Oxidative degradation of Remazol Turquoise Blue G 133 by soybean peroxidase

Article abstract of DOI:10.1016/j.jinorgbio.2010.11.009

Reactive dyes are widely employed in textile industries and their removal from wastewaters is a relevant environmental problem. In addition to chemical and physical methods, several bioremediation approaches, involving intact micro-organisms or isolated enzymes, have been proposed to decolorize dye solutions. In this paper, we report the complete and fast decolourization of a Cu(II)-phthalocyanine based reactive dye (Remazol Turquoise Blue G 133) by means of the soybean peroxidase/H₂O₂ system. The oxidative degradation of the dye in aqueous solution at 25 °C was studied as function of pH, revealing a quantitative decolourization yield at acidic pH values with a maximum of activity at pH 3.3. The reaction products were identified and characterized by HPLC-diode array detector (DAD)-mass spectrometry (MS), ionic chromatography and EPR techniques. This analysis showed that the enzyme catalyses the breaking of the phthalocyanine ring producing sulfophthalimide as the main degradation product, and the release of stoichiometric amount of ammonium and Cu(II) ions.

Full text of DOI:10.1016/j.jinorgbio.2010.11.009

Journal of Inorganic Biochemistry 105 (2011) 321–327
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Oxidative degradation of Remazol Turquoise Blue G 133 by soybean peroxidase
Tatiana Marchis a, Paola Avetta b, Alessandra Bianco-Prevot b, Debora Fabbri b,
Guido Viscardi c, Enzo Laurenti a,
⁎
a
Università degli Studi di Torino, Department of Chemistry I.F.M., Via P. Giuria 7, 10125 Torino, Italy
Università degli Studi di Torino, Department of Analytical Chemistry, Via P. Giuria 5, 10125 Torino, Italy
Università degli Studi di Torino, Department of General Chemistry and Organic Chemistry, C.so M. D'Azeglio 48, 10125 Torino, Italy
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 August 2010
Received in revised form 9 November 2010
Accepted 12 November 2010
Available online 26 November 2010
Reactive dyes are widely employed in textile industries and their removal from wastewaters is a relevant
environmental problem. In addition to chemical and physical methods, several bioremediation approaches,
involving intact micro-organisms or isolated enzymes, have been proposed to decolorize dye solutions. In this
paper, we report the complete and fast decolourization of a Cu(II)-phthalocyanine based reactive dye
(Remazol Turquoise Blue G 133) by means of the soybean peroxidase/H2O2 system. The oxidative degradation
of the dye in aqueous solution at 25 °C was studied as function of pH, revealing a quantitative decolourization
yield at acidic pH values with a maximum of activity at pH 3.3. The reaction products were identified and
characterized by HPLC–diode array detector (DAD)–mass spectrometry (MS), ionic chromatography and EPR
techniques. This analysis showed that the enzyme catalyses the breaking of the phthalocyanine ring
producing sulfophthalimide as the main degradation product, and the release of stoichiometric amount of
ammonium and Cu(II) ions.
Keywords:
Reactive dyes
Soybean
Peroxidase
Remazol Blue
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
processes have already replaced the traditional ones in some textile
industries [7].
Reactive dyes are extensively used in dyeing activities and their
use increases every year owing to capacity to bind on textile fibres by
covalent bonds. This favourable characteristic facilitates their inter-
action with the fibre, enhancing the fixation rate and reducing the
energy consumption [1]. On the other hand their use is problematic
because 10–15% is released in the wastewaters and dispersed in the
environment with toxic, mutagenic and carcinogenic effects [2].
Moreover, synthetic dyes are usually designed to be resistant to
degradation by light, chemicals and micro-organisms, and therefore
the development of efficient and economic technologies for the
treatment of wastewaters is a priority for textile industries. Despite
the existence of several chemical and physical methodologies [3,4],
bioremediation of the effluents is an attractive solution and several
studies have been already published about the use of intact micro-
organisms or isolated enzymes. In particular some species of white-
rot fungi have been demonstrated to be able to decolorize solutions
of dyes by means of oxidative processes catalysed by their
ligninolytic enzymes, laccases and peroxidases [2,5,6]. Since isolated
enzymes are also able to perform decolourizing reactions without
the problems generated from the use of living organisms, enzymatic
All these systems have advantages and disadvantages that must be
carefully assessed in view of their practical use. Some of the factors to
be considered are: cost, efficiency and environmental compatibility of
the degradation products. Although white-rot fungi are generally
inexpensive and often able to totally break down the pollutants, they
are also rather slow in their action and the decolourization of dye
solutions generally require some days of treatment [8–11]. On the
other hand, isolated laccases and peroxidases are generally more
expensive but allow to obtain high reaction rates and good yields
[6,12,13]. In addition, these enzymes can be used in working
conditions not suitable for living organisms, or immobilized on solid
supports and reused for several reaction cycles [14–18].
