International Journal of Biological Macromolecules 116 (2018) 463–471

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Guar gum blended alginate/agarose hydrogel as a promising support for

the entrapment of peroxidase: Stability and reusability studies for the
treatment of textile effluent
Misha Ali, Qayyum Husain ⁎
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, UP, India

a r t i c l e i n f o a b s t r a c t

Article history: Ginger peroxidase (GP) was entrapped into the hydrogels of guar gum (GG)-alginate/agarose and these
Received 30 October 2017 immobilized GP preparations were employed for the treatment of textile effluent. GG is a natural hydrophilic
Received in revised form 6 April 2018 polysaccharide, the average size of which increases in its hydrated form that helps in retaining the enzyme inside
Accepted 8 May 2018
the entrapping support. Therefore, the activity retention by alginate-guar gum (ANGG) and agarose-guar gum
Available online 8 May 2018
(AGG) was higher than that of alginate and agarose alone. ANGG-GP and AGG-GP were highly stable against var-
Keywords:
ious physical and chemical denaturants during the decolorization of textile effluent. As compared to free GP, both
Peroxidase the immobilized preparations were more efficient in the decolorization of textile effluent in batch processes.
Guar gum After 10th repeated use in batch processes, ANGG-GP and AGG-GP was quite effective in removing up to 68%
Decolorization and 55% of the color from textile effluent, respectively. Continuous packed bed reactors containing ANGG-GP
and AGG-GP were able to decolorize around 80% and 69% of the effluent color, respectively, even after 30 days
of their continuous operation at room temperature (30 °C). Genotoxicity of textile effluent was significantly re-
duced after GP mediated decolorization.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction choice among various techniques employed for the immobilization of

Continually increasing aromatic pollutants, especially synthetic dyes
from textile industries are polluting the water bodies. These dye-
comprising waste effluents induce undesirable effects, such as toxicity,
mutagenicity, carcinogenicity and teratogenicity on the living ecosys-
tem [1,2]. Therefore, an effective treatment technology is required for
the degradation and detoxification of such compounds. These synthetic
textile dyes are excessively stable and are highly resistant to physical,
chemical and biological treatments. Hence, enzymatic method is
gaining much attention for the degradation of these dyes to overcome
the limitations posed by other conventional methods [3,4].
In recent years, peroxidases from plants and fungi have been
employed for the treatment of colored wastewater [5–7]. However, sol-
uble enzymes are not very effective in degradation and detoxification of
environmental pollutants as several limitations are faced by them, such
as instability towards pH, temperature, non-reusability, incomplete
degradation of pollutants due to denaturation of enzymes, and also
they cannot be used in continuous reactors [8]. To overcome such limi-
tations, enzymes should be immobilized on solid supports with their
long term utilization at a large scale [9–11]. Entrapment is a better
⁎ Corresponding author.
E-mail address: qayyumbiochem@gmail.com (Q. Husain).
enzymes due to its non-reactive aqueous microenvironment inside
the matrix and also it does not affect the native structure of enzyme
[12]. Earlier, a variety of matrices have been employed for the entrap-
ment of enzymes such as gelatin, agar-agar, chitosan agarose, polyacryl-
amide, carrageenan, alginate, and pectin [13].
However, some disadvantages are often associated with these matri-
ces, including leakage of entrapped enzyme from the support during its
application and low mechanical strength [14]. To resolve these
problems such polymeric matrices have been blended with other
polysaccharides such as gelatin, starch, pectin, chitosan, etc. [15–17].
Guar gum (GG) is one of the natural polysaccharides derived from
the seeds of Cyamopsis tetragonolobus. It is hydrophilic in nature and
consists of linear chains of (1 → 4) β D mannopyranosyl units with
α D galactopyranosyl units attached by (1 → 6) linkages [18]. Due to
its hydrophilic properties, it can entrap a large amount of water be-
tween its chains and branches [19]. The hydrophilicity of GG prevents
the loss of water from entrapping support thus obstructing the leaching
of enzyme [18].
In the present study hydrogels of alginate/agarose mixed with GG
was prepared for the entrapment of GP. Several parameters were opti-
mized for the maximum entrapment of enzyme within the polymeric
support. A comparative stability study was done for free GP, agarose-
guar gum GP (AGG-GP) and alginate-guar gum GP (ANGG-GP) against

