Synergistic action of flavin containing NADH dependant azoreductase and cytochrome P450 monooxygenase in azoaromatic mineralization

Article abstract of DOI:10.1039/c2ra21389c

An alkaliphilic strain Bacillus lentus BI377 was isolated from contaminated soil of the textile area of Solapur, India. The strain was able to degrade almost 98% of recalcitrant azoic compounds by a mutually regulated process of azoreductase and a monooxygenase system. An enzyme activity study and a periodical carbon monoxide (CO) binding spectra study on a UV-visible spectrophotometer revealed that the intermediate amines formed by typical azoreduction (NN cleavage), subsequently underwent hydroxylation by the cytochrome P450 monooxygenase (CYP450) system. Azoreductase was purified by chromatographic techniques and characterization by MALDI-TOF substantiated its identity as FMN containing NADH dependent azoreductase of 32 kDa in size. Surprisingly, purified azoreductase showed the highest activity at 80 °C and pH 8.0. An increase in the activity of superoxide dismutase after decolorization confirmed the signature of oxidative stress and its involvement in the dismutation of reactive metabolites. Intermediate metabolite analysis by HPLC, GC-MS and FTIR and the removal of total organic carbon (TOC) suggested the azoaromatics' degradation leads to mineralization via a TCA cycle.

Full text of DOI:10.1039/c2ra21389c

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Synergistic action of flavin containing NADH dependant
azoreductase and cytochrome P450 monooxygenase in
azoaromatic mineralization3
Cite this: RSC Advances, 2013, 3,
3062
Chetan C. Oturkar,a Munif A. Othman,b Mahesh Kulkarni,c Datta Madamwarc
and Kachru R. Gawai*b
An alkaliphilic strain Bacillus lentus BI377 was isolated from contaminated soil of the textile area of
Solapur, India. The strain was able to degrade almost 98% of recalcitrant azoic compounds by a mutually
regulated process of azoreductase and a monooxygenase system. An enzyme activity study and a
periodical carbon monoxide (CO) binding spectra study on a UV-visible spectrophotometer revealed that
the intermediate amines formed by typical azoreduction (NLN cleavage), subsequently underwent
hydroxylation by the cytochrome P450 monooxygenase (CYP450) system. Azoreductase was purified by
chromatographic techniques and characterization by MALDI-TOF substantiated its identity as FMN
containing NADH dependent azoreductase of 32 kDa in size. Surprisingly, purified azoreductase showed
the highest activity at 80 uC and pH 8.0. An increase in the activity of superoxide dismutase after
decolorization confirmed the signature of oxidative stress and its involvement in the dismutation of
reactive metabolites. Intermediate metabolite analysis by HPLC, GC-MS and FTIR and the removal of total
organic carbon (TOC) suggested the azoaromatics’ degradation leads to mineralization via a TCA cycle.
Received 18th April 2012,
Accepted 16th December 2012
DOI: 10.1039/c2ra21389c
www.rsc.org/advances
anaerobic conditions to yield aromatic amines which are in
turn likely to be removed by aerobic treatment.1,2,5,6 The
typical reduction of these azoaromatic compounds is mainly
attributed to azoreductase, which cleaves the azo bond by a
Introduction
Azoic compounds constitute the largest group of mutagenic
and carcinogenic xenobiotics that are commonly discharged
into the ecosystem.1,2 They are characterized by aromatic
moieties, generally benzene or naphthalene groups, linked
together with one or more azo groups (–NLN–), which may
contain different substituents, such as chloro (–Cl), methyl
(–CH3), nitro (–NO2), amino (–NH2), hydroxyl (–OH), carboxyl
(–COOH) or sulfonic (–SO3H) groups.3 Apart from the conven-
tional approaches to dye removal,2 the increasing demand for
economically feasible enzyme associated bioprocesses seems
to offer an enormous potential to provide whole cell
biocatalysts with great applicability.2,4 In the last two decades,
there has been a tremendous upsurge in the exploitation of
ecofriendly alternatives to deal with azoaromatics’ bioreme-
diation. The redox potential generated by an anaerobic–
aerobic process accelerates site specific cleavage in complex
aromatics so that electron withdrawing azo linkages, which are
very resistant to oxidative reactions, can be reduced under
ping pong mechanism, using NADH or NADPH as
a
cofactor.1,2,7 The attainment of azo bond cleavage leads to
another problem as it generates toxic amines or reactive
oxygen species, which are intermediate metabolites in
biomass.8 This unexpected bioactive interference creates a
burden on cells and hinders the degradation/mineralization
process by affecting the growth of biomass or by enzyme
repression.9 To overcome this problematic intermediate
saturation, a sequential anaerobic–aerobic bioreactor has
been increasingly employed as an ecofriendly alternative.6,7
In light of recent developments in bioremediation, aerobic–
anaerobic processes will be difficult to justify because of their
time consuming nature and high cost.
