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Applied Clay Science 163 (2018) 92–99
Contents lists available at ScienceDirect
Applied Clay Science
journal homepage: www.elsevier.com/locate/clay
Research paper
Charge controlled immobilization of chloroperoxidase on both inner/outer
wall of NHT: Improved stability and catalytic performance in the
degradation of pesticide
Xueting Fana, Mancheng Hua,b, Shuni Lia,b, Quanguo Zhaia,b, Fei Wangc,
⁎⁎,1
, Yucheng Jianga,b,
T
⁎,1
a
School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, PR China
Key Laboratory of Macromolecular Science of Shaanxi Province, Shaanxi Normal University, Xi'an 710062, PR China
c
Advanced Catalysis and Green Manufacturing Collaborative Innovation Center, School of Petrochemical and Engineering, Changzhou University, Changzhou 213164, PR
China
b
A R T I C LE I N FO
A B S T R A C T
Keywords:
Halloysite nanotubes
Chloroperoxidase
Immobilization
Improved stability and activity
Application
Halloysite nanotubes (HNT) is a kind of kaolin clay consisting of one alumina octahedron sheet and one silica
tetrahedron sheet formed by rolling flat sheets into a hollow tubular structure. It is an ideal carrier for immobilization of enzyme due to its large surface area, biocompatibility, and chemical and mechanical stability
besides abundance and low-cost. In order to make the most of this carrier for enzyme immobilization, in this
work, two strategies were proposed for entrapping and embedding chloroperoxidase (CPO) on both inner/outer
wall of HNT rather than in the hollow space alone by pH modulated electrostatic adsorption. Besides the enhanced loading amount, the thermal stability and tolerance to organic solvents of immobilized CPO (I-CPO) was
greatly improved compared to the free enzyme. The free CPO can retain only 11.66% activity after 1 h incubation at 80 °C, while the I-CPO remained 87.63% activity at the same condition. Even after 1.5 h incubation
at 90 °C, when the free CPO lost all its activity, the I-CPO can still remain 40.3% activity; Moreover, in the
presence of organic solvent (ethyl acetate, acetonitrile, methanol, and DMF) with volume fraction of 10%, almost
no loss of activity of I-CPO was observed, but free CPO can only remain 41.6%, 38.2%, and 23.5% of its initial
activity in ethyl acetate, acetonitrile, methanol-water mixed system respectively, and even inactivated completely in DMF in the same condition. Furthermore, the enzymatic kinetic parameters (Km, and kcat/Km) suggested the affinity and specificity of I-CPO to the substrate was improved.
I-CPO was very efficient when applied in the degradation of isoproturon in wastewater. The isoproturon with
initial concentration of 26.7 μmol·L−1 can be completely degraded only in 10 min, indicating a potential practical application of I-CPO in treatment of wastewater containing pesticide.
1. Introduction
An ideal carrier for immobilization of enzyme is generally considered to be porous with huge surface area, modifiable surface, biocompatibility, and chemical and mechanical stability. Halloysite clay
seems to be one of the good candidates. It is a layered aluminosilicate
nanotube consisting of one alumina octahedron sheet and one silica
tetrahedron sheet in 1:1 stoichiometric ratio formed by rolling flat
sheets of kaolin clay [Yuan et al., 2015; Tully et al., 2016]. Halloysite
nanotube (HNT) possess hollow nanotubular structure in the submicrometer range and large specific surface area, with a diameter of
50 nm, an inner lumen of 15 nm and a length of 600–900 nm [Lvov
et al., 2016]. In contrast with other nanoparticles, such as carbon nanotube (CNT), naturally occurring HNT are cheap, durable and available in abundance in many province of China as well as other locations
around the world [Zhai et al., 2010]. Dispersion of HNT to single particles is much easier compared with platy kaolin, montmorillonite, or
bentonite particles because they are not stacked together [Zhang et al.,
⁎
Corresponding author at: School of Chemistry & Chemical Engineering, Shaanxi Normal University Xi'an, No. 620 West Chang'an Road, Chang’an District 710119,
PR China.
⁎⁎
Corresponding author.