Although the partial decolourization of industrial dyes solutions and
textile effluents by means of horseradish peroxidase (HRP) [19–21] and
soybean peroxidase (SBP) [20] has been recently reported, the
application of plant peroxidases to the problem of reactive dyes
degradation has been rather limited until now. On the other hand, it
was also shown that SBP is able to oxidize a broad range of substrates
[22–24] and is highly resistant to thermal and chemical denaturation
[25–27], all features that make this enzyme really suitable for industrial
applications. Therefore, we decided to use SBP to catalyse the
degradation of Remazol Turquoise Blue G 133 (RTB), a reactive dye,
based on a copper(II) tetrasulfonated phthalocyanine derivatized with
N-(4-(2-sulfatoethylsulfonyl)phenyl)sulfonamide groups, taken here
as an example of a widely used and commercially available reactive dye.
⁎
Corresponding author. Tel.: +39 011 670 7951; fax: +39 011 670 7855.
E-mail address: enzo.laurenti@unito.it (E. Laurenti).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.11.009

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T. Marchis et al. / Journal of Inorganic Biochemistry 105 (2011) 321–327
In this work, dye solutions were degraded in the presence of
Only the samples representing the initial and the final point of
degradation were taken into consideration. In the former case (t0
sample), RTB solution (200 mg L−1 at pH=5) was incubated with SBP
for 30 s while, in the latter (tdeg sample), H2O2 was added to the same
solution and left to react until maximum decolourization (~91%) was
obtained. Before HPLC analysis every solution was eluted through a
size exclusion PD-10 column in order to separate the enzyme from the
mixture and to stop the reaction. The efficiency of the separation was
verified by determining the SBP activity into eluted fractions and
monitoring their UV–VIS spectra. Then, the more representative
fractions, where the enzyme is not present anymore and the analyte
and its products are eluted, were collected together in a final volume
and the dilution factor was taken into account in later calculations.
The quantification of SPI and APSES was obtained by means of
HPLC–MS analysis in selected ion monitoring (SIM) mode through
standard addition calibration. The addition standard curves were
prepared under four different added concentrations in the range 2–
6×10−6 M for APSES and 2–6×10−5 M for SPI.
catalytic amount of SBP and the reaction products were analysed by
means of HPLC–diode array detector (DAD)–mass spectrometry (MS),
ion analysis and EPR techniques.
2. Materials and methods
2.1. Enzymes and chemicals
Soybean peroxidase was purchased from Bio-Research Products Inc.
(Iowa, USA) with RZ=1.97 and used as received. Remazol Turquoise
Blue G 133 was kindly supplied by DyStar Textilfarben (Frankfurt, D).
The structure and percentage of purity of this commercial dye are not of
public domain, but it is referable to Reactive Blue 21, a copper
phthalocyanine with a sulfato ethyl sulfone as reactive group, from
which the chemical formula CuC40H24N9O17S6Na4 was obtained.
Hydrogen peroxide (30% aqueous solution), phenol (99%), ammo-
nium chloride, K2CO3 and NaHCO3 were from Sigma-Aldrich. Acetoni-
trile was a Scharlau AC0331 Supergradient HPLC grade eluent, while
ammonium acetate (98%) was from Fluka. Sodium hypochlorite
solution (6–14%) and sodium nitroprusside (99%) were purchased
from Riedel de Haen. 2-(4-Aminophenylsulfonyl)ethyl hydrogen
sulphate (APSES) was from Maybridge (Fisher Scientific). Sulfophtha-
limide (SPI)was synthesized from 4-sulfophtalic acid (Aldrich, technical
grade, with up to 25% of the 3-isomer), according to a method described
elsewhere [10].