CYP450 is the main component of the mixed function
oxidase system with great catalytic versatility and plays an
important role in the biotransformation of a wide variety of
xenobiotics.10 A literature survey reveals that its induction in
response to aromatic amines10–12 continues as nucleophilic
reactions so that the hydrophilicity of these reactive metabo-
lites will be utilized as a source of energy via the central
metabolic pathway (TCA cycle) in bacteria.12,13 The current
experimental practice of utilizing lacasse enzymes for the
oxidative clevage of azo dyes reflects the single step nature of
aBRD School of Biosciences, Sardar Patel University, Vallabh Vidyanagar, India.
E-mail: chetanvc@gmail.com
bDepartment of Chemistry, University of Pune, India.
E-mail: chetanvc2000@yahoo.com; Fax: +91-020-25691728; Tel: +91-020- 2569606
cCenter for Material Characterization, National Chemical Laboratory, Pune, India
Electronic supplementary information (ESI) available: Details of UV Visible
3
spectra of dye decolorization, GC-MS fragmentation and FTIR frequencies,
substrate specificity study, MALDI-TOF MSMS ion peaks of azoreductase.
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the degradation, which results from its ability to use single
molecular oxygen as an electron acceptor, despite the cofactor
being present, non-specific oxidation,4 highly non-specific free
radical mechanism and the formation of phenolic type
compounds.9 Moreover, various oxidoreductase activities have
been tested using consortia with the highest reductase and
oxygenase activity e.g. from Pseudomonas putida MET 94 and
Bacillus subtilis for ecofriendly degradation or mineralization
respectively.5,14 It has been observed that bacteria contain the
flavin-diffusible monooxygenase (FDM) system in which free,
reduced flavin is first generated by the flavin reductase
component of the enzyme and then transferred to the
oxygenase component for activation.15 However, bacterial
biotransforming enzymes have significant limitations due to
physicochemical factors like temperature, concentration and
the nature of xenobiotics.16 Hence, it is a prerequisite to
isolate a strain with superior redox machinery to facilitate
microbial reactions even under extreme conditions. Recently,
research on thermostable azoreductases has come into the
limelight and focuses on the reduction of azoaromatics at high
temperature e.g. azoreductase from G. stearothermophilus was
found to be active at 60 uC.17 Previously, we reported the
isolation and identification of potential soil isolate Bacillus
lentus BI377 (B. lentus BI377) and its considerable catabolic
diversity towards sulphonated dyes.18
With this in mind, a mechanistic study of azoic miner-
alization was carried out using a wide range of azo dyes viz.
Red120 (RR120), Red141 (RR141), Red2 (RR2), Red11 (RR11)
Sunset Yellow (SY) and Tartrazine (TAR), and the intermediate
moieties formed during biodegradation were analyzed by gas
chromatography–mass spectroscopy (GC-MS), Fourier trans-
form infrared (FTIR) and high-performance liquid chromato-
graphy (HPLC) to elucidate a plausible degradation pathway.
Also, in the present study, it is the first time that a successful
attempt has been made to purify and characterize azoreduc-
tase from the gram positive alkaliphilic B. lentus BI377. The
critical role of the CYP450 monooxygenase system and
superoxide dismutase (SOD) in the process of mineralization
was also studied by a carbon monoxide (CO) binding assay and
an enzyme activity study.