E-mail addresses: fxt1992@snnu.edu.cn (X. Fan), hmch@snnu.edu.cn (M. Hu), lishuni@snnu.edu.cn (S. Li), zhaiqg@snnu.edu.cn (Q. Zhai),
wangfei@cczu.edu.cn (F. Wang), jyc@snnu.edu.cn (Y. Jiang).
1
These two authors make an equal contribution to this work.
https://doi.org/10.1016/j.clay.2018.07.016
Received 30 May 2018; Received in revised form 23 June 2018; Accepted 11 July 2018
Available online 18 July 2018
0169-1317/ © 2018 Elsevier B.V. All rights reserved.
Applied Clay Science 163 (2018) 92–99
X. Fan et al.
by 50 mmol·L−1 buffer (pH = 3), and dried in a vacuum oven overnight.
The concentration of CPO was measured by the adsorption at
λmax = 398 nm. The loading amount of CPO was calculated as following:
2017]. More importantly, HNT are among the few nanotubes with a
different composition of inner and outer surfaces. The outer surface is
similar to the properties of SiO2 while the inner cylinder core could be
associated with Al2O3. The different inside/outside chemistry of HNT
allow for different strategy and modification methods for loading of
guest molecules [Shchukin et al., 2005; Shu et al., 2017].
Recently, there are a few references reported the immobilization of
enzymes on HNT. Besides the physical adsorption through electrostatic
interaction [Lvov et al., 2008; Tully et al., 2016], surface modification
was employed to link enzyme and HNT, such as dopamine[Chao et al.,
2013; Zhai et al., 2013; Pandey et al., 2017]. Yao et al. synthesized
porous organic/inorganic hybrid microspheres by HNT and chitosan to
immobilize laccase [Yao et al., 2015]; Wang et al. immobilized lysozyme on HNT and LDH via a LbL deposition process [Wang et al. 2015];
Kadam et al. functionalized HNT with ATPES, magnetic nanoparticles,
and then immobilized laccase on HNT through cross-linked by GTA
[Kadam et al., 2017]. The distinguishing feature of HNT is the positively charged inner lumen and the negatively charged surface. However, in all the above works, enzymes were immobilized on the outer
wall or the inner tube alone instead of both inside/outside of HNT.
In this work, two strategies for the immobilization of chloroperoxidase (CPO) on both sides of HNT was proposed by charge controlled
and pH modulated electrostatic adsorption or hydrogen bonding interaction. The catalytic performance of immobilized CPO (I-CPO) was
investigated and compared with free CPO, including the thermal stability, the tolerance to organic solvent and reusability. Moreover, the ICPO was applied to the degradation of pesticide, isoproturon, in three
different countryside water samples, which showed the I-CPO had potential application in treatment of wastewater.
enzyme loading (mg•g −1) =
ΔCVM
× 1000
m
(1)
where ΔC is the difference of enzyme concentration in supernatant
before and after immobilization, V is the volume of the solution, M is
the molar mass of CPO, which is 42,000, and m is the weight of I-CPO.
The second strategy for CPO immobilization on both side HNT is to
change potential of the surface of HNT by a positively charged polymeric electrolyte (chitosan). In this way, negatively charged CPO can be
immobilized on both inside and outside of HNT simultaneously by
simply physical adsorption in one step.
Chitosan (CS) dissolved in acetic acid solution was added to previously homogenized HNT suspension. The final mixture (204 mL, 2%
HNT, 0.5% chitosan, 2% acetic acid) was stirred for 8 h to obtain HNTCS nanocomposites. HNT-CS were separated by centrifugation, washed
by deionized water till pH 7, and then dried for 24 h at 60 °C. 10 mg
HNT-CS was uniformly suspended into phosphate buffer, and then incubated with CPO solution for 12 h at 20 °C under shaking at 200 rpm.
CPO@HNT-CS was collected by centrifugation, then washed and dried.
2.3. Characterization of preteated HNT, I-CPO and CPO@HNT-CS
The micro-structure of HNT was observed using transmission electron microscopy (TEM) (JEM-2100). The distribution of pore size of the
samples was measured by an N2 adsorption desorption analyzer
(ASAP2020). The zeta-potential of the HNT is determined by Coulter
Backman (Delsa Nano C). The loading of CPO on the carrier was confirmed by laser confocal microscope (LSCM, FV1200).
