Enzymatic removal of chlorophenols using horseradish peroxidase immobilized on superparamagnetic Fe3O4/graphene oxide nanocomposite

Article abstract of DOI:10.1016/S1872-2067(15)60856-7

Magnetic Fe₃O₄ nanoparticles were successfully deposited on graphene oxide sheets by ultrasound-assisted coprecipitation. The nanoparticles were characterized using transmission electron microscopy, vibrating sample magnetometry, and X-ray photoelectron spectroscopy. The synthesized material was used as a support for the immobilization of horseradish peroxidase (HRP). The removals of 2-chlorophenol, 4-chlorophenol, and 2,4-dichlorophenol using the immobilized HRP were investigated. Batch degradation studies were used to determine the effects of the initial solution pH values, reaction temperature, reaction time, H₂O₂ and chlorophenol concentrations, and immobilized enzyme dosage on the removal of chlorophenols. The different numbers and positions of electron-withdrawing substituents affected the chlorophenol removal efficiency; the order of the removal efficiencies was 2-chlorophenol < 4-chlorophenol < 2,4-dichlorophenol. The oxidation products formed during chlorophenol degradation were identified using gas chromatography-mass spectrometry. The biochemical properties of the immobilized HRP were investigated; the results indicated that the storage stability and tolerance to changes in pH and temperature of the immobilized HRP were better than those of free HRP. The nanoparticles were recovered using an external magnetic field, and the immobilized HRP retained 66% of its initial activity for the first four cycles, showing that the immobilized HRP had moderate stability. These results suggest that the immobilized enzyme has potential application in wastewater treatment.

Full text of DOI:10.1016/S1872-2067(15)60856-7

Chinese Journal of Catalysis 36 (2015) 961–968
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c h n j c
Article
Enzymatic removal of chlorophenols using horseradish peroxidase
immobilized on superparamagnetic Fe3O4/graphene oxide
nanocomposite
Qing Chang a, Guodong Jiang b,*, Heqing Tang a, Na Li a, Jia Huang a, Laiyan Wu a
a Key Laboratory of Catalysis and Materials Science of the State Ethnic Affairs Commission and Ministry of Education, College of Chemistry and Material
Science, South-Central University for Nationalities, Wuhan 430074, Hubei, China
b College of Chemistry and Chemical Engineering, Hubei University of Technology, Wuhan 430068, Hubei, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Magnetic Fe3O4 nanoparticles were successfully deposited on graphene oxide sheets by ultra-
sound-assisted coprecipitation. The nanoparticles were characterized using transmission electron
microscopy, vibrating sample magnetometry, and X-ray photoelectron spectroscopy. The synthe-
sized material was used as a support for the immobilization of horseradish peroxidase (HRP). The
removals of 2-chlorophenol, 4-chlorophenol, and 2,4-dichlorophenol using the immobilized HRP
were investigated. Batch degradation studies were used to determine the effects of the initial solu-
tion pH values, reaction temperature, reaction time, H2O2 and chlorophenol concentrations, and
immobilized enzyme dosage on the removal of chlorophenols. The different numbers and positions
of electron-withdrawing substituents affected the chlorophenol removal efficiency; the order of the
removal efficiencies was 2-chlorophenol  4-chlorophenol  2,4-dichlorophenol. The oxidation
products formed during chlorophenol degradation were identified using gas chromatography-mass
spectrometry. The biochemical properties of the immobilized HRP were investigated; the results
indicated that the storage stability and tolerance to changes in pH and temperature of the immobi-
lized HRP were better than those of free HRP. The nanoparticles were recovered using an external
magnetic field, and the immobilized HRP retained 66% of its initial activity for the first four cycles,
showing that the immobilized HRP had moderate stability. These results suggest that the immobi-
lized enzyme has potential application in wastewater treatment.
