Efficient transformation of phenyl urea herbicide chloroxuron by laccase immobilized on zein polyurethane nanofiber

Article abstract of DOI:10.1016/j.molcatb.2013.10.022

Laccases are widely distributed in plants, bacteria, and fungi, and are used for a wide range of applications, including bioremediation. In the present work laccase was immobilized on an electrospun zein polyurethane nanofiber via crosslinking with glutaraldehyde. FESEM and FTIR analysis clearly demonstrates the binding efficiency of laccase on the nanofiber. The relative activity of immobilized laccase was 85% that of free laccase. Immobilized laccase had a better pH and thermal stability than free laccase. The immobilized laccase completely degraded chloroxuron up to 25 reuse cycles in the presence of 1 mM HoBt. Paddy seeds soaked with solution containing chloroxuron treated with immobilized laccase showed a germination percentage closer to the distilled water control; whereas no damage or fatality of paddy seedlings were noticed in treated chloroxuron solution, which demonstrates the ability of immobilized laccase to detoxify the phenyl urea herbicide chloroxuron. Abbreviations* FESEM*field emission scanning electron microscopy*SEM*scanning electron microscopy*FTIR*Fourier transform infrared spectroscopy*PUHs*phenyl urea herbicides*PU*poly urethane*HoBT*1-hydroxybenzotriazole*ABTS*2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate)*DMF* dimethylformamide*THF*tetrahydrofuran*GA* glutaraldehyde*CX*chloroxuron control*ILCX*immobilized laccase treated chloroxuron*IL*immobilized laccase*DW* distilled water

Full text of DOI:10.1016/j.molcatb.2013.10.022

Journal of Molecular Catalysis B: Enzymatic 99 (2014) 156–162
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Efficient transformation of phenyl urea herbicide chloroxuron by
laccase immobilized on zein polyurethane nanofiber
Thayumanavan Palvannana,1,2, Thiyagarajan Saravanakumara,2, Afeesh R. Unnithanb,
Nam-Jin Chungc, Dae-Hyuk Kimd, Seung-Moon Parka,∗
a Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 570-752, South Korea
b Bionano Systems Engineering Department, Chonbuk National University, Jeonju 561-756, South Korea
c Department of Crop Science and Biotechnology, College of Agricultural and Life Science, Chonbuk National University, Jeonju 561-756, South Korea
d Department of Molecular Biology, College of Natural Science, Chonbuk National University, Jeonju 561-756, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Laccases are widely distributed in plants, bacteria, and fungi, and are used for a wide range of applica-
tions, including bioremediation. In the present work laccase was immobilized on an electrospun zein
polyurethane nanofiber via crosslinking with glutaraldehyde. FESEM and FTIR analysis clearly demon-
strates the binding efficiency of laccase on the nanofiber. The relative activity of immobilized laccase
was 85% that of free laccase. Immobilized laccase had a better pH and thermal stability than free laccase.
The immobilized laccase completely degraded chloroxuron up to 25 reuse cycles in the presence of 1 mM
HoBt. Paddy seeds soaked with solution containing chloroxuron treated with immobilized laccase showed
a germination percentage closer to the distilled water control; whereas no damage or fatality of paddy
seedlings were noticed in treated chloroxuron solution, which demonstrates the ability of immobilized
laccase to detoxify the phenyl urea herbicide chloroxuron.
Received 11 February 2013
Received in revised form 28 October 2013
Accepted 28 October 2013
Available online 5 November 2013
Keywords:
Laccase
Immobilization
Zein
Chloroxuron
Phenyl urea
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Several methods have been proposed for the treatment of the pol-
luted water by herbicides or pesticides, including laccase-based
Phenylurea herbicides (PUHs), such as chloroxuron: IUPAC
name 3-[4-(4-chlorophenoxy)phenyl]-1,1-dimethylurea, are used
in agriculture for the control of weed growth in several crops where
they act as inhibitors of the photosynthesis and cause growth inhi-
bition, chlorotic, and necrotic effects on foliage. These herbicides
are either endocrine disruptors, or have eco-toxic or genotoxic
effects. The residues of PUHs and their transformation products
were found in aquatic environments causing risk to aquatic flora
and fauna [1–5]. Water contaminated with PUHs can be more
toxic than the herbicides themselves, as the by-products gener-
ated during water disinfection processes based on strong oxidants
like chlorine or ozone can be more toxic to human health [3].