2. Materials and method
2.1. Enzyme and regents
CPO was purified from the culture medium of the fungus, C. fumago,
according to the method reported by Morris and Hager [Morris and
Hager 1966], except acetone was used instead of methanol in the step
of solvent extraction. Then, CPO was further purified by column
chromatography which was packed by DEAE-Sephadex A-50 cation ion
exchange resin. The catalytic activity of CPO was measured through the
chlorination of monochlorodimedon (MCD) to DCD, which was
5538 U·mL−1 [Hager et al., 1966]. The purity of CPO expressed by Rz
value was 1.11 (Rz = A398/A280 = 1.44 regarded as pure CPO solution).
The collected CPO was concentrated to 14.1 mg·mL−1, and stored in
phosphate buffer at pH 5.5 at 4 °C. The CPO solution was freshly prepared by appropriately dilution.
Halloystie nanotube was obtained from Sigma-Aldrich. Isoproturon
was purchased from Sinopharm. All the other regents were analytical
reagent and from Xi'an Chemical Co. Ltd. The countryside water samples were taken from artificial lake, Heihe, and Zaohe in north China
with isoproturon added range from 26.67 μmol·L−1 to 133.33 μmol·L−1.
2.4. Stability of I-CPO at elevated temperatures and in the presence of
organic solvent
The thermal stability of immobilized CPO was investigated in the
temperature range of 25 °C–100 °C. The remained activity was measured according to the chlorination of MCD after incubation at fixed
temperature for 1 h. The remained activity of free CPO with the same
amount as I-CPO was also determined at the same condition, and
compared with the I-CPO. Then, at 90 °C, after 0.5 h, 1 h, 1,5 h, 2 h,
2.5 h, and 3 h incubation, the remained activity of immobilized and free
CPO was measured and compared.
N, N-dimethyl formamide (DMF), methanol, acetonitrile and ethyl
acetate were selected to examine the tolerance of I-CPO in the presence
of organic solvent at different volume ratio between 0% - 30% after 1 h
incubation, which was expressed as relative activity and compared with
free CPO.
2.2. Immobilization of CPO on both inside/outside of HNT
2.5. Application of immobilized CPO in the degradation of isoproturon in
wastewater
The HNT was pretreated by H2SO4 (2 mol·L−1) using the method
reported previously [Spepi et al., 2016]. 10 mg HNT were suspended
into phosphate buffer by ultrasonic treatment for 1 h until the aggregates is invisible. Then, CPO was embedded into the inner space of
HNT under vacuum at pH = 4.5 (which was above the isoelectric point
of CPO) so as to link the positively charged inner lumen and the negatively charged CPO by simply physical adsorption for 30 mins. This
process of vacuuming was repeated three times. Then, the buffer pH
was adjusted below the CPO isoelectric point (pH =3) to adsorb CPO
on the outer wall of HNT owing to the negatively charged outerside and
positively charged CPO at this pH on standing for another 12 h with
shaking at 200 rpm. The I-CPO was collected by centrifugation, washed
he concentration of isoproturon was determined by HPLC and the
degradation efficiency (ƞ) is calculated according to eq. (2).
η=
Co − Ct
× 100%
Co
(2)
where C0 is the initial concentration of the substrate, Ct is the concentration at anytime.
Isoproturon wastewater was prepared using three kinds of countryside water sample, Heihe river, Zaohe river and artificial lake with
26.67 μmol·L−1 - 133.3 μmol·L−1 isoproturon added.
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Applied Clay Science 163 (2018) 92–99
X. Fan et al.
Fig. 1. FESEM image of HNT (pristine) (a,b) and HNT (H2SO4)(c,d).
3. Results and discussion
collection of substrates, making the access of substrates to the active
site in enzyme more easier.
