 ml of 20 mM
sodium phosphate buffer, pH 7.0, lysed by two cycles of freezing at
80 °C and thawed at 37 °C followed by sonication (15 s, 70%
output, 10×). The supernatants from a 9000 × g, 30 min centrifugation were used directly for enzyme assay or SDS-PAGE analysis.
Purification of Recombinant Azoreductase by Red-Sepharose
CL-6B--
The cells from 200 ml of culture were suspended in 20 ml of
20 mM sodium-phosphate buffer, pH 7.0 and lysed by freezing
and thawing followed by sonication (15 s, 70% output, 10×). After centrifugation at 9000 × g for 30 min, the supernatant
was applied to a Red-Sepharose CL-6B column (Amersham Pharmacia
Biotech) followed by washing with 20 mM sodium phosphate
buffer, pH 7.0. The recombinant azoreductase was eluted from the column
with 10 mM -NADH. The eluate was dialyzed against two
changes of 1000 volumes of 20 mM sodium phosphate buffer,
pH 7.0 and used for enzyme assay.
Enzyme Assays--
Azo dye-reducing activity was analyzed by
measuring the decrease in optical density at suitable wavelengths with
a Hitachi U 3300 spectrophotometer at various temperatures basically
according to Pasti-Grigsby et al. (15). The reaction mixture
in a total volume of 1.0 ml consisted of various concentrations of azo
dyes (Roccelin, Solar Orange, Sumifix Black B; shown in Fig.
3) in 20 mM sodium phosphate
buffer, pH 7.0, and bacterial lysates or purified enzyme. The reaction
mixture was preincubated for 5 min at the assay temperature, and the
reaction was started by the addition of 25 µl of various
concentrations of -NADPH. The enzymatic activities were measured by
the decrease in optical density at optimal wavelengths. The enzyme
activity was expressed as the amount of reduced dye per min with 1 mg
of enzyme. Kinetic parameters for the reduction of each dye by native
and recombinant azoreductases were estimated by nonlinear regression
analysis according to Shimada et al. (16).
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RESULTS AND DISCUSSION |
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We have been interested in biological reduction of azo dyes under
aerobic conditions. The utilization of azo dye-degrading microorganisms
is very important for generating an efficient bioreactor for
waste-water treatment plants in industries that use azo dyes. In
addition, these enzymes can be applied for white discharge printing of
cloths. In the course of our study, we have isolated three bacterial
strains that can reduce azo dyes under aerobic conditions. A
constitutively expressed enzyme in Bacillus sp. OY1-2 (one
of the isolated strains) was purified from bacterial cell lysates and
characterized with respect to reduction of different kinds of azo dyes
in the presence of -NADPH (10). Subsequently, we cloned and
characterized the gene encoding this protein activity.
Cloning of the Gene Encoding the Azoreductase--
To clone the
azoreductase gene of Bacillus sp. OY1-2, the N-terminal
amino acid sequence of purified azoreductase was determined. Because
there was neither significant amino acid nor nucleotide homology to the
N-terminal amino acid sequence in the databases, this protein was
determined to be novel. We attempted to clone the gene encoding this
enzyme by means of screening the genomic library of Bacillus
sp. OY1-2 with digoxigenin-labeled probe amplified by PCR using primers
designed from the N-terminal amino acid sequence of azoreductase (Fig.
2A). A 0.1-kbp DNA fragment was amplified by PCR using
primers AZR-1 and AZR-2. The PCR product carrying the DNA fragment
encoding the N-terminal part of azoreductase was extracted from the
agarose gel, directly subcloned into the pT7Blue(R)T vector, and
sequenced (Fig. 2B). A Dig-labeled hybridization probe was
synthesized by PCR using the plasmid carrying the azoreductase N-terminal part as a template. A genomic library of Bacillus
sp. OY1-2 was constructed with the EcoRI-digested phage
vector gt10 by ligation with the completely
EcoRI-digested fragments of total genomic DNA. Transfection
of the host E. coli C600hfl by the in vitro
packaged library gave independent clones from 1.1 × 106 plaques. By using Dig-labeled probe, seven clones with
inserts of the same length were isolated from the 2 × 105 plaques. Inserts in these seven clones were amplified
by PCR and sequenced. One of the clones (
gt10-AZR5) was then
subcloned into the E. coli vector pUC18 (pUC18-AZR5-8) for
further analysis. The entire sequence of the 1.2-kbp insert was
determined according to the sequencing strategy shown in Fig.