2.4. Detection of ions formation
The release in solution of heteroatoms as sulphate, nitrate, nitrite
or ammonium ions, deriving from the breaking of the sulfophthalo-
cyanine dye, has been investigated. The anions present in the solu-
tions containing RTB and SBP were analysed with suppressed ion
chromatography. A Dionex DX500 instrument equipped with Anion
Self-Regenerating Suppressor-Ultra (ARSR-ULTRA, 4 mm, Dionex) a
conductimeter detector (ED 40, Dionex) and GP40 pump (Dionex)
was used. Injections of 20 μL were performed into AS4A-SC column
Dionex IonPack (200 mm×4 mm i.d.) with isocratic conditions:
90% of H2O and 10% of K2CO3 12 mM and NaHCO3 5 mM (flow=
0.5 mL min−1). The amount of sulphate, nitrate, and nitrite anions
present in the samples was determined by standard addition
calibration mode.
Ammonium cations were determined performing a colorimetric test
[30] in which indophenol is produced by the reaction between
ammonium ions, phenol and hypochlorite, catalysed by sodium nitro-
prusside. The tests were conducted onto 3 mL volumes of appropriately
diluted samples adding 0.12 mL of phenol 4.3×10−2 M (in 95% ethanol),
0.12 mL of sodium nitroprusside (0.5% w/v), 0.24 mL of alkaline citrate
solution (trisodium citrate 0.68 M, in 0.25 M of NaOH) and 0.06 mL of
6–14% hypochlorite solution. The colour was detected at 640 nm after 2 h
from the beginning of the reaction. The quantification was obtained with a
calibration curve (slope 0.72, intercept 0.04, R2=0.99) realized with
ammonium chloride standard solutions between 0.05 and 0.4 mg L−1
(each measure was repeated three times at least).
2.2. Remazol Turquoise Blue G 133 enzymatic degradation
The ability of SBP to decolourize aqueous solutions of RTB was
initially investigated varying dye concentration from 20 to 200 mg L−1
(concentrations by weight referred to the weight of powder), hydrogen
peroxide concentration from 6.4×10−7 to 2.4×10−3 M and pH values
from 2.0 to 9.0. Solutions were prepared in a triple buffer: borate 50 mM,
phosphate 50 mM and acetate 50 mM, and the decolourization
isotherms were recorded at 621 nm and 25 °C, with an SBP concentra-
tion of 2.06×10−7 M and H2O2 9.98×10−5 M. The concentration of the
enzymesolutionswasdetermined by UV–visible (UV–VIS) spectroscopy
at 403 nm, (ε403 nm=9.64×104 M−1 cm−1 [25]).
The initial rate of decolourization was also investigated as function
of pH using the following concentrations: H2O2 6.4×10−5 M, SBP
2.06×10−8 M and RTB 70.8 mg L−1. The same experiment was
repeated replacing RTB with 2,4,6-trichlorophenol (2,4,6-TCP) at the
concentration of 1.17×10−5 M. In this case we investigated the
oxidative dechlorination of the 2,4,6-TCP following the formation of
the final reaction product, 2,6-dichloro-1,4-benzoquinone, at 272 nm
(ε272 nm =1.4×104 M−1 cm−1 [28]).
2.5. EPR spectroscopy
All UV–VIS measurements were acquired with a UNICAM UV300
Thermospectronic double beam spectrometer, equipped with a Peltier
cell for temperature control.
EPR spectra of RTB solutions were recorded at 77 K by an ESP300E
Bruker X-band spectrometer equipped with a 4103TM cylindrical
cavity. Samples were placed in quartz tubes and the experimental
parameters were as follows: frequency 9.40 GHz, power 4 mW,
modulation frequency 100 KHz, modulation amplitude 2 G, gain
4×104, time constant 8.091×10−2 s.
2.3. HPLC–DAD–MS measurements
Thermofinnigan Surveyor MSQ equipped with a photodiode array
detector and an electrospray ionization (ESI) probe with a single
quadrupole Mass Spectrometer was employed to follow the RTB fate.
The separation was obtained with a C-18 reversed-phase
encapped column [29]. The column was equilibrated with 30% of
acetonitrile (A) and 70% of ammonium acetate 5 mM solution (B), and
a 20 μL volume was injected and eluted with the following gradient:
0 min 30% A, 70% B and 30 min 100% B with 0.5 mL min−1 flow rate.
Photodiode array and Mass Spectrometer were used in parallel. Ion
source conditions were optimized through direct infusion technique
to maximize the signal: 400 °C of temperature, 3 kV of needle voltage
and 50 V of cone voltage in negative-ion mode.