3.1. The lumen etching
3.2. Strategies for immobilization of CPO on HNT
The two-component of HNT allows the separate and controlled acid
etching for the inside wall of alumina or base decomposition of outside
of silica [Spepi et al., 2016]. In this work, HNT was firstly treated by
2 mol·L−1 H2SO4 for 48 h in order to increase the lumen’ diameter to
enhance the loading amount of enzyme. The morphology of HNT
(H2SO4) characterized by Field Emission Scanning Electron Microscope
(FESEM) showed HNT remained tubular structure after acid etching
(Fig. 1). The Transmission Electron Microscope (TEM) gave a clearer
morphology of inside wall of HNT, which indicated that HNT (pristine)
had a tubular structure with a smooth passage in the middle. After
etching by H2SO4, though the lumen’ diameter was not increased as
expected, however, some regular circular defects appeared on the inner
wall, in which the substrates can be concentrated and the mass transfer
resistance can be reduced accordingly. If the enzyme amount in immobilized CPO was kept same, the activity of immobilized CPO on HNT
treated by H2SO4 was higher than that before treating. The converting
efficiency of 0.25 μmol MCD by CPO (0.27 mg) immobilized on HNT
before or after acid etching was 90.36% and 98.43% respectively.
Meanwhile, the opening of the tubes kept unobstructed and the hollow
tubular structure remained (Fig. 2). The change of HNT morphology by
acid etching was caused by dissolution of aluminum oxide in the acidic
medium [Lvov et al., 2016]. Energy Disperse Spectroscopy (EDS) (Fig.
S1) showed that the values of Al/Si decreased from 98.6% to 77.9%
after acid etching, indicating some of the aluminum elements were
removed by H2SO4 treatment. Besides, the N2 adsorption/desorption
measurements also supported this selective etching (Fig. 3), which
showed the hollow size did not changed obviously (around 13.5 nm),
however, the BET surface area increased from 37.43 m2·g−1 to
57.53m2·g−1 due to the appearance of the regular circular defects on
inside wall with the pore size around 4 nm. This pore size was not big
enough to accommodate enzyme, but can serve as a center for
3.2.1. Simply adsorption by pH modulation through two steps
The surface electrical ζ-potential of HNT is about −30 mV at
pH 2–8, which is less than the typical ζ-potential for pure silica particles
(−50 mV). This is may be due to the superposition of HNT negative
outermost surface charges by positive charge on inner wall [Lvov et al.,
2016]. Based on the different charge distribution inside or outside walls
of HNT, we proposed a charge controlled and pH modulated strategy
for immobilization of CPO on both inside/ouside of HNT simultaneously. Firstly, the buffer pH was controlled at pH 4.5 so that CPO was
negatively charged owing to the buffer pH > pI (isoelectric point). The
negativlye charged CPO was entrapped into the inner tube by electrostatic attraction under vacuum condition. In this way, the negative
charge on the outside wall of HNT would increased due to that the
positive charges on innermost of the HNT was neutralized by the entrapped and negatively charged CPO [Lvov et al., 2016], which was also
supported by the ζ-potential measurements (Table 1). The increase of
negative charge on outside wall of HNT was also contributed by some
alumina was removed by acid etching. Then, the buffer pH was adjusted
to 3.0, at which pH < pI, so the positively charged CPO can be loaded
onto the outside wall of HNT simply by adsorption through the electrostatic attraction. In this way, CPO can be immobilized both on inside
or outside walls of HNT simultaneously. This strategy is summarized in
Scheme 1. Compared with the immobilization only in the inner hollow
of HNT reported in reference [Martin and Kohli 2003], the loading
amount of enzyme in this work increased from 11.6 mg·g−1 to
27.4 mg·g−1.
3.2.2. Modification by chitosan for One-step immobilization of CPO on
HNT
Chemical modification of HNT can generate a nanoarchitecture with
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Applied Clay Science 163 (2018) 92–99
X. Fan et al.
Pore volume distribution(m3/g.nm)
Fig. 2. TEM image of HNT (pristine) (a,b) and HNT (H2SO4) (c).
0.6
was proved by the ζ-potential listed in Table 1. So, when the buffer pH
was controlled at 4.5 (pH > pI), the negatively charged CPO can be
embedded on both inside/outside walls of HNT in one step by electrostatic interaction. This strategy is summarized in Scheme 2. However, we found if CPO was entrapped into the inner hollow before the
coating of chitosan, the CPO loading amount was 18.3 mg·g−1. This
data was a little higher compared with 14.6 mg·g−1 obtained by this
one-step method. This phenomenon indicated some opening of the
hollow may be blocked due to the coating of chitosan.