https://doi.org/10.1016/j.ijbiomac.2018.05.037
0141-8130/© 2018 Elsevier B.V. All rights reserved.
464 M. Ali, Q. Husain / International Journal of Biological Macromolecules 116 (2018) 463–471
various physical and chemical denaturants during the decolorization of
industrial textile effluent. Dye decolorization efficiency of both AGG-GP
and ANGG-GP in batch and continuous reactors were also evaluated. Fi-
nally, genotoxic assessment of textile effluent was done before and after
treatment by GP.
2. Materials and methods
2.1. Materials
Bovine serum albumin, Histopaque 1077, Roswell Park Memorial In-
stitute 1640 (RPMI 1640) media and o dianisidine HCl were the prod-
ucts of Sigma Chemical Co. (St. Louis, MO, USA). 1-
Hydroxybenzotriazole (HOBT), agarose, sodium alginate and GG were
obtained from SRL Chemicals (Mumbai, India). Ginger was purchased
from local vegetable market surrounding the AMU Aligarh, India.
Other chemicals and reagents used were of analytical grades and were
employed without any further purification.
2.2. Ammonium sulphate fractionation of GP
The extraction of GP was done according to the procedure described
earlier [20].
2.3. Optimization of different parameters for the maximum activity reten-
tion of entrapped GP
2.3.1. Effect of sodium alginate and calcium chloride concentration
Different concentrations of sodium alginate (1.0–3.5%) and calcium
chloride (0.5–3.0%) were used to attain the maximum activity retention
of immobilized GP.
2.3.2. Effect of curing time
The influence of curing time on percent activity retention of enzyme
was examined by keeping beads in 2% calcium chloride solution for dif-
ferent time intervals ranging from 20 to 180 min.
2.3.3. Effect of agarose concentration
To achieve the maximum activity retention of entrapped GP, con-
centration of agarose was varied from 1.0 to 6.0%.
2.4. Entrapment of GP into ANGG beads under optimized conditions
The GP (50 U) was mixed with sodium alginate (2.5%, w/v) and GG
(1%, w/v) solution prepared in 20.0 mL of assay buffer. The resulting
mixture was slowly extruded as droplets into 2% (w/v) calcium chloride
solution and further gently stirred for 2 h. The obtained ANGG-GP beads
were washed with 100 mM sodium acetate buffer, pH 5.0 and stored in
the assay buffer at 4 °C for its further use. GP was also entrapped alone
within calcium alginate beads using the same procedure as above to
find out the effect of GG on the activity retention of GP into alginate.
2.5. Entrapment of GP within AGG support under optimized conditions
Agarose (3%, w/v) and GG (1%, w/v) solution (20.0 mL) was pre-
pared in assay buffer by vigorous shaking at 100 °C and was brought
at 40–45 °C. Afterwards, GP (50 U) was mixed thoroughly with the pre-
pared solution. This mixture was directly poured into glass plates and
was kept at 4 °C for one hour to completely solidify the agarose. Then
the enzyme containing gel matrix was cut into small pieces of 5 × 5
× 5 mm3 size. Similarly, GP was also entrapped in agarose alone in
order to evaluate the effect of GG on the activity retention of GP into
agarose gel.
2.6. Scanning electron microscopy (SEM) analysis
Surface morphology studies of alginate/agarose alone, ANGG/AGG
and ANGG-GP/AGG-GP were carried out by SEM. Gel of alginate/agarose
alone, ANGG/AGG and ANGG-GP/AGG-GP were dried at 37 °C overnight.
Dried gels were placed on carbon tape over copper stubs and coated
with 25 nm thick gold layer using a sputter coater. SEM micrographs
of all the samples were taken by using JSM-6510 LV scanning electron
microscope at 15 kV [21].
2.7. Effluent processing and dilution
