Decolorization of the phthalocyanine dye reactive blue 21 by turnip peroxidase and assessment of its oxidation products

Article abstract of DOI:10.1016/j.molcatb.2011.12.006

Peroxidases can be used in decolorization processes and the treatment of textiles effluents. This study evaluates the potential of the turnip peroxidase enzyme in the decolorization of the phthalocyanine textile dye Reactive Blue 21 (RB21). Some factors such as pH, the amount of H₂O₂ and the enzyme were evaluated in order to determine the optimum conditions for the enzyme performance. The reaction products formed during the decolorization of the RB21 dye were analyzed by high-performance liquid chromatography-mass spectrometry coupling (LC-ESI/MS). LC-ESI/MS analysis showed that the decolorization of the dye RB21 by turnip peroxidase is due to the breaking up of the chromatogenous system. The tests for toxicity towards lettuce seeds showed an increase of the toxicity after enzymatic treatment of the dye. This study verifies the viability of the use of the turnip peroxidase enzyme in the biodegradation of textile dyes.

Full text of DOI:10.1016/j.molcatb.2011.12.006

Journal of Molecular Catalysis B: Enzymatic 77 (2012) 9–14
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Decolorization of the phthalocyanine dye reactive blue 21 by turnip peroxidase
and assessment of its oxidation products
Maria Cristina Silvaa,∗, Angelita Duarte Corrêaa, Maria Teresa Sousa Pessoa Amorimb, Píer Parpotc,
Juliana Arriel Torresa, Pricila Maria Batista Chagasa
a Federal University of Lavras, Department of Chemistry, 37200-000, Lavras, MG, Brazil
b University of Minho, Department of Textile Engineering, 4800-058 Guimarães, Portugal
c University of Minho, Department of Chemical, 4710-057, Braga, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 June 2011
Received in revised form 23 October 2011
Accepted 12 December 2011
Available online 20 December 2011
Peroxidases can be used in decolorization processes and the treatment of textiles effluents. This study
evaluates the potential of the turnip peroxidase enzyme in the decolorization of the phthalocyanine
textile dye Reactive Blue 21 (RB21). Some factors such as pH, the amount of H2O2 and the enzyme
were evaluated in order to determine the optimum conditions for the enzyme performance. The reac-
tion products formed during the decolorization of the RB21 dye were analyzed by high-performance
liquid chromatography–mass spectrometry coupling (LC–ESI/MS). LC–ESI/MS analysis showed that the
decolorization of the dye RB21 by turnip peroxidase is due to the breaking up of the chromatogenous
system. The tests for toxicity towards lettuce seeds showed an increase of the toxicity after enzymatic
treatment of the dye. This study verifies the viability of the use of the turnip peroxidase enzyme in the
biodegradation of textile dyes.
Keywords:
Environmental biocatalysis
LC–ESI/MS
Turnip peroxidase
Decolorization
Textile dyes
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
The inherent properties of reactive phthalocyanine dyes, such
as color fastness, stability, and resistance towards oxidative degra-
The removal of dyes from textile wastewater prior to its dis-
charge or reuse is a challenging task. The presence of color hinders
the absorption of solar radiation, which can modify photosynthetic
activity, causing changes in aquatic biota. Moreover, many of these
dyes present acute or chronic toxicity on the ecosystems [1].
Unfortunately, the exact data on the quantity of dyes produced
in the world or discharged in the environment are not available.
It is assumed a production of 10,000 tons per year, while a loss of
1–2% in production and 1–10% loss in use are a fair estimate [2].
Phtalocyanine (PC) dyes are among the dyes which resist to bac-
terial degradation. These dyes constitute the main category of the
reactive dyes which are one of the most important class of textile
dyes [3].
dation, have made color removal from textile wastewaters a
particularly difficult task. Reactive phthalocyanine dyes are highly
water-soluble, resistant to biological degradation under aerobic
conditions, and are not effectively removed by adsorption to the
biomass in wastewater treatment plants, resulting in colored efflu-
ents [4].
Researchers have been focusing their attention to study enzy-
matic pretreatment as
a potential and viable alternative to
conventional methods, due to its highly selective nature [5–7].
Enzymes can act on specific recalcitrant pollutants to be removed
by their precipitation or transformation into other innocuous prod-
ucts [8,9].