Results and discussion
Whole cell decolourization assessment
The decolourization percentage and the shift in the original
absorbance of each dye were monitored every 2 h till the
complete decolorization under static conditions was achieved
(ESI Fig. S1 ), and the relevant enzyme activity studies of the
3
control as well as the decolorized sample were measured as
shown in Table 1. Almost 75% decolorization was observed in
the case of all azo dyes within 4 h, whereas, eight hours after
the culture was supplemented with azoic dyes, 99% decolor-
ization was established. Significant azoreductase activity was
noted at 4 h, which was independent of NADH or NADPH1,2
since the reduction reaction with the purified enzyme takes
place in the presence of such cofactors. Maximum decoloriza-
tion was achieved with a linear growth in biomass at optimum
pH 8.0. Whereas, a gradual decrease in the original absor-
bance with an increasing activity of azoreductase confirmed its
involvement in the reduction of the chromophoric group
(NLN) of all the dyes.19
In other cases, the content of CYP450 and the specific
activities of the superoxide dismutase were significantly
increased compared to the control and surpass azoreductase
activity after
4 h of decolorization. Plausible bioactive
metabolites could have suppressed the activity of azoreductase
and repressed the production of CYP450 and the activity of
SOD.20–22
Purification and characterization of azoreductase
The induced intracellular azoreductase for azo dye degrada-
tion was purified from B. lentus BI377 and precipitated with
(NH4)2SO4 (80%), followed by anion exchange column chro-
matography on DEAE-cellulose and size exclusion chromato-
graphy on Sephadex G-100 which allowed the recovery of 2.8 U
in 2.6 mg ml21 of protein. The initial total amount of protein
(34.8 mg ml21), equivalent to an activity of about 0.41 U of
azoreductase, was obtained from the crude extract of disrupted
cells. The results of the purification of azoreductase from B.
lentus BI377 are summarized in Table 2.
The effort has been successfully made to achieve 52.09 fold
purification with 40% recovery (approximately) and 26.57 U
mg21 of the total specific activity. The presence of a single
band on both native and SDS-PAGE and a single band during
Table 1 The whole cell activities during the decolourization of azo dyesa
Decolorization percentage Azoreductase activity (U mg21
)
CYP450 content (nmol mg21 protein)
SOD (U mg21
)
Substrates lmax (nm) 4 h
6 h
0 h
4 h
6 h
0 h
4 h
6 h
0 h 4 h 6 h
RR120
RR141
RR5
RR11
SY
530
540
550
550
460
75.2
85.4
76.7
79.6
81.7
85.7
97.7
98.9
95.8
97.7
98.4
97.2
0.051
0.044
0.046
0.048
0.024
0.033
0.071
0.062
0.074
0.068
0.079
0.084
0.074
0.065
0.078
0.071
0.080
0.088
0.031
0.031
0.037
0.025
0.021
0.024
0.032
0.041
0.040
0.031
0.030
0.029
0.062
0.066
0.058
0.054
0.066
0.071
422 492 762
431 484 766
437 474 858
425 461 854
421 473 866
424 489 871
Tartrazine 470
a
Note: All activities were recorded in triplicate and average values are mentioned in the table. Zeroth hour reading values are from control
flasks.
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Table 2 Summary of the purification of azoreductase from B. lentus BI377
Purification step
Protein (mg ml21
)
Total activity (U)
Specific activity (U mg21
)
Purification fold
Crude extract
80% (NH4)2SO4
Dialysis
Ion exchange
Gel filtration
24.8
3.16
2.82
2.6
0.41
1.26
1.52
2.8
0.51
12.05
16.22
23.33
26.57
1.0
23.62
31.8
46.39
52.09
0.6
2.65
gel filtration on a Sephadex G-100 column indicate the
electrophoretic homogeneity (Fig. 1).
theoretical values of azoreductases from various bacterial
isolates which have been used for azoic dye degradation.23a,b,24
A specific activity of 26.57 U mg21 for azoreductase was
Characterization and substrate specificity study of the purified
azoreductase
noted after purification which corresponded to
a 40%
improvement as compared to the crude extract. Protein
separation by SDS-PAGE resulted in a single band equivalent
to a molecular mass of 32 kDa in agreement with previous
To observe the effect of pH, the activity of the purified enzyme
was monitored over a wide pH range (4–10). It was found that
the enzyme azoreductase showed significant activity in the pH
range of 7–9 as compared to other pH ranges, however, the
optimum pH was established to be pH 8.0 (Fig. 2a).