Received 26 January 2015
Accepted 8 April 2015
Published 20 July 2015
Keywords:
Magnetic nanoparticle
Immobilized enzyme
Horseradish peroxidase
Biodegradation
Chlorophenols
Wastewater treatment
© 2015, Dalian Institute of Chemical Physics, Chinese Academy of Sciences.
Published by Elsevier B.V. All rights reserved.
1. Introduction
In particular, some of them have carcinogenic, teratogenic, and
mutagenic effects. In 1987, the US Environmental Protection
Chlorophenols, which are chlorinated aromatic compounds,
are widely used in the manufacture of plastics, pharmaceuti-
cals, printing and dyeing materials, pesticides, wood preserva-
tives, and petrochemicals [1]. They are a major group of pollu-
tants of environmental concern because of their high toxicities.
Agency categorized chlorophenols as priority pollutants and
set an upper permissible limit of 0.5 mg/L in public water sup-
plies [2,3]. Chlorophenols in aquatic environments must there-
fore be continuously monitored. Many techniques, including
physical adsorption [4,5], catalytic oxidation [6,7], and biodeg-
* Corresponding author. Tel/Fax: +86-27-59750481; E-mail: jianggd66@gmail.com
This work was supported by the National Natural Science Foundation of China (21107143, 21207033 and 21307164) and the Fundamental Research
Funds for the Central Universities, South-Central University for Nationalities (CZY15003).
DOI: 10.1016/S1872-2067(15)60856-7 | http://www.sciencedirect.com/science/journal/18722067 | Chin. J. Catal., Vol. 36, No. 7, July 2015

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Qing Chang et al. / Chinese Journal of Catalysis 36 (2015) 961–968
radation [8,9], have been used for the removal of chlorophe-
nols. Enzymatic degradation has been extensively used because
it has the merits of high specificity, selectivity, and catalytic
activity, mild reaction conditions, and few byproducts [10]. The
degradation of chlorophenols using horseradish peroxidase
(HRP), a widely used catalyst in enzymatic reactions [11−13],
proceeds through generation of phenoxy radicals, which can
react with other substrate molecules to give oligomers or pol-
ymers that are much more water insoluble than the original
monomers [12,14]. However, the use of free HRP is limited
because it has poor stability, and it is expensive because its
recovery and reuse are difficult. These drawbacks can be over-
come by immobilizing the enzyme [15,16]. The immobilized
enzyme should retain the same functionality and have the ad-
vantages of better storage stability, thermal stability, and ease
of operation compared with those of the free enzyme in solu-
tion [17].
Graphene oxide (GO), a derivative of graphene, is a two-di-
mensional planar material of one-atom thickness and has a
large specific surface area. It is therefore a good candidate for
supporting metal oxide nanoparticles and the immobilization of
a large number of enzymes [18,19]. GO contains a range of re-
active oxygen functional groups on the surface, therefore it can
be well dispersed in water and easily modified. The large
amount of surface functional groups on GO enables rapid en-
zyme immobilization through electrostatic interactions [20,21].
It has been reported that GO is an ideal platform for accepting
electrons from an enzyme and transferring them to a substrate
[22].
The superparamagnetism of magnetic nanoparticles (MNPs)
is attractive in a broad range of biomedical application because
the nanoparticles can be easily separated using an external
magnetic field and redispersed rapidly after removing the
magnetic field [23]. Fe3O4 MNPs are biocompatible, are not
hemolytic or genotoxic, and are superparamagnetic; they
therefore have potential application in areas such as separation
of biochemical products [24], magnetic resonance imaging [25],
targeted drug delivery [26], and biosensing [27]. The use of
solid enzyme supports and immobilized enzymes in degrada-
tion using enzymes has attracted our interest. In the present
work, Fe3O4 nanoparticles were deposited on GO sheets using
an ultrasound-assisted coprecipitation method, and HRP was
then immobilized on the Fe3O4/GO magnetic nanocomposite.