enzymatic remediation for removing phenolic contaminants from
the environment [6–9]. Laccases (p-diphenol oxidase, EC 1.10.3.2)
belong to the family of multicopper oxidases that catalyze the oxi-
dation of aromatic substances, such as diphenols, arylamines, or
aminophenols, with the concomitant reduction of O2 to H2O. Lac-
cases are widespread in nature and have been found in fungi, in
plants, and in some bacteria [10,11]. The laccases have the ability to
catalyze a plethora of reactions, including the degradation of poly-
mers, oxidative coupling of phenolic compounds, functionalization
of polymers, and ring cleavage; these abilities position laccases as
significant industrial enzymes [12–14]. Immobilization technology
has been proven to be an effective and most straightforward way
to implement efficient and continuous application of enzymatic
oxidation [6,15,16]. Similar to the free enzyme, immobilized lac-
case can be applied in various industrial processes, especially in
environmental applications. The search for inexpensive supports
and the recovery of activity during the immobilization process is
constantly on increase to potentiate the application of laccase in
immobilized systems [17]. Biopolymer from renewable resources
has gained much attention for economical and environmental rea-
sons. Zein, the major protein of corn and a by-product of the
bioethanol industry, is a non-toxic, biocompatible, biodegradable
polymer which in recent years had gained much attention because
Abbreviations: FESEM, field emission scanning electron microscopy; SEM, scan-
ning electron microscopy; FTIR, Fourier transform infrared spectroscopy; PUHs,
phenyl urea herbicides; PU, poly urethane; HoBT, 1-hydroxybenzotriazole; ABTS,
2,2ꢀ-azino-bis-(3-ethylbenzothiazoline-6-sulfonate); DMF, dimethylformamide;
THF, tetrahydrofuran; GA, glutaraldehyde; CX, chloroxuron control; ILCX, immo-
bilized laccase treated chloroxuron; IL, immobilized laccase; DW, distilled water.
∗
Corresponding author. Tel.: +82 63 850 0837; fax: +82 63 850 0834.
E-mail address: smpark@chonbuk.ac.kr (S.-M. Park).
Permanent address: Laboratory of Bioprocess and Engineering, Department of
1
Biochemistry, Periyar University, Salem, Tamil Nadu 636011, India.
2
These authors contributed equally to this work.
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.10.022

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157
of the electrospinning of zein nanofibers [18–21]. High molecular
2.4. Physicochemical characterization of laccase–Zein–PU
weight substances, which are either natural or artificial with differ-
ent structures, can be used for enzyme immobilization; one of the
best supports for this is polyurethane (PU). The unique properties
of PU are its elasticity, toughness, durability, and good resistance to
environmental factors [22]. These properties of PU, when combined
with the biodegradable polymer zein, can function as efficient and
novel support for immobilization of enzymes for use in commercial
interests.
In the present study, the novel Zein-based nanofiber was
developed by co-spinning with polyurethane followed by immobi-
lization of laccase from Tinea versicolor for effective transformation
of the PUH chloroxuron.
nanofiber
2.4.1. Scanning electron microscopy (SEM) and field emission
scanning electron microscopy (FESEM)
The surface topography of the electrospun fibers and laccase
immobilized zein–PU nanofiber was analyzed by field emission
scanning electron microscopy (FESEM). For FESEM, a thin layer of
iridium was coated (2 nm) onto the sample to prevent charging.
The image characterization was performed using JSM-6700F (JEOL
Ltd., Tokyo, Japan).
2.4.2. Fourier transform infrared measurements (FTIR)
The zein–PU nanofiber and the laccase immobilized zein–PU
nanofiber were characterized by Fourier transform infra red spec-
trometer (FTIR Nicolet 5700). Small amounts of samples were
mixed separately with KBr and the prepared pellet was used for
FTIR spectra.
2. Experimental
2.1. Materials
Laccase (EC 1.10.3.2: p-diphenol:dioxygen oxidoreductase;
20 U mg−1) from T. versicolor, 1-hydroxybenzotriazole (HoBT),
2,2ꢀ-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), ethyl
acetate, zein, and glutaraldehyde solution (25%, w/w) were
obtained from Sigma–Aldrich Chem. Co., St. Louis, MO, USA. All
other chemicals were of analytical grade and were used as received,
without further purification.