The removal of phthalocyanines dyes in aqueous solu-
tion by peroxidase has been widely reported, in last years,
especially by white-rot fungi. Peroxidases can catalyze degrada-
tion/transformation of aromatic dyes either by precipitation or by
opening the aromatic ring structure [10].
Phthalocyanine reactive dyes are metallic complexes used to
produce blue and green shades. Most of these dyes are copper
phthalocyanines. They are potentially mutagenic and of special
toxicity concern because of their metal Cu content [4].
The decolorization of two reactive PC dyes, Reactive blue 15
(RB15) and Reactive blue 38 (RB38), by Bjerkandera adusta, Trametes
versicolor and Phanerochaete chrysosporium was shown by Hein-
fling et al. [11]. Meanwhile, the PC dye RB21 (Reactive Blue 21) was
oxidized by Horsehadish peroxidase. The decolorization of the dye
RB21 in this case was approximately 59% [12]. The degradation of
dye RB21 was also evaluated by Marchis et al. [13], using soybean
∗
Corresponding author. Tel.: +55 32 88736085; fax: +55 35 3829 1628.
E-mail addresses: crisiria@yahoo.com.br (M.C. Silva), angelita@dqi.ufla.br
(A.D. Corrêa), mtamorim@det.uminho.pt (M.T.S.P. Amorim),
parpot@quimica.uminho.pt (P. Parpot).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.12.006

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M.C. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 9–14
for 30 s. The homogenate was filtered in organza cloth and cen-
trifuged at 10,000× g for 15 min, at 4 ◦C [18]. The obtained solution
was subjected to precipitation by adding cold acetone until reach-
ing 65% (v/v). After a rest from 12 to 14 h, at −18 ◦C, the homogenate
was centrifuged at 11,000× g for 15 min, at 4 ◦C. The supernatant
was collected and acetone was subsequently recovered by distilla-
tion in a rotary evaporator, at controlled temperature of 56 ◦C. The
obtained precipitate after the removal of the acetone by a treatment
in gridge during 72 h was redissolved in 15 mL sodium phosphate
buffer, pH 6.5 and then used in the dye removal studies.
Fig. 1. The molecular-structure of reactive blue 21 dye.
2.3. Determination of enzyme activity
The activity was determined according to Khan and Robinson
[19], using as reaction medium: 1.5 mL of guaiacol (Vetec; 97%, v/v)
1% (v/v); 0.4 mL of H2O2 (Vetec, PA) 0.3% (v/v); 0.1 mL of enzyme
and 1.2 mL of 0.05 mol L−1 phosphate buffer pH 6.5. The reaction
was monitorized during 5 min at 30 ◦C using a Spectrovision spec-
trophotometer coupled to a thermostatic bath.
One unit of peroxidase activity represents the oxidation of ␮mol
of guaiacol during 1 min in the assay conditions and it was calcu-
lated using data relative to the linear portion of the curve.
peroxidase as biocatalyst. The decolorization obtained was 95–96%
after 4 h of reaction at pH 3.0.
Despite these previous investigations have shown that PC dyes
can be decolorized by white-rot fungi or by plant peroxidases, the
degradative pathway and potential metabolites, mostly, remain
unknown. The knowledge of the metabolites formed during the
decolorization of textile dyes by plant peroxidases is a way for the
understanding of the break up mechanism of complex structures
chemically stable, by the enzymes [11].
Many treatments can be efficient in the decolorization, but it
is essential to know if there is formation of toxic products during
the process. A valuable technique to evaluate the toxicity of the
reaction products is the use of bioindicators [14].
In general it becomes very important for a bioremediation tech-
nology to assess the toxicity of the pollutants and metabolites
formed after their degradation in order to test out the feasibility
of the technique [15].
There are few studies to assess the toxicity of dyes and the
products formed during their enzymatic degradation. Da Silva and
coworkers [16] observed the reduction of Artemia salina mortality
after decolorization reactions of Drimarene Blue X-3LR (DMBLR),
Drimarene Rubinol X-3LR (DMR), and Drimarene Blue CL-R (RBBR)
by horsehadish peroxidase.
The metabolites formed after degradation of remazol red by
Pseudomonas aeruginosa BCH were more toxic than the parental
molecule [15].
In this work study, the use of the turnip peroxidase in the decol-
orization of the PC dye RB21 is studied. In this context, the effect
of parameters such H2O2, dye and enzyme concentrations, as well
as contact time has been investigated to optimize the system con-
ditions. The toxicity of the dye both before and after the enzymatic
dye was evaluated by utilizing lettuce seeds (Lactuca sativa) as a
bioindicator.