The textile effluent was collected from the industrial site situated in
Panki, Kanpur, UP, India. The effluent was centrifuged at the speed of
5000 ×g for 10 min and clear supernatant obtained was diluted by
100 mM sodium acetate buffer, pH 5.0 till the optical density of 0.550
(approx) at 600 nm was exhibited by textile effluent. The λmax of the ef-
fluent was determined by using UV-1700 Pharma Spec UV–vis
spectrophotometer.
2.8. Effect of various parameters on the decolorization of effluent by using
free and ANGG/AGG immobilized GP
2.8.1. Effect of pH
Free and immobilized GP, each preparation 2.0 U, was taken inde-
pendently to determine the decolorization of textile effluent in the
buffers of different pH (2.0–9.0) in the presence of 0.75 mM H2O2 and
1.0 mM HOBT for 1 h at 40 °C. The buffers used were glycine-HCl
(pH 2.0 and 3.0), sodium acetate (pH 4.0 and 5.0), sodium phosphate
(pH 6.0 and 7.0) and Tris–HCl (pH 8.0 and 9.0).
2.8.2. Effect of temperature
The effect of temperature on the decolorization of textile industrial
effluent by free GP, AGG-GP and ANGG-GP was investigated at various
temperatures (20–80 °C) in sodium acetate buffer, pH 5.0 in the pres-
ence of 0.75 mM H2O2 and 1.0 mM HOBT for 1 h.
In another set of experiment, all three GP preparations were incu-
bated at 60 °C for varying time intervals in 100 mM sodium acetate
buffer, pH 5.0. After each incubation period the enzyme was quickly
chilled in crushed ice for 5 min. Each GP preparation was brought at
room temperature and the decolorization of textile effluent was mea-
sured in sodium acetate buffer, pH 5.0 in the presence of 0.75 mM
H2O2 and 1.0 mM HOBT at 40 °C for 1 h. The decolorization of textile ef-
fluent by enzyme without incubation at 60 °C was taken as control
(100%) for the calculation of remaining percent effluent decolorization
activity.
2.8.3. Effect of urea
Free GP, AGG-GP and ANGG-GP were incubated with 4.0 M urea for
varying times in 100 mM sodium acetate buffer, pH 5.0 at 40 °C for 2 h.
After each incubation period, the decolorization efficiency of enzyme
was measured in sodium acetate buffer, pH 5.0 in the presence of
0.75 mM H2O2 and 1.0 mM HOBT at 40 °C for 1 h. The decolorization
by enzyme without incubation with urea was taken as control (100%)
for the calculation of remaining percent effluent decolorization activity.
2.8.4. Effect of water-miscible organic solvent
The effect of water-miscible organic solvent on the decolorization of
textile industrial effluent by free GP, AGG-GP and ANGG-GP was inves-
tigated with varying concentrations of DMSO (10–80%, v/v) in sodium
acetate buffer, pH 5.0 in the presence of 0.75 mM H2O2 and 1.0 mM
HOBT at 40 °C for 1 h.
2.8.5. Effect of detergent
The effect of detergent on the decolorization of textile industrial ef-
fluent by free GP, AGG-GP and ANGG-GP was investigated with varying
 M. Ali, Q. Husain / International Journal of Biological Macromolecules 116 (2018) 463–471
concentrations of Triton X-100 (0.5–5.0%, v/v) in sodium acetate buffer,
pH 5.0 in the presence of 0.75 mM H2O2 and 1.0 mM HOBT at 40 °C for
1 h.
2.9. Effluent decolorization in batch processes and reusability of ANGG/AGG
immobilized GP
Textile effluent (250 mL) was independently treated by free and
immobilized GP preparations (20 U) in the presence of 1.0 mM HOBT
and 0.75 mM H2O2 at 40 °C for 3 h with constant stirring in batch pro-
cess. Aliquots were collected from the reaction mixtures of each set of
experiments at different time intervals and a reduction in absorbance
was recorded by UV–vis spectrophotometer at 600 nm, using the fol-
lowing equation;
 