0.4
3.3. Characterization of the immobilization of CPO on HNT
0.2
The Laser confocal microscope (LSCM) was employed to investigate
the loading of CPO on the HNT, in which CPO was labeled by fluoresceine isothiocyanate (FITC). Fig. 4 showed a clear green fluorescence,
confirming CPO was immobilized on HNT by both the two strategies.
Besides, Fig. 3 also showed the inner hollow (around 13.5 nm) almost
disappeared after enzyme immobilization, which can be attributed to
the occupation of CPO in the hollow space. Moreover, the loss of weight
between 200 °C–400 °C on TG curve (Fig. S2) was assigned to the decomposition of CPO, supporting the conclusion of CPO immobilized on
HNT. The catalytic activity of I-CPO was determined according to the
changes of absorbance at 278 nm caused by chlorination of MCD converted to DCD. Fig. 5 showed the completely disappearance of the peak
at this position. The recovered activity of I-CPO was 97.9% by physical
adsorption and 93.3% by chitosan modification compared with the
activity of free CPO regarded as 100% in the same condition, indicating
the immobilization process had little influence on the catalytic activity.
The loss of catalytic activity was possibly due to the difficulty for
substrate to access to the active site of CPO in HNT and the little deform
of the required conformation of enzyme after immobilization.
The enzymatic kinetic parameters was determined using
Lineweaver-Burk double reciprocal method [Blaza et al., 2017], and
showed in Table 2. The Michaelis constant Km of I-CPO was small than
that of free CPO, suggested I-CPO had better affinity to substrates;
Moreover, the two order kinetic constant kcat/Km is often used to
evaluate the specificity of enzymes to substrates. The increased kcat/Km
of I-CPO compared with free CPO (about 20 times) indicated that the
specificity of I-CPO was greatly improved. But the Vmax decreased after
CPO immbilization possibly caused by the difficulty of access of substrate to I-CPO than to free CPO.
HNT(H2SO4)
1.4
HNT(pristine)
1.2
I-CPO
1.0
0.8
0.0
0
10
20
30
40
50
60
Pore size(nm)
Fig. 3. Pore size distrubution of HNT (pristine), HNT (H2SO4) before and after
CPO immobilization.
Table 1
ζ-potential of HNT in the buffer with different pH.
ζ-potential
pH = 3
pH = 4.5
HNT (pristine)
HNT (H2SO4)
HNT (CPO)
HNT-CS
−31.2 mV
−40.76 mV
−46.35 mV
12.34 mV
−24.56 mV
−42.72 mV
−49.22 mV
11.69 mV
targeted affinity through outer surface functionalization. Here, in order
to strengthen the bonding of CPO with HNT, chitosan was employed to
be coated on the surface of HNT to supply some functional groups, such
as eNH2 and eOH. CPO is a heavily glycosylated heme protein, and
carbohydrate accounts for about 19% of the total molecular weight
[Sundaramoorthy et al., 1995]. So, there is plenty of eOH group on
CPO to bond with HNT through hydrogen bonding formed between
these polar groups. Moreover, the charge on HNT surface turned to
positive due to the protonation of coated chitosan in acidic medium. In
this case, both the inside/ouside of HNT were positively charged, which
Scheme 1. Strategy for two-step immobilization of CPO on HNT by simply adsorption through pH modulation.
95
Applied Clay Science 163 (2018) 92–99
X. Fan et al.
Scheme 2. Strategy of chitosan modification for one-step immobilization of CPO on HNT.
3.4. Stability and reusability of I-CPO
1.5
HNT
I-CPO
The free enzymes generally unstable at elevated temperatures,
which limited greatly its practical application. The immobilization of
enzymes on a carrier can often improve its thermal stability. The I-CPO
was investigated at range of 20 °C–100 °C, and the results were showed
in Fig. 6. The activity of free CPO dropped rapidly with the increase of
temperature, and can retain 14.2% and 11.66% activity after 1 h incubation at 70 °C and 80 °C respectively, while the activity of I-CPO
decreased more slowly. I-CPO can remain 87.63% activity at 80 °C and
even no activity loss at 70 °C at the same condition. When temperature
was elevated to 90 °C, the free CPO and I-CPO can keep 8.72% and
50.76% of its original activity respectively after 1 h incubation. Even
after 1.5 h incubation at 90 °C, when the free CPO lost all its activity,
the I-CPO can still remain 40.3% activity. Obviously, the thermal stability of I-CPO was improved greatly compared to that of the free enzyme due to that the stable HNT can play a shielding effect to prohibit ICPO from inactivation. At the same time, HNT limited the area for
catalytic action of CPO, so, its extension and specific aggregation at
high temperature was limited to avoid the change of its three-dimensional structure.