Moreover, we here report the identification of the major
metabolites of the treatment of the PC dye RB21 with turnip perox-
idase by liquid chromatography–mass spectrometry (HPLC–MS).
2.4. Dye removal studies
Experiments were conducted to assess the turnip peroxidase
catalyzed removal of phthalocyanine dye in aqueous phase. The
experiments were carried out at a constant temperature (30 ◦C) by
varying the process parameters such as dye, H2O2 and enzyme con-
centrations [20]. Initially the enzymatic reactions were conducted
in sodium phosphate buffer, 0.05 mol L−1, pH 7.0 (1.2 mL), contain-
ing: (1) H2O2 100 ␮mol L−1 (0.4 mL), (2) the dye Remazol Turquoise
G 133%, at concentration of 50 mg L−1 (1.5 mL) and 0.1 mL of enzy-
matic solution for estimated the optimum contact time.
The reaction mixture was incubated in a spectrophotometer
coupled to a thermostatic bath. The monitorization of the substrate
consumption was carried out at 624 nm which corresponds to the
maximum absorption of Remazol Turquoise G 133%. The calcula-
tion to determine the color removal percentage of the dyes was
made according to the equation:
absorbancyinitial − absorbancyfinal
× 100
absorbancyinitial
Subsequent series of experiments were performed by varying the
concentrations of dye concentration (from 10 to 50 mg L−1), H2O2
dose (from 50 to 500 ␮mol L−1) and enzyme concentration (from
1.62 to 26.16 U mL−1) to understand the optimum conditions for
dye removal. Reactions were performed also using multiple or sin-
gle additions of H2O2.
2. Material and methods
2.5. HPLC–MS
2.1. Dye
The HPLC–MS analyses were performed using a liquid chro-
matographic system (Thermo Surveyor with gradient pump, auto
sampler and diode array detector – DAD) coupled to a mass spec-
trometer Thermo LXQ Linear Ion Trap with electrospray ionization
(ESI+) and a diode array detector.
The textile dye RB21 was kindly provided by DyStar (Brasil)
and were used for degradation experiments without any further
purification. The molecular-structure of the dye is shown in Fig. 1
[17].
The samples were filtered through ultrafiltration membranes
(Millipore) with molecular weight cut 50 kDa, before injection into
the chromatograph.
2.2. Obtention of the enzymatic extract
A volume of 20 ␮L of sample was injected and the chromato-
graphic separation was performed on a Hypersil GOLD column
(100 mm × 4.6 mm). Methanol/water (acidified 1%, v/v) was used
The enzyme was extracted from turnip roots purchased from
local market. The roots (with peel) were washed in water and cut
into small uniform pieces. Turnip roots (300 g) were homogenized
in a blender with 100 mL of 0.05 mol L−1 pH 6.5 phosphate buffer
as mobile phase at a flow rate of 0.4 mL min−1
.

M.C. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 9–14
11
Fig. 3. Effect of H2O2 dose on the decolorization of RB21 by turnip peroxidase.
Fig. 2. Effect of reaction time on the decolorization of RB21 by turnip peroxidase.
2.6. Acute toxicity test with L. sativa
portion of H2O2 was added, so that in the end, the reagent concen-
tration was 100 ␮mol L−1. The percentage for color removal after
multiple addition of H2O2 in reaction was 57 0.83%, demonstrat-
ing that the concentration of H2O2 has no significant effect on the
overall enzyme catalyzed reaction. In addition, it was observed that
in the absence of this coadjutant there was no decolorization.
The lettuce seed root growth inhibition test was performed
with 20 seeds in a polystyrene Petri dish, containing a filter paper
embedded in 2 mL of each sample dilution (100, 75, 50, and 25%).
Root lengths were measured after 72 h and the LC50 was calculated
[21]. The samples used consisted of the dye before and after enzy-
matic treatment and, as negative control, distilled water. Controls
were carried out in parallel using only a Cu2+ solution at the con-
centration of 0.037 ␮mol mL−1 (Cu2+ content theoretical for RB 21
40 mg L−1). The tests were carried out in triplicate.
3.1.3. Optimum concentration of dye
The concentration of substarate is a key factor which affects the
rate of the enzyme catalyzed oxidation.