Absi−Absf
D ð%Þ ¼  100
Absi
where D is the dye decolorization (%), Absi is the initial absorbance of
the reaction mixture and Absf is the final absorbance of the reaction
mixture. In order to evaluate the operational stability of AGG-GP and
ANGG-GP, after completion of first batch of experiment, immobilized
enzyme was separated from the reaction mixture and washed thrice
with 0.1 M sodium acetate buffer, pH 5.0 and used for the second
batch of experiments for the decolorization of textile effluent. The pro-
cess was repeated for 10 successive batch processes by using same
immobilized GP. Each batch was done in triplicates. Decolorization per-
cent was calculated after each reuse.
2.10. Decolorization of textile effluent in continuous vertical bed-reactors
filled with ANGG/AGG immobilized GP
Two packed bed-reactor systems were constructed and used for the
continuous decolorization of textile effluent. The columns (10 × 2 cm)
were filled independently with AGG-GP (4.8 g, 50 U) and ANGG-GP
(3.67 g, 50 U) and equilibrated with 100 mM sodium acetate buffer,
pH 5.0. The working volume of both the reactors was 11.2 mL. Void vol-
ume of AGG-GP and ANGG-GP reactors was 6.07 mL and 4.5 mL, respec-
tively. The textile effluent containing 0.75 mM H2O2 and 1.0 mM HOBT
was continuously passed through the reactors at room temperature (30
°C). The flow rate and residence time of both the columns were main-
tained as 10 mL h−1 and 12 min, respectively. Samples from both the
column outlets were collected regularly at a gap of 5 days and percent
decolorization was measured for over 1 month and readings were
taken by using UV–visible spectrophotometer for the remaining color
at 600 nm.
2.11. Genotoxicity assessment of textile effluent and its degraded products
by comet assay
2.11.1. Isolation of lymphocytes
Heparinized blood sample (10 mL) was obtained from single healthy
donor (first author) and consecutively diluted in phosphate buffered sa-
line. The lymphocytes from blood were isolated by using Histopaque
1077 and finally suspended in RPMI 1640.
2.11.2. Experimental procedure
Comet assay was done under alkaline conditions with slight modifi-
cations as the procedure described earlier [22]. Fully frosted microscopic
slides were pre-coated with 1% normal melting agarose. Mixture of di-
luted lymphocytes and 2.0% low melting agarose in 1:1 ratio was pipet-
ted over the first layer of agarose. Quickly the slides were covered by
cover slips and kept over ice for 15 min to solidify the agarose. After so-
lidification, cover slips were removed and lymphocytes were treated
with textile effluent and its degraded products for 1 h at 4 °C. Treatment
was followed by lysis of lymphocytes which was done in cold lysis
465
solution containing 100 mM EDTA, 2.5 M NaCl, 10 mM Tris pH 10 and
1% Triton X-100 (added just before use) for 1 h at 4 °C. Unwinding of
DNA was done in alkaline electrophoretic solution containing 300 mM
NaOH, 1 mM EDTA, pH 13 at 4 °C for 30 min and then electrophoresis
was carried out at 300 mA current. After electrophoresis, neutralization
of DNA was done with 0.4 M Tris buffer, pH 7.5. Ethidium bromide (20
μg mL−1) was used for the staining of slides which were then washed
with distilled water, covered by cover slips and ultimately stored in a
humidified chamber.
2.11.3. Visualization of slides and scoring
An image analysis system (Komet 5.5; Kinetic Imaging, Liverpool,
UK) connected to an Olympus (CX41) fluorescent microscope (Olympus
Optical Co., Tokyo, Japan) and a COHU 4910-integrated CC camera
(equipped with 510–560 nm excitation and 590 nm barrier filters)
(COHU, San Diego, USA) was used for the visualization of slides. Images
of 50 cells (25 from each replicate slide) were analysed for the measure-
ment of tail length (migration of DNA from the nucleus in μ meter). DNA
damage was quantified in terms of tail length.
2.12. Measurement of peroxidase activity
Peroxidase activity was assayed by the change in the optical density
(460 nm) at 37 °C by estimating the initial rate of oxidation of 6.0 mM
o dianisidine HCl by 18.0 mM H2O2 [23].
2.13. Determination of protein concentration
The concentration of protein was determined by using the proce-
dure described by Lowry et al. [24]. The used standard protein was bo-
vine serum albumin.
3. Results and discussion
3.1. Entrapment of GP into ANGG and AGG
The entrapment conditions of GP into calcium alginate beads has
been optimized at various concentrations of sodium alginate and CaCl2
solution for varying times. It was observed that the maximum activity
was retained at 2.5% sodium alginate and 2% CaCl2 solution when
beads were stirred for 2 h (Fig. 1a–c). Lower activity was retained by
the beads obtained at a higher concentration of alginate and CaCl2 incu-
bated for the longer duration. Because these beads are more compact in
terms of higher crosslinking which may decreases the pore size of the
beads and thus restrict the movement of substrate and products from
the beads, consequently decreasing the activity of the entrapped en-
zyme. Similarly, at the lower concentration of alginate and CaCl2 and
with shorter stirring time the beads formed retained lower activity of
enzyme which may be attributed to larger pore size resulting in the
leakage of enzyme. These beads were also found to be fragile, were
not suitable for repeated uses and cannot be employed in continuous re-
actors for a longer duration. [25–28].
The maximum activity retention (78%) of GP into agarose was ob-
tained at its 3% concentration (Fig. 1d). Porosity of gel and substrate dif-
fusion inside the matrix depends upon the concentration of agarose
which is very critical for the entrapment of enzyme. Gel obtained at a
lower concentration of agarose retained low activity of enzyme which
may be due to large pore size of the gel resulting in the leaching of en-
zyme. Nevertheless, high concentration of agarose leads to the forma-
tion of gel with small pores which restricts the diffusion of substrate
to the active site of enzyme and finally decrease the activity recovery
yield [29].
The immobilization yield was found to be high in the ANGG (81%)
and AGG (78%) as compared to alginate (68%) and agarose (66%), re-
spectively (Table 1). Loss of water from the beads during initial gelling
process before the gel is completely polymerized causes the leaching
466 M. Ali, Q. Husain / International Journal of Biological Macromolecules 116 (2018) 463–471