2.5. Activity assays of free and immobilized laccase
Laccase activity was measured using ABTS as substrate at 30 ◦C
[23]. Free laccase activity was assessed by initiating the reac-
tion with 50 ␮L (0.5 U) of enzyme solution and 0.5 mM of ABTS
in 100 mM of sodium phosphate buffer (pH 4.5). The activity of
immobilized laccase was assayed by incubating five 0.5 cm × 0.5 cm
pieces of support containing immobilized laccase with 0.5 mM
ABTS in 100 mM of sodium phosphate buffer (pH 4.5) under con-
stant shaking (100 rpm). After 3 min, the membrane was removed
using forceps and substrate oxidation by free and immobilized
laccase was monitored at 420 nm (ε420 = 36.0 mM−1 cm−1) using
a UV–vis spectrophotometer (Shimadzu UV-1800). One unit (U)
of activity was defined as the amount of enzyme needed to oxi-
dize 1 ␮mol of ABTS per minute. Enzyme activity was expressed in
2.2. Electrospinning of zein–PU nanofibers
Zein (Sigma–Aldrich, USA) and polyurethane (MW = 110,000,
Cardio Tech. Intern., Japan) were used in making the solution. PU
(10%) solution with 5% and 10% concentration of zein to PU was
used to prepare the composite nanofiber mats. A mixed solvent,
DMF:THF (1:1) was used to prepare the 5% zein–10% PU and 10%
zein–10% PU polymer solution and the composite polymer solu-
tion was stirred for 1 h prior to electrospinning. Polymer solution
was fed to the 5 mL syringe with a plastic micro-tip. A high voltage
power supply (CPS-60 K02V1, Chungpa EMT, South Korea) of 16 kV
was supplied to the syringe micro-tip in order to electrospin the
nanofibers; whereas, a ground iron drum covered by a polyethylene
sheet served as the counter electrode. The solution was kept in the
capillary by adjusting the inclination angle. The tip-to-collector dis-
tance was kept at 15 cm. Finally, the Zein–PU composite nanofiber
mats were vacuum dried in an oven at room temperature for 24 h to
remove the residual solvent, and this sample was used for further
characterizations.
U mg−1 or mL−1
.
2.6. Protein estimation
Protein estimation was performed according to manufacturer’s
instruction of the Thermo® Scientific Pierce Coomassie Plus (Brad-
ford) Protein Assay Kit (Product No. 23236), which uses bovine
serum albumin as a standard. Concentration of the immobilized
protein in the ZP nanofiber was estimated inversely by measuring
the decrease in dye absorbance at 465 nm, rather than conventional
protein estimation by measuring increase in absorbance at 595 nm.
The amount of immobilized protein could be directly quantified by
this method.
2.3. Immobilization of laccase on zein–PU nanofiber
2.7. Optimum pH and temperature
All the immobilization steps were carried out by using 5 mg
weighed standard pieces of zein–PU nanofiber. Prior to immobi-
lization, the nanofibers were activated with various concentrations
of glutaraldehyde (GA) water solution (5, 10, 15, 20, and 25%, w/v),
and the mixture was put on a rocker (100 rpm) at 4 ◦C overnight.
The GA activated nanofibers were transferred to new vials, and
washed extensively by decanting with 10 mM sodium phosphate
buffer, pH 4.5 (buffer A) to remove the excess GA solution. Then,
the washed zein–PU was incubated with laccase at 4 ◦C overnight
in a rocker (50 rpm). The excess enzyme from the nanofibers was
washed extensively with buffer A until no leaching of enzymes was
observed in the washing solution. This washing process took 1 h,
with 5 washings. After washing, further cross linking of the immo-
bilized enzymes were achieved with 5% GA solution for 3 h at 20 ◦C.
Again the excess of GA was removed by extensive washing with
buffer A. The enzyme coatings on polymer nanofibers were stored
in buffer A at 4 ◦C.
The optimum pH for laccase activity (free and immobilized) was
investigated using 50 ␮M ABTS in a 0.1 M sodium phosphate buffer
(pH 2.5–7). The relative activity was calculated as the ratio between
the activity at each pH and the maximum attained.
The effect of temperature (20–90 ◦C) on laccase activity (both
free and immobilized) was determined by measuring activity at
the corresponding temperature under standard conditions. The rel-
ative activity was calculated as the ratio between the activity at
each temperature and the maximum attained.