These observations were quite similar to those for other
azoreductases near to neutral pH.17,25,26 Moreover, alkaliphilic
strain Bacillus lentus always regulates its intracellular pH in
such a way that it will remain near neutral.27 Studies
concerning the thermostability were performed at different
temperature points of 40–100 uC at pH 8.0 (Fig. 2b). The
azoreductase activity linearly increased in the temperature
range from 40 to 70 uC but the optimum activity was found at
80 uC. The optimum pH and temperature conditions for
purified enzyme were 8.0 and 80 uC respectively, nevertheless
the maximum stability and maximum relative activity (¢70%)
were observed in the range of 50 to 70 uC for three days. Very
few scientific reports are available on thermostable alkaliphilic
azoreductase at 80 uC, therefore this uniqueness could be
beneficial for bioremediation even under extreme condi-
tions.25 To obtain the Km and Vmax value electron donor study,
the enzyme activity was measured by varying the concentra-
tions of one of the substrate dyes, NADH and NADPH, and
keeping the concentration of another substrate fixed. Their
individual values were determined by double reciprocal plots
as shown in Table 3.
Fig. 1 Molecular mass estimation of purified azoreductase by SDS-PAGE (lane 1:
marker, lane 2: enzyme).
Fig. 2 Effect of a) pH & b) temperature on purified azoreductase activity.
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Table 3 Kinetics parameters of purified azoreductase with TAR and electron donorsa
Substrates
Azoreductase activity (U mg21
)
Km (mM)
Vmax (mmol min21 mg21
)
kcat (s21
)
Tartrazine
NADH
NADPH
0.123 ¡ 0.007
0.056 ¡ 0.005
0.042 ¡ 0.004
0.20 ¡ 0.08
0.32 ¡ 0.11
0.42 ¡ 0.04
11 ¡ 2
20 ¡ 5
13 ¡ 2
4.5 ¡ 0.12
4.6 ¡ 0.14
2.2 ¡ 0.13
a
Note: All assays were done in triplicate. Values are mean ¡ standard deviation.
It was shown that the Vmax values are significantly close to
each other, however, the catalytic efficiency of azoreductase in
the presence of NADH was found to be higher than with
NADPH. Hence, it was established that NADH dependant
azoreductase is involved in the TAR reduction process.24 The
catalytic efficiency of the purified azoreductase was screened
using azo dyes viz. RR120, RR141, RR5, RR11, SY and TAR
which were used in whole cell decolorization previously.
Eventually, study revealed that the purified enzyme azoreduc-
ylamino) naphthalene-1,3,6-triol [339.13 m/z] (5), 2-amino-
naphthalene-1,6-diol [175 m/z] (7), and 1,4-diaminobenzene
[108 m/z] (8) was established from GC-MS fragmentation (m/z)
ions for the metabolites isolated after 4 h of incubation with
RR141, RR11, RR5, SY and TAR respectively. Broad peaks were
observed in the FTIR spectra at 4 h. Those at 3452.3 and 3500
cm21 were due to N–H stretching, those at 2921.0 to 2852.0
cm21 were due to C–H stretching and those at 1045.0 and 1032
cm21 were due to S–O stretching.30 This helps to understand
that the metabolites at 4 h viz., 1, 3, 5, 7 and 9 are the cleaved
products of azoreduction and simultaneous transformation.30–32
Eventually, the enzyme activity study and spectroscopic data
confirmed the involvement of azoreductase in the cleavage of the
azo bond (NLN) thereby leading to the formation of aromatic
amines under static conditions.30,33 After 6 h, the major
metabolites were found to be 8-(methylamino) naphthalene-
2,3,6-triol [205.21 m/z] (2), 8-aminonaphthalene-1,3,6-triol
[191.18 m/z] (4), naphthalene-1,3,6,8-tetraol [207.18 m/z] (6), as
established by the GC-MS fragmentation m/z ion peaks and by
FTIR frequencies at 2923, 2926, and 1660, cm21 for RR141, RR11
and RR5 respectively. After six hours, FTIR frequencies were
observed at 2923, 2926, 2853 and 2850 cm21 for the C–H
aliphatic stretch for RR141, RR11, SY and TAR metabolites
respectively. A FTIR frequency at 3404 cm21 corresponding to the
O–H stretch for metabolites of tartrazine confirmed the forma-
tion of lower molecular weight phenolic and/or aliphatic
metabolites by an aerobic multistep conversion process.