Fig. 1. Optimization of different parameters for the maximum activity retention of GP inside ANGG and AGG. (a) Effect of alginate concentration, (b) Effect of CaCl2 concentration, (c) Effect
of curing time and (d) Effect of agarose concentration.

of enzyme from gel beads [30]. The high activity retention in ANGG
compared to alginate alone demonstrates the poor loss of water during
gel formation which led to decrease in enzyme leakage. This may be due
to water holding capacity of GG which helped in the retention of higher
enzyme activity within polymeric structure, and prevented the leakage
of GP from the gel [18]. Similarly, the hydrogel of agarose alone has
water losing tendency due to the process of syneresis [31]. Therefore,
the high activity retention in AGG than agarose alone is also due to
the water holding capacity of GG. On the other hand, the average size
of GG increases in its hydrated condition which provides a dense barrier
to leakage of the enzyme from the entrapping support (Fig. 2) [32].
3.2. Surface morphology studies of alginate/agarose alone, ANGG/AGG and
ANGG-GP/AGG-GP beads
It can be clearly observed in Fig. 3 that GG has significant effect on
the surface morphologies of alginate/agarose gels. Alginate beads have
quite porous structure while ANGG beads are somewhat smooth in na-
ture with some ridges. This might be due to covering of pores by GG
(Fig. 3a & b). Surface morphology exhibited by ANGG-GP beads was
similar to the ANGG beads with some additional ridges confirming the
entrapment of GP inside the ANGG beads (Fig. 3c). Such pores covering
transition was also reported by De Matteis et al. [33] in which pores of

Table 1
Immobilization yield of different supports.

Immobilization Protein Protein Enzyme Enzyme activity Enzyme activity Specific activity Effectiveness Immobilization Enzyme activity
support loaded, W entrapped, activity in washes, Z, (U) bound bound factor, B/A = η yield (%), B/A × expressed per gram
(mg) X (mg) loaded, Y, (U) 100 of support (U/g)
Theoritical Actual Theoritical Actual
Y-Z = R, S, (U) R/X = A, S/X = B,
(U) (U/mg) (U/mg)

Agarose 20.8 13.72 50 17 33 21.91 2.40 1.59 0.66 66 36.51

Agarose-guar 20.8 16.64 50 10 40 31.20 2.40 1.88 0.78 78 52.00
gum
Alginate 20.8 14.14 50 16 34 23.20 2.40 1.64 0.68 68 46.40
Alginate-guar 20.8 17.47 50 8 42 34.02 2.40 1.95 0.81 81 68.04
gum

Each value represents the mean for three independent experiments performed in duplicates, with average standard deviation of ≤5%.
 M. Ali, Q. Husain / International Journal of Biological Macromolecules 116 (2018) 463–471 467

Fig. 2. Schematic representation of GP entrapment in ANGG/AGG.