4, and an ORF was found in this fragment
(Fig. 5) using the specific nucleotide
sequence as a probe. An inframe initiation codon (ATG) was located at
nucleotide 32, as indicated in Fig. 5 (boxed), and a
putative Shaine-Dalgarno sequence GGAG (17) (Fig. 5, double
underline) was found in its upstream region. The deduced amino
acid sequence from this initiation codon showed good agreement with the
N-terminal amino acid sequence determined from the purified native
azoreductase (Fig. 5, underline). In addition, the molecular
mass of the azoreductase product calculated from the gene encoding
azoreductase was 19,423 Da, which exhibited good agreement with the
molecular mass of 20 kDa of the purified azoreductase (10). Thus we
concluded that the ORF found in this 1.2-kbp EcoRI DNA
fragment coded for the azoreductase. However, as the upstream region of
this clone was only 31 bp, the sequence of the putative promoter region
could not be identified. When we searched for homologs from other
organisms in the DNA and protein databases, an ORF of Bacillus
subtillis (function unknown) was obtained with an amino acid
sequence homology of 52.8% (GenBankTM/EBI database,
accession no. Y14079), although it was not present in the protein
databases. Thus, the azoreductase gene cloned and characterized in this
study was a member of a novel family of enzymes. The deduced amino acid
sequences from the azoreductase gene and the B. subtillis
ORF are aligned in Fig. 6. Although the
overall homology between them was 52.8%, only 2 of 17 C-terminal amino
acids were identical, suggesting that the 17 C-terminal amino acid
sequence may not be essential for azoreductase activity. This should be
further investigated by examining the activities of truncated
recombinant proteins. The NADH binding motif
(GXGXXG) found in NADH-dependent
enzymes such as lactate dehydrogenase (18), alcohol dehydrogenase (19),
and hydrotransferase (20) was found in the deduced amino acid sequences
of these two genes (Fig. 6, underlined).
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Southern Blot Hybridization--
Fragments of genomic DNA
generated by digestion with restriction enzymes were separated on a
0.7% agarose gel, transferred onto the nylon filter, and hybridized
with the 1.2-kbp EcoRI fragment of genomic DNA carrying the
entire open reading frame of azoreductase. The probe hybridized to DNA
fragments of length 8.0, 20, 25, 6.6, 5.0, 1.2, and 11 kbp cut by
restriction enzymes BamHI, EcoRI, HindIII, PstI, SalI, SmaI
and XbaI, respectively (Fig.
7). The probe hybridized to only one band
for each restriction enzyme tested, indicating that the azoreductase
gene exists as a single copy gene on the chromosome.
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Expression of the Azoreductase in E. coli--
The entire open
reading frame of the azoreductase gene was amplified by PCR using
pUC18-AZR5-8 as a template, was inserted into expression vectors
pTrx-Fus and pTrcNd, and was transformed into E. coli to
express recombinant azoreductase (Fig.
8A). The expression of
azoreductase was observed not in pTrcNd but in pTrx-Fus (data not
shown). The reason that the azoreductase was expressed only by the
pTrx-Fus system is not clear although it might be attributed to the
different promoters as follows: the PL-Trp promoter used in pTrx-Fus
can be controlled strictly by cI repressor and tryptophan
starvation, whereas the control of the trc promoter (fusion promoter of
T5 phage promoter and lactose operator, inducible by addition of IPTG)
used in pTrcNd is more leaky than the PL-Trp promoter. The expressed
azoreductase may have a negative effect on the growth or survival of
E. coli and inhibit the colony formation of transformed
E. coli in the pTrcNd system.
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The expressed azoreductase in plasmid (pTrx-Fus) in E. coli GI724 and GI618 was analyzed by SDS-PAGE to identify the protein with a molecular mass of 20 kDa (Fig. 8B). The 20-kDa protein on SDS-PAGE was then transferred onto a polyvinylidene membrane and subjected to N-terminal amino acid sequence determination. The resulting N-terminal amino acid sequence corresponds to the N-terminal amino acid sequence of native azoreductase. These results indicate that the recombinant protein obtained in this study was the same as the native azoreductase obtained from Bacillus sp. OY1-2.
Azo Dye-degrading Activity of Crude Recombinant
Azoreductase--
The E. coli GI724 and GI618 expressing
recombinant azoreductase were lysed by freezing and thawing followed by
sonication. Recovery of supernatant fractions following centrifugation
and SDS-PAGE analysis revealed larger amounts of recombinant
azoreductase in the GI618 supernatant than the GI724 samples (data not
shown). The activity of azoreductase in 5 µl of the sonic supernatant (containing 0.1 mg of protein) of GI618 was compared with that of
original Bacillus sp. OY1-2. The azo dyes Roccelin, Solar
Orange, and Sumifix Black B were used as substrates for this assay.
These compounds were reduced into a colorless mixture by the
azoreductase in the sonic supernatants. The reaction mixture consisted
of 20 µM substrate, 20 mM sodium phosphate
buffer, pH 7.0, 250 µM -NADPH. The azoreductase
activity in the GI618 supernatant was ten times or more greater than
that of Bacillus sp. OY1-2 (Fig.