Metabolites 2, 4 and 6 were generated by the oxidation
reaction.18,31,34,35
tase has a wide range of substrate specificity (ESI Table S1 ).
3
Nevertheless, it has shown maximum activity for TAR dye and
minimum activity for RR11 dye. The highest activity against
TAR as compared to other dyes may be due to the COONa
group ortho and SO3Na group para to the azo bond. Both
highly electronegative groups would withdraw the electrons
from the azo bond via resonance, leading to the azo bond
being more electrophilic and reductive for color removal.28
Identification of the purified azoreductase by MALDI-TOF
The protein band corresponding to the zone of clearing on the
native PAGE assay was analyzed by trypsinolysis, MALDI-TOF
mass spectrometry and database searching. Utilizing this
approach, the principal protein in the band was identified as
FMN containing NADH dependant azoreductase NCBI data-
base information. After a database search, the purified protein
was found to have a high sequence homology to FMN-
dependent NADH-azoreductase of Bacillus thuringiensis
(Accession No. ZP_04075052.1) and Bacillus cereus AH1273
(Accession No. ZP_04177368.1). The identification of the
azoreductase was based on matching 9 of 16 experimental
ion masses which were assigned from the most intense peaks
The CO binding spectra exhibited a characteristic induction
in response to azo dyes and their intermediate metabolites
(Fig. 3).
(ESI Fig. S2 ).
3
The magnitude of the peak at 450 nm increased just after
the addition of RR120, RR141 and SY whereas those for RR5,
RR11 and TAR appeared after 2 h of incubation till the process
of mineralization was complete (8 h). Thus, the level of
induction and content of CYP450 in the case of all dye
degradations suggests its possible involvement in desulphona-
tion as well as deamination of aromatic intermediate
metabolites via hydroxylation.2,12,18 The induction of SOD
activity marked the generation of oxidative stress and
indicated their subsequent action in the dismutation of
reactive intermediate metabolites.9,21 Hence, the entire pro-
cess revealed the imperative redox potential of the strain B.
lentus BI377 to carry out nonspecific azoaromatic mineraliza-
tion by a mutually regulated process in the proximity of its
outer membrane.13 In due course, these biotransforming
enzymes along with azoreductase were solely responsible for
the degradation of the toxic dyes into aromatic amines and the
Spectrophotometric, bioanalytical, and enzyme analysis of
azoaromatic degradation
UV-visible spectra analysis of the decolorized samples revealed
that maximum decolorization was achieved within 8 h in the
case of all substrate dyes. A significant decrease in the original
absorbance maxima of the dye within two hours, and the
appearance of new intense peaks at 450 nm after four hours
indicated the formation of lower molecular weight aromatic
metabolites.1,2,29
Based on the GC-MS (ESI Fig. S3 ) and FTIR (ESI Fig. S4 )
3
3
analysis of the intermediate metabolites, possible major
fragmented products are illustrated in Table 4. In correlation
to this, the formation of 5-(4-amino-6-chloro-1,3,5-triazinyla-
mino)-3-aminonaphthalene-2,7-diol [318.72 m/z] (1), sodium 4-
(4,6-dichloro-1,3,5-triazin-2-ylamino)-5-hydroxynaphthalene-
2,7-disulfonate [511.22 m/z] (3), 8-(4,6-dichloro-1,3,5-triazin-2-
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Table 4 Summary of the major intermediate metabolites and % TOC during 4 to 6 h of decolorization/degradationa
Four hours of incubation
Major intermediate metabolites
Six hours of incubation
Major intermediate metabolites
Substrate dyes
RR141
%TOC
11.3
%TOC
2.24
RR11
9.2
1.29
RR5
9.7
1.58
SY
8.2
0.42
TAR
7.9
0.51
a
Note: The metabolites 8 and 10 were not detected due to an insufficient quantity of sample for characterization. They are proposed plausible
intermediates during the degradation process of SY and TAR respectively.
further opening of all aromatic nuclei to aliphatic form and
their subsequent transformation to CO2 and H2O under
microaerophilic conditions.12,13,18 HPLC analysis of the
degradation product of TAR showed major peaks at retention
times of 3.15 and 3.69 min.