silica was covered by chitosan. Fig. 3d–f demonstrates the sequential
changes in the surface morphology of the agarose gel. Surface morphol-
ogy of agarose alone was found to be smooth while AGG gel was quite
rough due to presence of GG in the agarose gel. The extent of roughness
was increased when GP was entrapped in AGG gel. Similar surface mor-
phology changes were also reported earlier [34].
3.3. Effect of various parameters on the decolorization of effluent by using
free and ANGG/AGG immobilized GP
as compared to AGG-GP and free GP. The ANGG-GP decolorized 79% of
the effluent at pH 9.0, whereas AGG-GP & free GP decolorized 40% and
9.4% of the effluent under similar experimental conditions, respectively.
Earlier reports also showed that effluent decolorization was maximum
at the acidic pH (pH 3.0–4.0) and declined significantly in the alkaline
pH range [27]. The broadening in pH activity profile of immobilized
GP might be due to change in the state of protonation of enzyme mole-
cules which depends upon the microenvironment provided by gel
beads [35].

3.3.1. Effect of pH 3.3.2. Effect of temperature

Fig. 4a demonstrates the effect of pH on the decolorization of textile
effluent by free and immobilized GP. Both immobilized preparations
and free GP showed same pH-optima for effluent decolorization at
pH 5.0. However, immobilized preparations showed a notable broaden-
ing in pH-activity profiles as compared to free GP. ANGG-GP showed
relatively higher effluent decolorization at alkaline side of pH-optima
Both immobilized preparations and free GP exhibited same
temperature-optima, at 40 °C (Fig. 4b). ANGG-GP and AGG-GP showed
notably high effluent decolorization activity at temperatures below and
above the optimum temperature as compared to free GP. ANGG-GP and
AGG-GP exhibited 63% and 58% effluent decolorization at 80 °C, respec-
tively, while free GP decolorized only 19% of the effluent at this

Fig. 3. SEM images of (a) alginate alone, (b) ANGG, (c) ANGG-GP, (d) agarose alone, (e) AGG, and (f) AGG-GP.
468 M. Ali, Q. Husain / International Journal of Biological Macromolecules 116 (2018) 463–471

Fig. 4. Effect of pH, temperature and thermal denaturation on the decolorization of effluent by free and ANGG/AGG immobilized GP. (a) Decolorization of effluent in the buffers of varying
pH, (b) Decolorization of effluent at different temperatures, and (c) Effect of thermal denaturation at 60 °C for varying times on effluent decolorization activity. Data represents mean ± S.D.
of three individual experiments, p-value ≤ 0.05.

temperature. Our results are much better than earlier reported
findings in which calcium alginate entrapped turnip peroxidase
retained b40% of the enzyme activity [36]. Activity exhibited by
immobilized preparations at higher temperatures may be attributed to
the structural rigidity of enzyme provided by hydrophobic interactions
between enzyme and support. Moreover, the high diffusion rate of sub-
strate and products at higher temperatures may also account for this
high activity [37]. Also the heat energy may get absorbed by entrapping
support which provides resistance to immobilized enzyme against ther-
mal denaturation.
Free and immobilized GP were incubated at 60 °C for various
time intervals. Incubation of free GP at 60 °C for 2 h resulted in a
loss of 65% of initial effluent decolorization activity. However,
ANGG-GP and AGG-GP retained 77% and 72% of the effluent decoloriza-
tion activity under similar incubation conditions, respectively (Fig. 4c).
It is well known that at higher temperatures protein gets denatured
into an irreversible unfolding form resulting in aggregated and distorted
structures [38]. Such kind of unfolding of protein molecules is resisted
by immobilized GP may be due to multipoint attachment of GP to the
GG [16].
3.3.3. Effect of urea
Fig. 5a demonstrates the effect of 4.0 M urea on the effluent decolor-
ization by GP. ANGG-GP and AGG-GP retained 83% and 72% of their de-
colorization efficiency after 2 h incubation in 4.0 M urea, whereas free
GP retained around 60% decolorization activity under identical
exposure. Matto and Husain [38] reported that calcium alginate-starch
hybrid beads entrapped bitter gourd peroxidase retained around 70%
of its initial activity after 2 h incubation.
3.3.4. Effect of organic solvent
The effect of increasing concentrations of DMSO (10–80%, v/v) on
the effluent decolorization efficiency of free and immoblilized GP is
shown in Fig. 5b. ANGG-GP and AGG-GP decolorized around 72% and
63% of the textile effluent in the presence of 80% (v/v) DMSO, respec-
tively. However, free GP decolorized only 42% of the textile effluent
after exposure to 80% (v/v) DMSO. It has been reported that low
water requirement and enhanced structural rigidity of immobilized en-
zyme is responsible for its increased stability against various organic
solvents [39].
3.3.5. Effect of detergent
Fig. 5c demonstrates the effect of Triton X-100 on the decolorization
activity of free and immobilized GP. ANGG-GP and AGG-GP decolorized
around 70% and 65% of the textile effluent when exposed to 5.0% (v/v)
Triton X-100. However, free GP was sensitive to Triton X-100 exposure
and it decolorized only 15% of the textile effluent under similar expo-
sure. ANGG-GP and AGG-GP are quite stable against the high concentra-
tion of detergent as compared to calcium alginate entrapped Con A-
pointed gourd peroxidase which retained only 40% of its initial activity
in the presence of 5.0% (v/v) Triton X-100 [40].
 M. Ali, Q. Husain / International Journal of Biological Macromolecules 116 (2018) 463–471 469