The Fe3O4/GO MNPs with immobilized HRP were used to re-
move chlorophenols in the presence of H2O2. The effects of the
pH values, reaction temperature, reaction time, and H2O2,
chlorophenol, and immobilized enzyme concentration on the
removal of chlorophenols were investigated. The stability and
reusability of the immobilized enzyme were also evaluated. The
oxidation products formed during chlorophenol degradation
were identified.
purification. KMnO4, FeCl3·6H2O, FeSO4·7H2O, NH3·H2O, H2O2
(30% m/m), 2-chlorophenol, 4-chlorophenol, 2,4-dichloro-
phenol, 1-ethyl-3-(3-dimethylaminopropy)carbodiimide (EDC),
NaH2PO4·2H2O, Na2HPO4·12H2O, potassium ferricyanide, and
4-aminoantipine (4-AAP) were obtained from the Sinopharm
Chemical Reagent Co., Ltd. (China). HRP (specific activity 250
units/mg, RZ ≥ 3) was purchased from the Tianyuan Biologic
Engineering Corp. (China). Natural flake graphite (99%) was
obtained from the Qingdao Lihaofeng Graphite Co., Ltd. (China).
Doubly distilled water was used.
The product morphology was examined using transmission
electron microscopy (TEM; FEI Tecnai G2 20, USA). The mag-
netic properties were evaluated using an ADE 4HF vibrating
sample magnetometer at 27 °C (USA). X-ray photoelectron
spectroscopy (XPS) was performed using a VG Multilab 2000
spectrometer (Thermo Electron Corporation, USA) with Al Kα
radiation as the excitation source. Ultraviolet-visible spectros-
copy was performed using an Evolution 201 spectrophotome-
ter (Thermo Scientific, USA). The chlorophenol oxidation in-
termediates were analyzed using a gas chromatography-mass
spectrometry (GC/MS) system (Thermo Scientific, USA)
equipped with a TR-5MS column (Thermo Scientific, USA).
2.2. Synthesis of nanostructured Fe3O4/GO nanoparticles
GO was obtained using the method described by Hummers
and Offeman [28]. The GO was added to distilled water and
ultrasonically exfoliated in a bath sonicator for 30 min to obtain
a GO dispersion. Fe3O4/GO MNPs were synthesized using an
ultrasound-assisted coprecipitation method [21]. Briefly,
FeSO4·7H2O (6.0  10−4 mol) and FeCl3·6H2O (9.0  10−4 mol)
dissolved in distilled water (20 mL) were mixed with the GO
dispersion. The mixture was added dropwise to ammonia wa-
ter (20 mL, 12.0 mol/L) at 60 °C and reacted for 1 h under ul-
trasound irradiation in an ultrasound cleaning bath
(KQ-200KDE, Kunshan Ultrasound Instrument Co., Ltd.). The
product was washed with ethanol and distilled water until
neutral and then dried to obtain GO/Fe3O4 MNPs.
2.3. Enzyme immobilization
GO/Fe3O4 MNPs (0.3 g) and HRP (3 mg) were dispersed in
phosphate buffer (pH = 6, 20.0 mL), followed by addition of
EDC (1.5 mL, 25 mg/mL). The mixture was incubated in a
shaker at 200 r/min for 12 h at 25 °C. The nanoparticles were
collected by magnetic separation and washed separately with
phosphate buffer and water to remove non-specifically un-
bound enzyme. The immobilized enzyme was dispersed in wa-
ter and stored at 4 °C.