After comparing these intermediate metabolites with stan-
dards it was found that the peak of 1,4-diaminobenzene
(retention time 3.18 min) corresponds to the chromatogram of
the 4 h intermediate metabolites of TAR degradation (Fig. 4),
which supported the strong possibility of the formation of 1,4-
diaminobenzene by the reduction mechanism of azoreductase
by cleaving the azo linkage (NLN) which was further confirmed
by GC-MS and FTIR and enzymatic studies (as discussed
earlier). Hence, under this notion, the proposed pathway of
tartrazine reduction could be illustrated as shown in Fig. 5.
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quinone related metabolites resulting from the tyrosinase
enzyme activity (data not shown). The activity of tyrosinase
remains unchanged during dye degradation, suggesting the
possible oxidative cleavage of catechol into aliphatic metabo-
lites via the cis-muconic acid pathway.18,38 Eventually, the trace
of aliphatic metabolites and decreasing % TOC (Table 4)
confirmed that the recalcitrant sulphonated dyes’ degradation
pathway leads to complete mineralization via the TCA cycle.2,30
Experimental
Chemicals
DEAE-cellulose and Sephadex G-100 were purchased from
Sigma Aldrich, USA. Precision plus Protein Standard 161-
0373EDU was purchased from Bio-Rad, USA and NADH was
procured from SRL Chemicals, India. All azo dyes viz. RR120,
RR141, RR2, RR11, SY and TAR were procured from a textile
plant in Solapur, India (see the molecular structure in ESI Fig.
S 5 ). All other chemicals were of the highest grade of purity.
3
Microorganism, growth medium and culture condition
The soil isolate B. lentus BI377 was grown in nutrient broth
(NB) containing (g l21) peptone, 5; yeast extract, 5; and NaCl, 5,
aerobically at 40 uC for 24 h in Erlenmeyer flasks (500 ml
capacity).18 The media pH 8.0 was adjusted by sodium
phosphate buffer using a EUTECH-510 pH meter.18
Fig. 3 The CO-binding cytochrome P450 spectra of A) RR120, B) RR141,C) RR5,
D) RR11, E) SY and F) TAR decolorization/degradation [——— (0 h),
(4 h), (6 h), (8 h)].
(2 h),
Decolourization assessment with whole cell activity
Analysis could not succeed in isolating the intermediate
catechol during each dye degradation but the resemblance of
most the metabolites (7 and 9 in Table 4) strongly suggested its
existence (8 and 9)18,35,36 and its further utilization by the
central metabolic pathway.36,37 The progress of catechol
metabolism by B. lentus BI377 was monitored by measuring
To study the whole cell activity during decolorization, the
strain was grown in 100 ml of the same medium in 500 ml
Erlenmeyer flasks for 12 h at 40 uC under static conditions
before the addition of substrate azo dyes to a final concentra-
tion of 10 mg l21. A control sample measurement was done
Fig. 4 HPLC chromatogram of TAR degradation at 4 h and 1,4-diaminobenzene standard (inset).
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Fig. 5 Proposed pathway of TAR degradation by B. lentus BI377.
before the addition of the dye at 0 h. Decolorised sample
aliquots were withdrawn from the control and the decolorised
sample flask at 2 h intervals and centrifuged at 10 000g for 15
min using a DuPont Sorvall RC-5B refrigerated centrifuge and
the same procedure was repeated till the maximum decolor-
ization was obtained. The supernatant was used to determine
the percentage of decolorization by spectrophotometric
measurement19 and the pelleted cell mass was suspended in
50 mM sodium phosphate buffer (pH 7.2) for enzyme study.