Most of the enzymatic reactions need to be carried out in aqueous
solution. However, most of the substrate are hydrophobic. So, it is
usually necessary to add some organic solvent as a co-solvent to
278nm
Absorbance
1.0
0.5
0.0
200
250
300
350
400
450
500
Wavelength/nm
Fig. 5. The absorbance of MCD in the presence of HNT and I-CPO respectively.
improve the solubility of the organic substrate in water. But the presence of organic solvents may have negative effect on activity of the
enzyme due to the destruction of the hydrated layer on the surface of
Fig. 4. Confocal microscopy image of HNT (a) and (d), I-CPO by adsorption (b), I-CPO by adsorption +HNT (c), I-CPO by chitosan modification (e), I-CPO by
chitosan modification+ HNT (f).
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Applied Clay Science 163 (2018) 92–99
X. Fan et al.
conventional methods can not remove these herbicides from water efficiently. Isoproturon is a typical herbicides. In this work, I-CPO immobilized by the two strategies was applied to the degradation of isoproturon in three real water sample to evaluate if I-CPO can be used to
treat wastewater directly at non-optimum reaction conditions and in
the presence of some inorganic or organic component which was not
beneficial to the CPO activity and stability. The degradation efficiency
of isoproturon was determined by HPLC. The results in Fig. 9 showed
that the degradation efficiency decreased with the increase of initial
isoproturon concentration. When the added isoproturon concentration
was 26.7 μmol·L−1, a complete degradation was achieved in 10 min and
by both I-CPO (physical adsorption) and I-CPO (chitosan modification).
Even the initial concentration of isoproturon reached 133.3 μmol·L−1,
the degradation efficiency by I-CPO (physical adsorption) in the three
wsterwater samples can still maintain 57.21%, 77.99% and 86.12%
respectively, while 63.02%, 84.64% and 89.06% respectively by I-CPO
(chitosan modification), indicating the I-CPO was very efficient in the
treatment of isoproturon in various real water samples, which ensure a
potential application of I-CPO. The reference [Yao et al., 2015] reported
that 76.1% of removal efficiency was achieved after 4 h by immobilized
laccase on HNT-chitosan micro-sphere for removal of phenol in wastewater,. Compared with this result, the degradation of isoproturon by
I-CPO is more efficient.
Table 2
Reaction kinetic constants of CPO and I-CPO.
CPO
I-CPO
Vmax
(mmol·L−1·s−1)
Km
(mmol·L−1)
kcat
(s−1)
kcat/Km
(s−1·L·mmol−1)
0.3285
0.1050
0.1732
0.0534
0.69 × 103
0.22 × 103
0.4 × 103
8.13 × 103
enzyme. Fig. 7 showed the residual activity of free CPO and I-CPO in
the presence of organic solvents, including N, N-dimethyl formamide
(DMF), methanol, acetonitrile and ethyl acetate, with a fixed volume
fraction in aqueous buffer after 1 h incubation. With the increasing of
the organic solvent volume fraction, both the catalytic activity of free
enzyme and I-CPO decreased gradually, however, I-CPO was more
tolerable than free CPO. For example, when the organic solvent volume
fraction was 10%, almost no loss of activity of I-CPO was observed in all
the four organic solvent-water mixed system, while free CPO can only
remain 41.6%, 38.2%, and 23.5% of activity in ethyl acetate, acetonitrile, methanol, respectively, and even inactivated completely in DMF
in the same condition. When the organic solvent volume fraction increased to 20%, I-CPO remained more activity compared with free CPO
in the presence of organic solvent with higher Log P. For example, ICPO remained 76.4% and 63.2% of activity in ethyl acetate and acetonitrile, respectively, however kept about only 20% of activity in methanol and DMF, which was related to the different stability hydrated
layer of the enzyme in the presence of these organic solvents.