2.4. Catalytic experiments
2. Experimental
Immobilized enzyme solution (1.2 mL) was added to an
aqueous solution of chlorophenol (40 mL, 0.5 mmol/L); the pH
was adjusted to 6.4 using sodium phosphate buffer. The mix-
ture was vibrated at a speed of 200 r/min at 25 °C. After 30 min
to achieve adsorption-desorption equilibrium, the reaction was
2.1. Materials and characterization
All reagents were analytical grade and used without further

Qing Chang et al. / Chinese Journal of Catalysis 36 (2015) 961–968
963
initiated by the addition of H2O2 (0.5 mmol/L). At given time
intervals, 2 mL aliquots of the reaction solution were removed
and the MNPs were immediately recovered by magnetic sepa-
ration. The concentration of chlorophenol in the supernatant
was determined spectrophotometrically [29].
nanoparticles with a narrow size distribution, some of which
aggregated to give larger particles. The magnetization curves of
the Fe3O4/GO nanoparticles and Fe3O4 nanoparticles were
measured at room temperature; the results are shown in Fig.
1(b). The hysteresis loops of the dried Fe3O4/GO and Fe3O4
nanoparticles are S-shaped curves; this shows that the samples
have negligible coercivity (Hc) and remanence, and are there-
fore typical superparamagnetic materials. The specific satura-
tion magnetizations (Ms) of the Fe3O4/GO and Fe3O4 nanoparti-
cles are 26.0 and 68.8 emu/g, respectively.
XPS is an effective technique for determining surface chem-
ical states. Fig. 2(a) shows the C 1s deconvolution spectra of
Fe3O4/GO MNPs; there are four peaks, centered at 284.5, 286.5,
288.2, and 289.0 eV, and these are assigned to non-oxygenated
carbon (C=C/C–C) in aromatic rings, C–O (epoxy and alkoxy),
C=O, and O–C=O groups, respectively. The spectrum shows that
non-oxygenated carbon (284.5 eV) and C–O (286.2 eV) are the
predominant carbon forms in the GO. The formation of Fe3O4
nanoparticles is confirmed by the high-resolution Fe 2p spec-
trum (Fig. 2(b)), in which the observed peaks are at 724.8 and
711.2 eV, characteristic of Fe 2p1/2 and Fe 2p3/2 of Fe3O4 [31],
rather than at 710.35 and 724.0 eV, from -Fe2O3 [32].
2.5. Assays of enzymatic activity
A colorimetric method was used to determine the activity of
the enzyme using phenol, 4-AAP, and H2O2 as substrates [30].
The catalytic reaction was monitored by recording the absorb-
ance of the red product at 510 nm. One unit of activity was de-
fined as the amount of HRP required to hydrolyze 1 × 10−6 mol
of H2O2 per minute under the conditions stated above.
2.6. Recycling experiments
The used immobilized enzyme was collected by centrifuga-
tion and magnetic separation, and washed with deionized wa-
ter to remove the reaction solution. The recovered catalyst was
reused in subsequent degradation reactions under the experi-
mental conditions used in the original reaction.
3. Results and discussion
3.2. Effect of reaction conditions
3.1. Characterization of Fe3O4/GO composites
3.2.1. pH values
The effect of pH values on the degradation of 2-chlorophe-
nol, 4-chlorophenol, and 2,4-dichlorophenol by the immobi-
lized enzyme was investigated. The results are shown in Fig. 3.
A TEM image of the prepared Fe3O4/GO composite (Fig.
1(a)) clearly shows GO sheets uniformly decorated with Fe3O4
80
(b)
(a)
(a)
60
40
(2)
(1)
20
0
-20
-40
-60
-80
-15
-10
-5
0
5
10
15
Applied field (kOe)
Fig. 1. (a) TEM image of Fe3O4/GO MNPs and (b) magnetization curves of Fe3O4/GO MNPs (1) and Fe3O4 MNPs (2) measured at 27 °C.
(a)
(b)
Fe 2p3/2
Fe 2p1/2
292
290
288
Binding energy (eV)
Fig. 2. XPS profiles of Fe3O4/GO MNPs. (a) C 1s; (b) Fe 2p.