Studies were carried out at different concentrations of the dye
(10–60 mg L−1), keeping all the other parameters constant (H2O2
100 ␮mol−1; reaction time 50 min; enzyme 10.83 U mL−1) and the
results are shown in Fig. 4. The increase in dye concentration until
40 mg L−1 provides an effective increase in color removal. Subse-
quent increase in dye concentration above 40 mg L−1 resulted in
negligible dye removal. Mohan et al. [20] studied the enzymatic
decolorization of the Acid Black 10BX dye by Horsehadish perox-
idase (HPR) at different dye concentrations, and it was concluded
that concentrations above 30 mg L−1 resulted in low efficiency of
the decolorization.
3. Results and discussion
3.1. Decolorization of RB 21 dye by turnip peroxidase
In this paper, the parameters were optimized separately (reac-
tion time, dye concentration, quantity of H2O2 and quantity of
enzyme), to obtain the maximum decolorization of the dye.
3.1.1. Optimum contact time
The efficiency of the decolorization of the RB 21 dye as a function
of contact time with the enzyme, is given in Fig. 2. The decoloriza-
tion was 57.00 0.45% in 50 min of contact with enzyme. After
50 min of reaction, the dye removal became negligible. Souza et al.
[12] have reported that 45 min is the reaction time required to cat-
alyzed the degradation of the RB 21 dye, with decolorization of
59% by HPR. Subsequent experiments were performed for 50 min
of reaction time.
3.1.2. Optimum concentration of H2O2
Hydrogen peroxide acts as a co-substrate to activate the enzy-
matic action of peroxidase radical. However, the excess of this
reagent in the reaction inhibits the enzyme activity and, when
present in small quantity, limits the reaction rate [20–22]. It was
observed in Fig. 3 that the peroxide concentration of 100 ␮mol L−1
showed a better enzyme performance (55.1 0.75% of decoloriza-
tion).
The concentration of peroxide in the reaction showed no sig-
nificant influence on the efficiency of color removal. However for
concentrations above 200 ␮ mol L−1 takes place an inhibitory effect.
Multiple H2O2 additions provide a low effective concentration
of peroxide in reaction medium, avoiding the loss of enzyme activ-
ity [23]. Therefore, after 30 min of the oxidation reaction a second
Fig. 4. Effect of the dye concentration on the decolorization of RB21 by turnip per-
oxidase.

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M.C. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 9–14
Fig. 6. Mass spectra of reaction products of RB21 incubated with turnip peroxidase
obtained by LC–ESI/MS analysis.
3.2. Formation and identification of metabolites
Fig. 5. HPLC chromatograph (a) control [dye] and (b) sample [treated dye].
In order to detect biodegradation intermediates or stable
metabolites yielded during oxidation of the RB21 by turnip per-
oxidase, HPLC–DAD and LC–ESI/MS analysis were carried out
simultaneously. HPLC–DAD profile on the control sample (dye
without treatment) showed two peaks with retention times of 2.80
and 3.67 min respectively. After enzymatic treatment the chro-
matogram shows two important peaks with retention times of
3.15 and 3.72 respectively. The peak at 2.80 min corresponding
to the control sample was not detected after enzymatic treatment
(Fig. 5). These results indicate the possible breakdown of the parent
molecule and the formation of the new products.
Chromatograms and mass spectra of the reaction products of
RB21 incubated with turnip peroxidase are displayed in Fig. 6. Mass
spectrometry data revealed at least 3 major products (Fig. 6), two
of which were identified as metabolite I: m/z = 437 and metabolite
II: m/z = 524 respectively.
3.1.4. Optimum concentration of enzyme
Normally the removal of the aromatic compound is dependent
on the amount of catalyst added since the catalyst has a finite
lifetime and also the conversion is found to be dependent on the
contact time [20]. Within the enzyme concentrations evaluated
(1.26–26.16 U mL−1) it was observed that when the concentra-
tion studied was 10.83 U mL−1, the decolorization of the dye was
54.5 0.5%; however, when the concentration increased approx-
imately twice (20.3 U mL−1) the decolorization was 57.7 0.3%.
From these results, it was concluded that using a higher concentra-
tion of enzyme, there was a slight decrease on the decolorization
of the dye. This observation is in agreement with the outcome
presented by Forgiarini et al. [12] which studied the enzymatic
decolorization of RB21 by HPR. The authors mentioned that a con-
centration of 14.885 U mL−1 corresponds to a decolorization of 58%
and the multiplication of the concentration by two gave only a
percentage of decolorization corresponding to 62%.