9). These results indicate that the
recombinant azoreductase expressed in E. coli contains a
large amount of azoreductase activity.
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Purification of Recombinant Azoreductase from E. coli
Lysate--
The recombinant azoreductase was purified from the
supernatant of GI618 bacterial lysate by means of affinity purification by Red-Sepharose column chromatography. The recombinant azoreductase was eluted with 10 mM -NADH (Fig.
10A) and was purified in one step of column chromatography (Fig. 10B). In contrast, our
standard protocol for purifying the azoreductase from the lysate of
Bacillus sp. OY1-2 required four steps of column
chromatography (DEAE-cellurofine, Blue-cellurofine, and
Red-Sepharose followed by gel filtration, Ref. 10). However, as the
amount of expressed recombinant azoreductase in E. coli in
this system was very large, only the Red-Sepharose column
chromatography was necessary to obtain purified azoreductase of the
same quality. Thus, the azoreductase expression system designed in this
study may make it possible to obtain large amounts of purified
azoreductase in a very simple manner.
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Azo Dye-reducing Activity of Recombinant Azoreductase--
The
eluate obtained by NADH elution from Red-Sepharose was then dialyzed
against 20 mM sodium phosphate buffer, pH 7.0 and used in
the azoreductase enzyme assay. The enzyme assay was performed at
different reaction temperatures from 20 to 85 °C in a reaction mixture containing 20 µM Roccelin and 250 µM -NADPH. The maximum specific activity of
recombinant azoreductase (7.60 µmoles/min/mg protein) was achieved at
50 °C whereas that of native enzyme (11.7 µmoles/min/mg protein)
was achieved at 70 °C (Fig. 11). The
maximal specific activity of recombinant azoreductase was 1.54 times
lower and the optimal temperature was 20 °C lower than that of the
native form. These results indicate that the temperature stability of recombinant azoreductase was lower than that of the native enzyme. The
reason why the temperature stability of recombinant enzyme declined was
not defined in this study, but will be elucidated in future
studies.
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Kinetic analysis was performed with different concentrations of
substrates at 25 °C in a reaction mixture consisting of 20 mM sodium phosphate buffer, pH 7.0, 20 mM
-NADPH, and native and recombinant enzymes. The value of
Vmax and Km for three dyes
were estimated by nonlinear regression analysis (Fig. 12). The
Vmax/Km value of recombinant
azoreductase for Rocceline was 3.03, which is very close to 2.78 of the
native enzyme. Similar observations were found with other substrates (Sumifix Black B, 0.15 and 0.14; Solar Orange, 0.013 and 0.014; by
native and recombinant azoreductase, respectively). The efficiency (Vmax/Km) for Rocceline was
about 20- and 200-fold greater than for Sumifix Black B and Solar
Orange, respectively, indicating that the former substrate was better
than the others. Considering the structure of dyes used in this study
(Fig. 3), the compounds with paired naphthalene groups coupled with the
azo group may serve as good substrates. This should be clarified in
future studies.
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Heiss et al. (21) described the cloning of DNA from a
Rhodococcus strain that confers the ability of decolorizing
azo dyes. However, no characterization of the gene was presented in his study. Recently, the azoreductase gene was cloned from an anaerobic bacteria Clostridium perfringens (gt11 genomic library)
by means of screening with an antibody raised against purified
azoreductase (10). Rafii and Coleman (10) observed azoreductase
activity in cell lysates of lytic and lysogenic E. coli
cultures infected with recombinant phage. In addition, they have
demonstrated the existence of a similar gene in some anaerobic bacteria
with Southern hybridization techniques. As the nucleotide sequence of
the azoreductase of C. perfringens was not presented in the
study, it is not clear whether the enzyme involved in the reduction of
azo dye in C. perfringens has some homology with the
azoreductase gene cloned in this study. This should be investigated in
the future. However, with regard to waste-water treatment plant
construction using azoreductases, reduction of azo dyes under the
aerobic conditions reported in this study is more practical. Thus, the
azoreductase gene cloned and characterized in this study may be a good
candidate for construction of a bioreactor for the treatment of azo
dye-containing waste-water.
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ACKNOWLEDGEMENTS |
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We thank Dr. Tsutomu Shimada for his kind discussions on the kinetic studies.
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FOOTNOTES |
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* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ GenBankTM/EBI Data Bank With accession number(s) AB002631.
§ To whom correspondence should be addressed. Tel.: 81-6-6972-1321(ext. 268); Fax: 81-6-6972-0772; E-mail: suzuki@iph.pref.osaka.jp.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M008083200
1 W. Sugiura, T. Mamashita, T. Yokoyama, and M. Arai, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; kbp, kilobase pairs; ORF, open reading frame; PCR, polymerase chain reaction; Dig, digoxigenin.
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