286
284
282
735
730
725
720
715
710
705
Binding energy (eV)

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Qing Chang et al. / Chinese Journal of Catalysis 36 (2015) 961–968
3.2.3. H2O2 concentration
100
80
60
40
20
0
The cosubstrate H2O2 activates the enzymatic reaction to
produce peroxidase radical intermediates, which attack the
chlorophenol compounds to form free radicals [33]. Optimiza-
tion of the conditions for the removal of chlorophenols was
performed using different concentrations of H2O2. Fig. 5 shows
that the removals of 2-chlorophenol, 4-chlorophenol, and
2,4-dichlorophenol increase significantly with increasing H2O2
concentration from 0.05 to 0.5 mmol/L. When the H2O2 con-
centration is higher than 0.5 mmol/L, the chlorophenol degra-
dation efficiency gradually decrease. It has been reported that
excess H2O2 inhibits the peroxidase activity by inactivating the
enzyme [34]; this is in agreement with the results of our study.
(3)
(2)
(1)
4
5
6
7
8
9
pH value
Fig. 3. Effect of pH values on chlorophenol degradation. (1) 2-chloro-
phenol; (2) 4-chlorophenol; (3) 2,4-dichlorophenol.
3.2.4. Reaction time
Fig. 6 shows the effect of time on the degradation of
2-chlorophenol, 4-chlorophenol, and 2,4-dichlorophenol using
the immobilized enzyme. The immobilized enzyme activates
H2O2, therefore chlorophenol removal initially increases rapid-
ly and then reaches a plateau after 120 min. The highest per-
centage of removal is observed for 2,4-dichlorophenol, fol-
lowed by 4-cholrophenol and 2-chlorophenol; 83% of the ini-
tial 2,4-dichlorophenol is degraded in 120 min. This observa-
tion is in agreement with the results of previous studies [35].
The percentage of removal is probably affected by the different
The optimum pH for the degradation of chlorophenol com-
pounds using the immobilized HRP is observed at around pH =
6.4, with maximum removals in 2 h of 83%, 46%, and 32% for
2,4-dichlorophenol, 4-chlorophenol, and 2-chlorophenol, re-
spectively. The catalytic activity of the immobilized enzyme
may be decreased by denaturation under acidic or basic condi-
tions.
3.2.2. Reaction temperature
An immobilized enzyme has an optimum temperature range
in which its activity is maximum. The degradation of
2-chlorophenol, 4-chlorophenol, and 2,4-dichlorophenol using
immobilized HRP was studied. Fig. 4 shows that the removals
of 2,4-dichlorophenol and 2-chlorophenol initially increase
with increasing temperature. A plateau appears in the temper-
ature range 25–30 °C, and the highest removals, i.e., 83% and
32% for 2,4-dichlorophenol and 2-chlorophenol, respectively,
are achieved. The degradation efficiency of 2,4-dichlorophenol
and 2-chlorophenol catalyzed by the immobilized enzyme de-
crease at temperatures higher than 30 °C; this is ascribed to
enzyme inactivation at high temperatures. For 4-chlorophenol,
the maximum removal is observed at 20 °C. It is concluded that
the immobilized HRP has different optimum temperatures for
different substrates. The optimum temperature range of the
immobilized enzyme is easily achieved, therefore this method
could be used in the treatment of industrial wastewater.
100
80
(3)
60
40
20
0
(2)
(1)
0.0
0.2
0.4
0.6
0.8
1.0
H2O2 concentration (mmol/L)
Fig. 5. Effect of H2O2 concentration on chlorophenol degradation. (1)
2-chlorophenol; (2) 4-chlorophenol; (3) 2,4-dichlorophenol.
100
100
80
80
60
40
20
0
(3)
(2)
(1)
(1)
60
(2)
40
20
(3)
0
0
30
60
90
120 150 180 210
15
20
25
30
35
40
Temperature (oC)
t (min)
Fig. 6. Effect of time on chlorophenol degradation. (1) 2-chlorophenol;
Fig. 4. Effect of temperature on chlorophenol degradation. (1) 2-chlo-
(2) 4-chlorophenol; (3) 2,4-dichlorophenol.
rophenol; (2) 4-chlorophenol; (3) 2,4-dichlorophenol.