3. Results and discussion
3.1. Decolourization of the Remazol Turquoise Blue G 133 solutions
Aqueous solutions of RTB have an electronic spectrum characterized
by the presence of a broad and intense band in the visible zone, which
gives the characteristic blue colour. Since the composition of the dye
mixture is not exactly known, we refer our concentration data to a
weighed amount of sample to avoid any possible error due to an
inaccurate estimate of the average molecular weight of the mixture. For

T. Marchis et al. / Journal of Inorganic Biochemistry 105 (2011) 321–327
323
Fig. 3. Decolourization percentage registered after 4 h of treatment at 25 °C of the
solution of RTB (200 mg L−1) with SBP 2.06×10−7 M and H2O2 9.98×10−5 M (white
columns), RTB only (black columns), RTB and H2O2 (grey columns).
Fig. 1. UV–VIS spectra of a RTB solution (35 mg L−1) in the pH range 2.0–9.0 (arrows
indicate the trend of absorption bands as pH increases). Inset: absorbance at 666 nm as
function of pH.
[31]). As well known, the catalytic cycle of peroxidases involves the
formation of two intermediates, Compound I and Compound II,
according to the following reactions (where SH indicates a generic
substrate):
the same reason the molar absorptivity was also referred to mg L–1 of RTB
rather than a molar concentration. As we can see from Fig. 1, the structure
of this band depends from the pH of the solution: at very low pH values
an absorption with a maximum at 621 nm predominates (ε621
=
nm
1.41×10−2 mg−1 L cm−1 at pH 7.0; 1.28×10−2 mg−1 L cm−1 at pH 5.0);
whereas, when the pH increases, a more intense absorption band appears
at 666 nm.
Peroxidase þ H2O2→Compound I þ H2O
Compound I þ SH→Compound II þ S•
Compound II þ SH→Peroxidase þ S• þ H2O
Since the variations in molar absorptivity are greater at 666 nm
than at 621 nm, we monitored for 4 h the decolourization trend
induced by hydrogen peroxide in the presence of SBP through the
decrease of absorbance at 621 nm. Moreover, as the pH of the batch of
reaction resulted unaltered during all the degradation reaction, it was
possible to calculate the percentage of decolourization as function of
time, as reported in Fig. 2.
These experiments show that the decolourization process is
strictly dependent from the pH of the solution: SBP activity increases
drastically changing the pH from 2 to 3, and in the pH range of 3–5, it
effectively and rapidly degrades about 2/3 of the RTB in just half an
hour. After 4 h (Fig. 3), at pH 3 approximately 95–96% of the dye
degradation occurred, and at pH 4 and 5 the results are slightly lower.
At higher pH values the percentage of decolourization decreases and
drops near to zero at pH 8. Since the stability of RTB solutions has been
verified in presence of hydrogen peroxide as well, we can affirm that
the dye degradation observed in these experiments is exclusively due
to the enzymatic activity of SBP.
Every step of the catalytic cycle is pH dependent: the Compound I
generation is favoured by the presence of a network of hydrogen
bonds between the Fe-heme/H2O2 adduct and the distal histidine and
arginine side chains [23], whereas the substrate oxidation may
depends on its protonation state [32,33].
The kinetic studies on the pH dependence of RTB degradation
allowed to obtain the bell-shaped curve showed in Fig. 4 (solid line)
with optimal pH=3.3 0.1. Two inflection points, at pH=3.0 0.1
and pH=3.9 0.1, can be extrapolated with a model derived from the
Henderson–Hasselbach equation applied to a system with a double
ionization process [32].
The proximity of the two inflection points, calculated for RTB
degradation, gives to the profile a very narrow shape, rarely detected
before with soybean peroxidase, and a optimum pH lower than those
previously reported with other substrates like ABTS (pH=5.5),
guaiacol (pH=5.5), p-cresol (pH=5.0) [26], or phenol (pH=6.4)
[22]. Although a notable exception was observed for veratryl alcohol
oxidation (pH=2.4) [34].
In order to better clarify the effect of pH on the enzymatic reaction, a
kinetic study has been carried out measuring the pH dependence of the
initial rate of the oxidation of RTB and 2,4,6-trichlorophenol, used as an
alternative substrate with a known dissociation constant (pKa=6.0
Fig. 2. Time course of the decolourization of Remazol Turquoise Blue G 133 solutions
Fig. 4. Initial rate of RTB 70.8 mg L−1 decolourization (●, solid line) and 2,4,6-TCP
1.17×10−5 M oxidation (○, dotted line) obtained with H2O2 6.4×10−5 M and SBP
2.06×10−8 M as function of pH.