Purification of the azoreductase enzyme
The crude cell free extract was subjected to ammonium
sulphate precipitation. In the resulting supernatant the
addition of ammonium sulphate was continued to get 80%
saturation. The precipitated protein was separated by centri-
fugation in cold conditions and was dissolved in phosphate
buffer pH 7.0 (0.1 M). The activity of azoreductase and the
protein concentration was determined at the end of each
purification step. Redissolved precipitate was dialyzed against
the same buffer and was stored at 4 uC for further purification.
After the dialysis, the sample was applied to a DEAE-cellulose
column (2 cm 6 45 cm) equilibrated and washed with 0.1 M
phosphate buffer (pH 7.0) and eluted enzyme The enzyme was
eluted from the DEAE- cellulose column by phosphate buffer
pH 7.0 (0.1 M) containing 0 to 0.5 M NaCl linear gradient at a
flow rate 4 ml for 15 min. The fractions with the maximum
activity of azoreductase were pooled together and loaded on a
Sephadex G-100 column. The protein was eluted by phosphate
buffer and the fraction of 4 ml each was collected at the rate of
1 ml min21. The fractions with the maximum activity were
pooled together and concentrated by reverse dialysis. The
purity of the enzyme was judged by polyacrylamide gel
electrophoresis according to Laemmli 1970 with slight
modification.43 The molecular weight of the enzyme was
determined by comparing the electrophoretic mobility of the
enzyme with Precision plus Protein Standards (Bio-Rad USA,
Precision plus Protein All Blue Standards 161-0373EDU).
Preparation of the cell free extract and enzyme assays
The pelleted cell mass was resuspended in the same buffer
and centrifuged three times to remove the residue of the dyes.
Cell disruption was carried out by using a probe sonicator (6 s
cycle and 60 Hz by Sonoplus HD 70, Bandelin, Berlin,
Germany). The resulting homogenate was centrifuged at
10 000g for 30 min at 4 uC and the supernatant was used for
enzyme purification. The protein concentration was estimated
by Lowry’s method.40 The azoreductase activity was deter-
mined according to the protocol of Zimmermann23b with
slight modification. In a typical procedure, the reaction
mixture containing 100 mM phosphate buffer (pH 7.4), 2
mM NADH, 150 mM of carmosine dye and 100 ml of the enzyme
was pre-incubated for 10 min. The reaction was then initiated
by addition of (1 mM) NADH and monitored for the decrease
in absorbance at 515 nm. One unit (U) of enzyme activity was
defined as the amount of the enzyme required to reduce 1
mmol of substrate dye per minute per milligram of protein.
CYP450 content was estimated using the method of Omura
and Sato, 1964.41 Superoxide dismutase activity was measured
spectrophotometrically according to Mishra and Fridovich42
on a spectrophotometer at 37 uC. The autooxidation of
epinephrine (formation of adrenochrome) was measured at
480 nm at the interval of 40 s for 5 min.
Characterization study of a purified azoreductase
The effect of pH on azoreductase activity was determined by
incubating the reaction mixture at pH values ranging from 4 to
10 using the standard buffer systems of acetate buffer for pH 4
to 6, phosphate buffer for 6 to 7 and tris buffer for 8 to 10.0.
Stability was determined by measuring the residual activity of
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the enzyme after 30 min of pre incubation in buffers of various
pH values from 4 to 9 at 35 uC. The optimum temperature for
enzyme activity was determined by conducting the assay at
various temperatures of 25, 30, 35, 40, 45 and 50 uC in 100 mM
phosphate buffer at pH 7.0.The residual activity was deter-
mined as per the standard assay procedure for the azo
reductase activity. A substrate specificity study was performed
by studying the reduction of six azo dyes viz., RR120, RR141,
RR2, RR11, SY and TAR as substrates with purified azoreduc-
tase from the strain B. lentus BI377. The electron donor study
was also performed using 0.5–5.0 mM solution concentration
of NADH and NADPH for 30 min at 35 uC. The values of
analysis using a C-18 reverse phase column with a solvent
system consisting of methanol and water (v/v). A linear
gradient from 20% methanol (isocratic for the initial 2 min)
increased to 100% over 10 min and then maintained at 100%
concentration for 10 min with a flow rate at 1 ml min .