Though CPO was immobilized on HNT only by simply adsorption
based on electrostatic interaction in the first strategy, the I-CPO still
showed a good reusability as Fig. 8a indicated. After 8 cycle of use, ICPO can maintain 50.6% of its original activity, which may be attributed to that HNT can prohibit CPO from dropping off, especially for
those CPO that was entrapped into the inner of tube. Comparably, ICPO immobilized on HNT by the second method showed better reusability (Fig. 8b). After 8 cycle of use, I-CPO can remain 62.2% of its
original activity because of the existing of hydrogen bond between
chitosan and CPO, besides the electrostatic attraction.
4. Conclusion
In this work, two strategies were proposed for entrapping and embedding chloroperoxidase (CPO) on both inner/outer wall of HNT simultaneously by pH modulated electrostatic adsorption.
The I-CPO was very efficient when applied in the degradation of
isoproturon in wastewater. The improved catalytic activity can be attributed to the following three aspects: (1) After etching by H2SO4,
some regular circular defects appeared on the inner wall, while the
opening of the tubes kept unobstructed and the hollow tubular structure
remained. This pore size was not big enough to accommodate enzyme,
but can serve as a center for collection of substrates, making the access
of substrates to the active site in enzyme more easier. (2) The loading
amount of enzyme was enhanced because of the increase of negative
charge on outer wall of HNT due to the dissolution of aluminum oxide
on inner wall and the positive charges was neutralized by the entrapping of negatively charged CPO into the hollow space. (3) Both the
affinity and specificity of I-CPO to substrates was greatly improved,
suggested by enzyme kinetic parameters. Moreover, the enhanced
3.5. Application of I-CPO in the degradation of isoproturon in wastewater
Herbicides are applied to a wide range of crops, including rice,
wheat, corn, barley, potato, soybean and cotton et al. But herbicides
may contaminate streams, rivers or lakes from drainage of agricultural
lands due to their high water solubility, and long half-life. However,
CPO
I-CPO
80
60
40
20
0
a
20
80
60
40
20
0
40
60
80
CPO
I-CPO
100
Remained activity(%)
Remained activity(%)
100
100
b
0
40
80
120
160
200
Time(min)
Tempreture(°C)
Fig. 6. Effects of temperature on the activity of free CPO and I-CPO (a) activity dependence on temperature after 1 h incubation (b) activity dependence on
incubation time at 90 °C.
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Applied Clay Science 163 (2018) 92–99
X. Fan et al.
I-CPO
CPO
80
60
40
20
0
a
80
60
40
20
0
0
5
10
15
20
25
I-CPO
CPO
100
Remained activity(%)
Remained activity(%)
100
b
30
0
5
10
V(DMF)%
I-CPO
CPO
25
30
80
60
40
20
c
I-CPO
CPO
100
Remained activity(%)
Remained activity(%)
20
V(methanol)%
100
0
15
80
60
40
20
d
0
0
5
10
15
20
25
0
30
5
10
15
20
25
30
V(ethyl acetate )%
V(actonitrile)%
Fig. 7. Effects of organic solvent on the activity of free CPO and I-CPO:
(a) DMF; (b) methanol; (c) acetonitrile; (d) ethyl acetate.
a
50
0
1
2
3
4
5
6
7
8
9
b
100
Remained activity(%)
Remained activity(%)
100
50
0
10
1
Recycle time
2
3
4
5
6
7
Recycle time
Fig. 8. Resuability of I-CPO prepared by the two strategy (a) physical adsorption; (b) chitosan modification.
98
8
9
10
Applied Clay Science 163 (2018) 92–99
X. Fan et al.
Fig. 9. The degradation of isoproturon in three water samples by I-CPO.
by physical adsorption; b. by chitosan modification.
(1. Artificial lake water; 2. Heihe water; 3. Domestic sewage)
reusability, the thermal stability and tolerance to harsh reaction condition, such as in the presence of large amount of organic solvents and
inorganic salts enable I-CPO a potential practical application in the
treatment of wastewater.
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Acknowledgments
This work is supported by the National Natural Science Foundation
of China (21176150) and the Fundamental Research Funds for the
Chinese Central Universities (GK201701003) to Y Jiang and National
Natural Science Foundation of China (21503023) to F Wang.
Conflicts of interest
There are no conflicts to declare.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.clay.2018.07.016.
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