(200 mg L−1
) M and H2O2
at different pH values in presence of SBP 2.06×10−7
9.98×10−5 M.

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T. Marchis et al. / Journal of Inorganic Biochemistry 105 (2011) 321–327
In the case of 2,4,6-trichlorophenol (Fig. 4, dotted line) two pK
SBP but not hydrogen peroxide (t0 sample) was also eluted through
the column before the HPLC analysis (Fig. 6, upper left).
values can be calculated at pH 3.1 0.1 and 6.0 0.1 respectively. The
former has essentially the same pK value registered for RTB
degradation, whereas the latter was shifted at higher pH values, in
good agreement with the 2,4,6-TCP ionization constant. The similarity
in activity decrease for RTB and 2,4,6-TCP in the acidic range with pK
value of about 3.0, presumably refers to an ionization process of the
enzyme.
As can be seen, A and C components were partially lost in the
passage through the column, highlighting the presence of component
B. On the other hand, as expected, the chromatogram of the final stage
of the degradation (tdeg sample) representing the 91% of decolouriza-
tion (Fig. 6, upper right), showed the almost complete disappearance
of the blue-coloured components (peak A and C) and an additional
growth of peak B, probably due to the coelution of the reaction
products.
In order to identify the nature of the decolourization products, the
same samples were also analysed with HPLC–ESI–MS technique. The
MS spectra of the 10 mg L−1 aqueous solution of RTB, t0 solution and
of the 91% degraded sample of RTB, confirmed the trend observed
with the PDA detector. In fact, in correspondence of the PDA
chromatographic peak B (tR =3.6 min), all the analysed samples
revealed the presence of one species with m/z 280, whereas in the case
of the degraded sample, another signal with m/z 226 appeared at the
same retention time, as highlighted by the SIM mode chromatogram
(Fig. 6) and the corresponding MS spectrum (Fig. 7, upper right).
Based on these two m/z values, for m/z 280 APSES was
hypothesized as possible precursor of the RTB in the commercial
mixture and, for m/z 226, SPI as possible reaction product [10]. These
m/z values corresponding to the anionic form [M–H]− of the parent
molecules with calculated mass equal to 281.0 g mol−1 for APSES and
227.0 g mol−1 for SPI, respectively. The structural assignment was
confirmed by means of HPLC–ESI–MS analysis of the corresponding
authentic standards (Fig. 7), which have a very close retention time,
similar to that of peak B.
As previously demonstrated by Nissum et al. [23], in the case of
SBP the Compound I formation shows the typical bell-shaped profile
and is favoured between the two pKa values of 3.2 0.3 and 9.9 0.2,
therefore the absence of RTB decolourization at very low pH values
and the presence of the first inflection point at pH 3.0 0.1 are in
agreement with such mechanism. On the other hand, the correspon-
dence of 2,4,6-TCP ionization constant with the calculated pK for the
descendant branch of the SBP catalysed oxidation curve suggests the
presence of a second process, likely correlated with the substrate
ionization also in the case of the rapid loss of activity observed above
pH 4, for RTB oxidation. The ionic nature of the dye is confirmed by
the variations observed in the shape of the UV–VIS spectrum
acquired at different pH values (see Fig. 1). Since the dissociation
constants for this molecule are not known, an extrapolation of the
absorbance values at 666 nm is plotted against pH (inset of Fig. 1)
and reveal a sigmoid trend characterized by an inflection point at
pH=4.3 0.6. This profile can be explained by the establishment of
an acid–base equilibrium in the surrounding of pH 4, essentially
corresponding to the second inflection point of RTB decolourization
profile observable at pH 3.9 0.1.
3.2. HPLC–DAD–MS
Successively, we carried out the addition of the two compounds to
the samples representing tdeg, t0, and to a 10 mg L−1 aqueous solution
of RTB; in all cases a significant enhancement of the peak B in the PDA
acquired chromatogram, was observed.
A RTB aqueous solution (10 mg L−1) was analysed by HPLC, before
proceeding with the enzymatic reaction. The photodiode array (PDA)
total scan chromatogram showed the presence of three main
components, indicated in the Fig. 5 (upper part) as peaks A, B, and C
respectively. The HPLC profile obtained at 240 and 621 nm revealed
that the species corresponding to peaks A (tR =2.5 min) and C
(tR =5.0 min) are the main responsible for the blue colour of RTB
solution, whereas the species included in the broadband B (tR =3.1–
3.6 min) are ascribable to less coloured or colourless components.