21 34 The
degradation products were monitored at 230 nm. Fourier
Transform Infrared spectroscopy (FTIR) studies of the meta-
bolites were carried out using the KBr pellet technique in the
wavenumber range of 400–4000 cm21 on a Shimadzu-8400
FTIR spectrophotometer. Gas chomatography–mass spectro-
scopy (GC-MS) analysis was performed using a GB5 column
with a 15–20 min runtime on a Shimadzu-GC-MS-QP5050. TOC
analysis was performed as described by the dichromate
method of Walkely and Black45 and % removal was calculated
using the protocol of Saratale et al.39
Michaelis–Menten constant (Km) and maximal velocity (Vmax
)
for the enzyme with TAR was obtained by Lineweaver–Burk
double reciprocal plots.
Protein identification by matrix assisted laser desorption/
ionization time of flight (MALDI-TOF)
Conclusion
MALDI MS/MS analysis was performed on a SYNAPT HDMS
system (Waters Corporation, Milford, USA). Protein sample
digests were reconstituted in 5% ACN containing 0.1% TFA
and premixed with an equal volume of 10% a-cyano-4-
hydroxycinnamic acid (CHCA) matrix and applied on to a 96
well MALDI plate. All samples were acquired on a 200 Hz solid
state UV laser in V mode by MassLynx 4.1. The quadrupole
profile was set to 500 m/z for 5% of the scan time and then
ramped to 1500 m/z for remaining period of the scan. The
instrument mass calibration was carried with polyethylene
glycol (PEG). Proteins were identified by the MALDI survey
method, involving a combination of PMF and tandem mass
spectrometric (MS/MS) approaches for the identification of
proteins. In the MS survey method spectra were recorded in
the mass range of 800 to 4000 m/z for 60 s. MS/MS analysis was
performed in a data dependent manner for the top 7 peptides
with higher relative intensity for 30 s each and the product ion
mass range was set to 100 to 1500 m/z. For protein
identification MS/MS data were processed by PLGS software,
searched against the UniProt Bacillus database with the mass
tolerance set to 100 ppm, carbamidomethylation as the fixed
modification and methionine oxidation as the variable
modification.26
The present study emphasizes the major utility of isolate B.
lentus BI377 for the bioremediation of hazardous recalcitrant
azoaromatic compounds. The astonishing catabolic ability of
the strain certainly provides unparalleled opportunities for
understanding the fundamental molecular mechanism of the
degradation of organic compounds and their utilization as an
energy source. The purified and characterized flavin contain-
ing alkaliphilic azoreductase provides rich opportunities to
investigate the nonspecific diverse substrate specificity and
mechanism of promiscuity among the family of flavoprotiens.
This study opens up a dependable and efficient way to use B.
lentus to accelerate the exploration of the feasibility of the
bioremediation reactions over a wide range of temperature
and pH. The symbiotic action of the enzyme azoreductase,
cytochrome P450 monooxygenase and superoxide dismutase
may provide a selective advantage to the degradation reaction
under various conditions of environmental stress. Therefore,
this bacterial strain provides the potential enzymatic machin-
ery to remove a wide variety of natural and man-made
aromatic compounds discharged through geochemical cycles
and urban and industrial activities as well as their subsequent
transformation into CO2 and H2O.
UV-visible, GC-MS, FTIR and HPLC study of the intermediate
metabolites
The culture media was modified with 50 mg of each substrate
dye for the spectroscopic observations of decolorization study
in 500 ml capacity Erlenmeyer flasks with a control flask
(without dye). Decolorized samples were collected from each at
two hour intervals till the maximum decolourisation was
observed. Absorption of the supernatant was monitored at the
appropriate wavelength for each dye. Percentage decolourisa-
tion was calculated using the difference between initial and
final optical density. Metabolites were extracted from the
supernatants using methanol, ethyl acetate or dichloro-
methane (DCM) and after rotaevaporation were dried over
anhydrous sodium sulfate.44 The extracted metabolites were
subjected to thin layer chromatography (TLC) using 15%
methanol : 15% ammonium hydroxide/acetonitrile as the
mobile phase. 25 ml of the sample was subjected to HPLC
Acknowledgements
Author Chetan C. Oturkar gratefully acknowledges the
University Grants Commission (UGC, New Delhi),
Government of India for research and Dr Kothari’s postdoc-
toral fellowship.
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