Fig. 5. Effect of urea, organic solvent and detergent on the decolorization of effluent by free and ANGG/AGG immobilized GP. (a) Effect of 4.0 M urea for varying times on effluent
decolorization activity, (b) Decolorization of effluent in varying concentrations of DMSO, and (c) Decolorization of effluent in varying concentrations of Triton X-100. Data represents
mean ± S.D. of three individual experiments, p-value ≤ 0.05.

3.4. Effluent decolorization in batch processes and reusability of ANGG/AGG
immobilized GP
Fig. 6a shows the effluent decolorization by free and immobilized GP
in batch processes. It was observed that ANGG-GP and AGG GP could
decolorize N95% textile effluent within 3 h of incubation, whereas free
GP decolorized only 84% of the dye effluent under similar experimental
conditions. Thus, immobilized GP was more efficient in the decoloriza-
tion of textile effluent as compared to free GP over long incubation pe-
riod. This is due to the binding of enzyme to GG, as polysaccharides

Fig. 6. Decolorization of textile effluent in stirred batch processes and reusability of ANGG/AGG immobilized GP. (a) Decolorization of textile effluent in the stirred batch processes by free
and ANGG/AGG immobilized GP, and (b) reusability of ANGG/AGG immobilized GP in the stirred batch processes. Data represents mean ± S.D. of three individual experiments, p-value ≤
0.05.
470 M. Ali, Q. Husain / International Journal of Biological Macromolecules 116 (2018) 463–471
Fig. 7. Schematic illustration of continuous reactor.
provides stability to the enzyme and retain their activity by forming
complex with them [25]. Bilal et al. [27] reported around 87.4% decolor-
ization of textile effluent by calcium alginate entrapped manganese per-
oxidase after 5 h of incubation. Therefore, ANGG-GP and AGG-GP were
more efficient in the decolorization of textile effluent.
Reusability of enzyme is an important aspect which can make the
enzyme catalyzed processes very cost effective. After 10th repeated
use the ANGG-GP retained 68% of the decolorization activity, whereas
AGG-GP showed 55% decolorization activity (Fig. 6b). Loss of enzyme
activity during its repeated use might be due to its inhibition by the gen-
erated product as it may block the active site of the enzyme. Several
times these products act as competitive inhibitor for the enzyme or
they may block the pores of the beads thus limiting the access of dye
to the enzyme [41].
3.5. Decolorization of textile effluent in continuous vertical bed-reactors
filled with ANGG/AGG immobilized GP
Considering the success of effluent decolorization by immobilized
GP in batch process, its efficiency at large scale was also evaluated by
using the continuous reactor systems (Fig. 7). Table 2 demonstrates
the percent decolorization of textile effluent by ANGG-GP and AGG-GP
in continuous reactor systems. The reactors containing ANGG-GP and
AGG-GP could decolorize N90% of the textile effluent during initial
10 days of operation. However, after 30 days of their operation, both
the reactors containing ANGG-GP and AGG-GP were still capable of re-
moving 80% and 69% of the color, respectively, from the textile effluent
(Table 2). It was notable that the reactor systems used in this study were
able to operate continuously with high decolorization efficiency. This
was may be due to retaining of the enzyme by GG inside the entrapping
support which led to such a high operational stability of the reactors for
Table 2
Decolorization of industrial wastewater by ANGG/AGG immobilized GP in the continuous
reactors.
Time (days) ANGG-GP AGG-GP
5 98.3 ± 2.61 97.9 ± 3.51
10 96.4 ± 2.58 94.9 ± 2.68
15 92.0 ± 1.75 89.4 ± 2.43
20 88.1 ± 3.24 82.5 ± 1.94
25 84.1 ± 2.37 76.6 ± 2.03
30 80.0 ± 2.93 69.8 ± 2.70
Each value represents the mean for three independent experiments performed in dupli-