Qing Chang et al. / Chinese Journal of Catalysis 36 (2015) 961–968
965
100
80
60
40
20
0
100
80
60
40
20
0
0.0
0.2
0.4
0.6
0.8
1.0
0.15 0.20 0.25 0.30 0.35 0.40 0.45
Concentration of catalyst (g/L)
Concentration of 2,4-dichlorophenol (mmol/L)
Fig. 7. Effect of initial 2,4-dichlorophenol concentration on degradation.
Fig. 8. Effect of catalyst concentration on 2,4-dichlorophenol degrada-
tion.
numbers and positions of the electron-withdrawing substitu-
ents [36]. Because it has a larger number of substituents on the
benzene ring, 2,4-dichlorophenol can be removed via a greater
variety of reaction pathways. In addition, the different phenoxy
radical intermediates, which are produced by enzymatic catal-
ysis, may have different toxicities to the enzyme.
between the removal efficiency and enzyme cost should be
optimized. The optimum catalyst dose is 0.30 g/L.
3.3. Biochemical properties of immobilized HRP
3.3.1. Optimum pH value and temperature
3.2.5. 2,4-Dichlorophenol concentration
Fig. 9(a) shows the effect of pH values on the relative activi-
ty of free and immobilized HRP. The optimum pH values for
free and immobilized HRP are both around 6.4. The relative
activity of the free and immobilized enzymes decreases when
the pH is too acidic or too alkaline. However, the immobilized
HRP retains about 85% of its activity at pH = 9.4, whereas free
HRP retains about 54% of its activity at this pH value. The pH
profile of the immobilized enzyme is broader than that of free
HRP, which indicates that the immobilization method pre-
serves the enzymatic activity over a wider pH range [11]. The
effects of temperature on the activity of free and immobilized
HRP are shown in Fig. 9(b). The relative activity of the free en-
zyme initially increases with increasing temperature, and then
declines with increasing temperature above 30 °C. The de-
crease in activity can be attributed to deactivation of the en-
zyme at high temperatures. The immobilized HRP also has
maximum activity at 30 °C, but the performance of the enzyme
at higher temperatures is improved by immobilization. The
immobilized enzyme tolerates a much wider range of temper-
atures. These results suggest that immobilization stabilizes the
enzyme and enables it to resist denaturation by various factors
The substrate concentration in the aqueous phase signifi-
cantly affects enzyme-mediated reactions. The effect of the
2,4-dichlorophenol concentration on the removal efficiency
was investigated, and the results are shown in Fig. 7. The
2,4-dichlorophenol removal efficiency decreases with increas-
ing 2,4-dichlorophenol concentration. These results enable the
initial concentrations of chlorophenols in wastewaters needed
to achieve optimum removal efficiency to be determined.
3.2.6. Optimum catalyst dose
The catalyst concentration is also a key factor in chlorophe-
nol removal. The optimum dose of immobilized HRP was de-
termined by performing experiments under specified experi-
mental conditions; the results are shown in Fig. 8. The results
clearly show that immobilized HRP can catalytically oxidize
2,4-dichlorophenol effectively. 2,4-Dichlorophenol removal
initially increases with increasing catalyst concentration, and
then remains almost constant above 0.30 g/L of catalyst. The
highest proportion of the cost of enzymatic treatment of
wastewater is derived from the enzyme, therefore the balance
100
100
(a)
(2)
(b)
80
80
(2)
(1)
60
60
(1)
40
20
0
40
20
0
5
6
7
8
9
10
20
30
40
50
60
pH value
Temperature (oC)
Fig. 9. Effect of (a) pH values and (b) temperature on activity of (1) free and (2) immobilized HRP.