Heinfling-Weidtmann et al. [24] studied the degradation prod-
ucts of reactive blue 15 and reactive blue 38 (phthalocyanine dyes)
by the white-rot fungus B. adusta. Sulfophthalimides (SPI; 3 and
4) were identified as major metabolites by comparison with syn-
thesized reference compounds and the release of Cu2+ from the
metal complex was proposed. This outcome is supported by other

M.C. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 77 (2012) 9–14
13
Table 1
Percentages of inhibition on the growth of radicles of lettuce to the dye RB21 after treatment.
Treatments
Concentration (%)a
100
LC50
75
50
0.0 0.0
25
0.0 0.0
Raw dye
Dye treated
7.42 1.6
61.50 2.3
0.0 0.0
50.26 1.0
–
74.60
30.3 4.9
12.3 2.5
a
Data are the mean of three replicates standard deviation.
authors who also studied the decolorization of phthalocyanine dyes
by white-rot fungi [11–25].
that after enzymatic treatment an increase of the toxicity was
observed.
According to data reported in literature and mass spectra
obtained, the structures of metabolites I and II were suggested. The
major products formed involving cleavage of the nitrogen bonds
in the inner ring of the phthalocyanine molecule and release of
Cu2+ from the metal complex. As a result, the mechanism of the
sulfophthalocyanine dye (RB21) degradation by turnip peroxidase
was proposed (Fig. 7).
Probably, the discrepancy of one unit mass, related to metabolite
II, is due to changes in side chain as the gain of protons for example.
The metabolite II is probably a fragment related to the metabolite of
m/z 569.56, whose structure was not identified. This fragmentation
can occur due to oxidative changes also in the side chain.
The increase of the toxicity after enzymatic treatment can be
attributed to the presence of Cu2+ in solution, or to the formation
of metabolites that are more toxic than the parent molecule.
Therefore, the acute toxicity test with L. sativa was also carried
out using a Cu2+ solution at the concentration of 0.037 ␮mol mL−1
(Cu2+ content theoretical for RB21 dye at 40 mg L−1). The inhi-
bition percentage of growth of lettuce’s radicles obtained was
15.91 4.30%. It was concluded that, in fact, the release of Cu2+
to the solution after enzymatic treatment contributes to increase
of toxicity.
Similar results were observed by Kunz et al. [26], who obtained
increase toxicity of the dye RB21, after treatment with ozone
according to the release of copper from the dye structure. The
removal of Cu2+ from the solution can be carried out using sim-
ple precipitation or by adsorption on activated clay and activated
carbon [27].
These results emphasize the importance of toxicological eval-
uation after enzymatic treatment. Considering the potential
application of enzymes for color removal and the present result
where the metabolites formed are more toxic than parental
molecule, the enzymatic treatment should be associated with
another type of treatment.
In this case, the enzymatic treatment can be associated with
microbial degradation, in which the enzyme break up the com-
plex chemical structure of dye and the bacteria then mineralize a
substantial portion of the breakdown products [27].
3.3. Acute toxicity test with L. sativa
The toxicity study of the untreated dye and the dye after the
enzymatic treatment was carried out with the purpose of evaluat-
ing the change of toxicity due to the treatment.
From the results shown in Table 1, the toxicity of the dye before
and after enzymatic decolorization treatment, it can be observed
that the non-treated dye shows lower toxicity. Therefore, it was
not possible calculate the average lethal concentration (LC50). How-
ever, after treatment the LC50 was 74.60%. These results showed
4. Conclusions
The results showed that reactive phthalocyanine dyes can be
decolorized after incubation with turnip peroxidase. However, the
degradation process efficiency seems to be dependent of param-
eters such as concentration of enzyme, dye and H2O2; reaction
time.
LC–ESI/MS analysis showed that the decolorization of the dye
RB21 by turnip peroxidase is due to the breaking up of the chro-
matogenous system.
The toxicity of the dye RB21 towards lettuce seeds increases
after incubation with turnip peroxidase. This can be attributed to
the presence of Cu2+ in solution, or due to formation of metabo-
lites with higher toxicity. However, the toxicity of the dye can be
removed by combination with other biologics process.
The enzymatic treatment may represent an important step for
complete microbial degradation of textile dyes, especially those
classified as phthalocyanines.
Acknowledgements
The authors would like to acknowledge FAPEMIG, CNPq, CAPES
for supporting this work.
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