In order to identify the degradation products resulting from RTB
decolourization, the enzyme must be separated from the batch of
reaction before the injection in the HPLC system. The elution through
a size exclusion chromatography column ensured the best results in
terms of SBP separation from the batch of reaction and representa-
tiveness of gathered fractions, even inducing some changes in the
composition of the mixture. Therefore a solution containing RTB and
The species with 280 m/z (Fig. 6, peak 1), present in all the samples
analysed and identified as the reactive arm of the copper phthalocy-
anine, APSES, was quantified through a standard addition calibration
method as 5–9% of the starting solution of Remazol Turquoise Blue G
133. Although the molecule was compatible with a degradation
structure of the dye, the 280 m/z peak area showed no remarkable
alteration imputable to SBP oxidation activity, therefore neither
accumulation nor abatement was observed also in the decolourized
sample. These results confirm that, even in little quantity, APSES is
present like precursor of the RTB in the commercial mixture and that
this compound remains substantially unaltered by the SBP/H2O2
system.
On the other hand, the SIM analysis of the m/z 226 (Fig. 6, peak 2)
showed that this species is not present in the starting mixture, but
that is produced during the RTB degradation. In agreement with the
previously reported results about the degradation of sulfonated
phthalocyanine dyes by the white-rot fungus Bjerkandera adusta
[10], this compound is ascribable to a sulfophthalimide molecule
deriving from the breaking of the phthalocyanine ring and the
oxidation of the substituted indole groups. The assignation of the peak
to the molecular anion of SPI was confirmed by standard addition of
the reference synthesized as previously described [10].
3.3. Monitoring of ions formation
The identification of SPI as product of reaction presuppose the
breaking of the conjugated ring of the phthalocyanine associated with
the elimination of nitrogen atoms. Therefore we analysed the fate of
nitrogen as nitrate, nitrite or ammonium ions release. The concen-
tration of anions in initial and degraded solutions was investigated by
means of ionic chromatography, while the presence of ammonium
cations was detected with the phenate method [30].
Fig. 5. HPLC–DAD chromatograms of the 10 mg L−1 aqueous solution of RTB, recorded
in total scan PDA and at 240 or 621 nm.

T. Marchis et al. / Journal of Inorganic Biochemistry 105 (2011) 321–327
325
Fig. 6. HPLC–DAD–MS chromatograms recorded in total scan PDA and in SIM mode at 280 and 226 m/z before (left) and after 91% decolourization (right).
Three main peaks were found into ionic chromatograms of the
samples, corresponding to nitrite, nitrate and sulphate ions, but no
significant variation in the concentrations was revealed after the
decolourization.
The determination of ammonium ions has been obtained by a
colorimetric test, carried out onto the diluted samples corresponding
to the starting and the final point of RTB degradation. The results
showed the release of 8.92 0.33 mg L−1 of ammonium ions
(4.95×10−4 M) into the sample with 91% of RTB degradation.
The concentration of ammonium ion obtained at the end of the
reaction is consistent with the release of four ammonium cations from
the breaking of each phthalocyanine ring, estimating the starting RTB
Fig. 7. HPLC–DAD–MS chromatograms recorded in total scan PDA (left) and MS spectra (right) of RTB sample after 91% of decolourization (peak B), and SPI or APSES standards.

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T. Marchis et al. / Journal of Inorganic Biochemistry 105 (2011) 321–327
Scheme 1. Hypothetical reaction scheme summarizing the degradation of RTB.
concentration as 1.46–1.52×10−4 M, on the basis of a hypothetical
molecular weight of RTB equal to 1246 g mol−1 (see Section 2.1), and
of the presence of 5–9% w/w of APSES in the starting mixture. Thus,
taking into account the uncertainty concerning the molecular weight
of the parent dye molecule, and the purity of the furnished product,
the results highlights that nitrogen atoms contained in the phthalo-
cyanine ring are substantially released as ammonium ions.
Finally, although further studies are needed to better understand
the mechanism of action that will enable the SBP to break
phthalocyanine ring, these results suggest that this enzyme can be
successfully employed in the preparation of biocatalysts useful in the
degradation of effluents of textile dyeing industries.
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77 K confirmed this mechanism because, at the end of the reaction, we
observed the presence of an axial spectrum (not shown) with
gt =2.41, At =120 G and g⊥ =2.08, quite different to the EPR
spectrum of RTB (gt =2.27, g⊥ =2.05) and clearly originated from
the interactions of Cu(II) ions, released after RTB degradation, with N
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