966
Qing Chang et al. / Chinese Journal of Catalysis 36 (2015) 961–968
100
(a)
100
80
60
40
20
0
(b)
(2)
(1)
80
60
(2)
40
(1)
20
0
30
40
Temperature (C)
Fig. 10. (a) Thermal and (b) storage stability of (1) free and (2) immobilized HRP.
50
60
70
0
3
6
9
12
15
t (d)
such as acids, alkalis, and heat.
block the active sites of the enzyme, leading to decreased activ-
ity. (2) The immobilized enzyme could be inactivated by free
radicals during enzymatic oxidation of chlorophenol, which
would contribute to the activity reduction in the next cycle
[11,34]. Use of the immobilized enzyme would greatly reduce
the cost of the enzymatic system in practical application be-
cause of the ease of separation and good stability.
3.3.2. Thermal and storage stability
The immobilized HRP showed good thermal stability during
its use in the oxidative removal of organic pollutants. The
thermal stability of the immobilized and free enzymes were
determined by measuring the residual activity of immobilized
HRP incubated at temperatures of 30–70 °C. Fig. 10(a) shows
that after heat treatment at 70 °C, the relative activity of free
HRP decreases to 9% of the initial value, but the immobilized
HRP retains 40% of its initial activity. These results suggest
that the immobilized enzyme is more resistant to the heat than
the free HRP; this can be attributed to limitation of the confor-
mational changes of HRP molecules in the support matrix.
The storage stability of free and immobilized HRP was also
investigated. As shown in Fig. 10(b), the activity of free HRP
gradually declines at room temperature. Under the same stor-
age conditions, the activity of the immobilized HRP decreases
more slowly than that of the free enzyme. The immobilized
HRP retains about 84% of its relative activity during storage for
15 d, whereas the relative activity of the free HRP declines to
30% of its initial value. The immobilized HRP is therefore more
stable than the free HRP. The Fe3O4/GO magnetic nanocompo-
site has a stabilizing effect, and the immobilized enzyme has a
longer shelf-life than its free counterpart [11].
3.5. Chlorophenol oxidation products
The chlorophenol oxidation products were identified using
GC/MS; the results are shown in Table 1. Several two-ring ar-
omatic chlorinated compounds were detected. Oxidative cou-
pling reactions of radicals with dimers could result in the gen-
eration of trimers, tetramers, pentamers, and so on. Alterna-
tively, the addition of radicals to dimers could lead to formation
of high-molecular-mass species. Both processes ultimately re-
sult in the generation of polymers, which precipitate from
aqueous media [37]. A possible 2,4-dichlorophenol degradation
pathway, based on the GC/MS analysis, is shown in Scheme 1.
There are two main pathways for 2,4-dichlorophenol degrada-
tion: generation of 2-chloro-1,4-benzoquinone and transfor-
mation to polymer sediments. HRP is present mainly on the GO
surface because the large amount of surface functional groups
on GO favor HRP immobilization. Additionally, EDC is used to
crosslink the HRP and GO to improve the stability of the immo-
bilized HRP. In the catalytic procedure, HRP activates chloro-
phenol oxidation by H2O2. The immobilized HRP donates a lone
3.4. Reusability of immobilized HRP
Compared with the free enzyme, the immobilized enzyme
can be easily separated from the reaction solution and reused.
The recovery and reusability of HRP immobilized on the
Fe3O4/GO magnetic nanocomposite were studied. The used
immobilized enzyme was recovered by magnetic separation,
washed with water to remove the reaction solution, and reused
in a subsequent reaction under the same experimental condi-
tions. The removal of 2,4-dichlorophenol using the recycled
immobilized enzyme is shown in Fig. 11. The immobilized HRP
retains 66% of its initial activity for the first four cycles, show-
ing that the immobilized enzyme has moderate stability and
can be recovered and reused. There are two possible reasons
for the reduction in the activity. (1) Polymers produced during
the enzymatic reaction may cover the immobilized enzyme and
100
80
60
40
20
0
1
2
3
4
Run
Fig. 11. Activity of reused catalyst.