cates, with average standard deviation of ≤5%.
a longer period. This study was in agreement with an earlier study in
which continuous reactor containing calcium alginate–starch
entrapped bitter gourd peroxidase was able to decolorize around 80%
of the textile effluent even after 30 days of operation [42].
3.6. Genotoxicity analysis by comet assay
To evaluate the genotoxicity of textile effluent and its degraded
products, comet assay was done on human lymphocytes. Genotoxicity
is measured in terms of single strand break (SSB) in DNA. More the
SSB in DNA it will migrate faster during gel electrophoresis forming a
tail resembling comet like structure. Genotoxicity of the test substance
is measured in terms of tail length of the comet. Fig. 8 depicts that aver-
age tail length of the textile effluent treated lymphocytes was 26 μm
which was significantly higher than the tail length of control which
was 5 μm. Such a DNA damage induced in textile effluent treated lym-
phocytes confirms the genotoxic effect of the industrial textile effluent.
Tail length of the lymphocytes treated with GP degraded textile effluent
was found to be 8 μm which is comparable to the control value. Karim
et al. [43] also reported the reduction in genotoxicity induced by textile
effluent after the treatment by β-cyclodextrin-chitosan complex
immobilized horseradish peroxidase. Genotoxicity of the textile effluent
was also abated by Lysinibacillus sp. RGS immobilized on Loofa [44]. This
concludes that GP was found to be efficient not only for degradation of
dye present in textile effluent but also for its detoxification.
4. Conclusion
Our findings have demonstrated that GG has greatly improved the
entrapment of GP into alginate and agarose polysaccharides. ANGG-GP
retained higher amount of activity compared to AGG-GP and found
more stable against both physical and chemical denaturants i.e. pH,
temperature, urea, organic solvent and detergent. Besides, both the
entrapped enzyme preparations were immensely efficient in the decol-
orization of textile effluent in batch process with excellent reusability.
Continuous packed bed reactors were also efficacious in the continuous
treatment of textile effluent by retaining a significant amount of activity
even after 30 days of their continuous operation. Genotoxicity of the
textile effluent was also successfully diminished by GP treatment.
Therefore, these entrapped biocatalyst systems have shown their high
potential in the decolorization and detoxification of textile effluents at
a large scale.
Fig. 8. Comet assay of textile effluent before and after degradation by GP. Data represents
mean ± S.D. of three individual experiments, p-value ≤ 0.05.
 M. Ali, Q. Husain / International Journal of Biological Macromolecules 116 (2018) 463–471
Ethical statement
Five millilitre of fresh human blood (self-donor) was taken for the
genotoxic assessment of the textile effluent and its degraded products
in ‘comet assay’. According to the ethical guidelines of Indian Council
for Medical Research, New Delhi, India, Chapter- II, page no.11-12, eth-
ical approval for this research work was not deemed to be necessary. Ac-
cording to their guidelines, proposals which present less than minimal
risks are exempted from the ethical review process.
Conflict of interest
The authors declare that there is no conflict of interest regarding the
publication of this work.
Funding
This research did not receive any specific grant from funding agen-
cies in the public, commercial, or not-for-profit sectors.
Acknowledgements
Misha Ali is thankful to U.G.C., New Delhi, India, for the award of
UGC-SRF fellowship. University sophisticated instrumentation facility
(USIF), AMU Aligarh, India, is gratefully acknowledged for SEM analysis.
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