Qing Chang et al. / Chinese Journal of Catalysis 36 (2015) 961–968
967
Table 1
Oxidation products formed during degradation of chlorophenols.
2-Chlorophenol
4-Chlorophenol
2,4-Dichlorophenol
2-Chloro-6-(2-chloro-phenoxy)-phenol
2-Chloro-4-(2-chloro-phenoxy)-phenol
Trimer
2-(4-Chlorophenoxy)-4-chlorophenol
3-(4-Chlorophenoxy)-4-chlorophenol
2-(4-Chlorophenoxy)-1,4-benzenediol
p-Benzoquinone
2,4-Dichloro-6-(2,4-dichloro-phenoxy)-phenol
2,4-Dichloro-5-(2,4-dichloro-phenoxy)-phenol
2-Chloro-6-(2,4-dichloro-phenoxy)-1,4-benzenediol
2-Chloro-1,4-benzoquinone
Hydroquinone
2-Chloro-1,4-benzenediol
Trimer
Biopolymer/trimer
OH
Cl
ered by magnetic separation, and the recovered nanoparticles
were reused as a catalyst for the enzymatic degradation of
chlorophenols.
Cl
HRP H2O2
References
OH
OH
OH
O
O
OH
Cl
Cl
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sedimentation
Scheme 1. Possible degradation pathways of 2,4-dichlorophenol with
immobilized HRP as catalyst.
pair of electrons to GO, and the electrons are transferred to
H2O2, resulting in formation of a high-oxidation-state interme-
diate from HRP [38]. The intermediate catalyzes the oxidation
of phenols, generating phenoxy radicals, and is itself recon-
verted to HRP by two one-electron reduction steps. These free
radicals spontaneously form insoluble polymers, which can be
removed from solution by sedimentation and filtration.
4. Conclusions
Magnetic Fe3O4/GO MNPs were synthesized using an ultra-
sound-assisted in situ precipitation method. HRP was covalent-
ly immobilized on the Fe3O4/GO composites using EDC as a
coupling agent. The immobilized HRP was used as an enzymat-
ic catalyst to activate H2O2 for degradation of 2,4-dichlorophe-
nol, 4-chlorophenol, and 2-chlorophenol. The optimum pH for
chlorophenol oxidation using the immobilized HRP was around
6.4. The temperature did not greatly influence chlorophenol
removal; the optimum temperature was in the range 25–30 °C.
When the molar ratio of H2O2/chlorophenol in the batch reac-
tion system was 1.0, maximum removal was achieved within 2
h. The order of the chlorophenol removal efficiencies was
2-chlorophenol  4-chlorophenol  2,4-dichlorophenol. The
immobilized enzyme had high storage stability. It was recov-
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968
Qing Chang et al. / Chinese Journal of Catalysis 36 (2015) 961–968
Graphical Abstract
Chin. J. Catal., 2015, 36: 961–968 doi: 10.1016/S1872-2067(15)60865-7
Enzymatic removal of chlorophenols using horseradish peroxidase immobilized on superparamagnetic Fe3O4/graphene oxide
nanocomposites
Qing Chang, Guodong Jiang *, Heqing Tang, Na Li, Jia Huang, Laiyan Wu
South-Central University for Nationalities; Hubei University of Technology
100
2,4-dichlorophenol
2-chlorophenol
4-chlorophenol
80
NH3·H2O
60
40
20
0
HRP
EDC
0
30 60 90 120 150 180 210
t (min)
Fe2+
Fe3+
Fe3O4
HRP
Horseradish peroxidase (HRP) was immobilized on superparamagnetic Fe3O4/graphene oxide and used for enzymatic removal of chlo-
rophenols. The immobilized enzyme had high stability and was recovered by magnetic separation.
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