2.8. Determination of kinetic parameters of free and immobilized
laccase
The Michaelis–Menten constant (Km) and maximum reaction
rate (Vmax) for free and immobilized laccase were determined
using ABTS as a substrate at 0.025 to 1 mM at 30 ◦C and pH 4.5.

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An Eadie–Hofstee plot was constructed to determine the kinetic
parameters.
2.11. Reusability
The reusability of the immobilized laccase was investigated in
repeated batch experiments using ABTS as the substrate and by
chloroxuron biodegradation.
2.9. Chloroxuron degradation
Chloroxuron stock was prepared in aqueous methanol (50%,
v/v), and appropriate dilutions of stock solution were used for the
experiments. Drug transformation experiments were carried out
using a 1-mL reaction volume containing chloroxuron (0.175 mM),
sodium acetate buffer (100 mM; pH 4.5) and immobilized laccase
(5 U) with or without HoBt (1 mM). The final methanol content of
the reaction was less than 2.0%, at which no laccase inhibition was
observed. Reaction vials were incubated in the dark for various
time periods at 30 ◦C while being agitating at 100 rpm. Appropri-
ate control samples with free laccase were included. Experiments
were performed in duplicate and each sample was analyzed
twice.
2.12. Toxicity test
Paddy seeds (plant strain used: Sindongjin, a japonica rice
cultivator) were treated with 2-fold dilution of non-degraded
chloroxuron solution (CX: chloroxuron control) and degraded
chloroxuron solution (ILCX: immobilized laccase treated chlorox-
uron) by immobilized laccase. Distilled water was used as a control.
The seeds (30 seeds for each test) were disinfected or fumigated
with 5 mL of these solutions in 15 mL test tube and incubated in
plant growth chamber at 25 ◦C for 14 days. Germination index was
calculated from the treated seeds.
Cormophytes of these seeds were selected after treatment with
the plant pesticides. Paddy seeds were soaked (imbibition) in CX
solution and ILCX without any dilution for 3 days. Then seeds were
sowed in nursery soil at a depth of 1 cm. The nursery soils sowed
with seeds were irrigated from the bottom of the pot (bottom
watering). The seedlings were kept in plant growth chamber at
25 ◦C for 15 days. The plants grown above 10 cm height and having
leaf age of 2.5 days were selected. The selected plants with leaves of
age 2.5 days were treated with distilled water, CX, and ILCX solu-
tion, by using a painting brush. The laccase treated chloroxuron
solution was further checked for plant growth damage and fatality
rate index.
2.10. Analytical methods
2.10.1. UV–vis spectral scanning analysis
The biodegradation of chloroxuron by immobilized laccases
was monitored by measuring the UV–vis spectra in 1 cm opti-
cal path length quartz cuvettes with a Shimadzu UV1800 UV–vis
spectrophotometer at a resolution of 1 nm between 200 nm and
1100 nm.
2.10.2. HPLC analysis
Chloroxuron degradation by laccase was analyzed by HPLC
(Shimadzu, Japan), which consisted of the following compo-
nents; pump LC-20A, auto-sampler model SIL-20A, column
ACE C18 (250 mm × 4.6 mm, ACE) in column oven CTO-20A at
40 ◦C, detector model SPD-M20A, and software LC solution. Elu-
tion was done with a gradient of water and acetonitrile as
follows: 0–2 min, isocratic 40% acetonitrile; 2–12 min, linear gra-
dient to 95% acetonitrile; 12–20 min, isocratic 95% acetonitrile;
22–29 min return to isocratic start conditions; 29–29.5 min, re-
equilibration time. The flow rate was 1.0 mL/min. The retention
time of chloroxuron was determined by monitoring the eluent at
280 nm.
3. Results and discussion
3.1. SEM and FESEM image of zein–PU nanofiber
Fig. 1 shows the SEM and FESEM micrographs of the 10% zein–PU
(ZP), 25% GA activated ZP and laccase immobilized on ZP nanofiber
cross linked with 5% GA. Among the various percentage of zein used
for electrospinning with polyurethane, 10% zein–PU was employed
for further studies involving immobilization, as it was tough and
flexible. It is evident from the micrograph that the surface modifi-
cation of ZP nanofiber was achieved with GA (Fig. 1b) and with the
subsequent laccase immobilization, a uniform and nicely adhered
enzyme layers was produced on the nanofiber (Fig. 1c). The FESEM
image of ZP nanofiber after GA activation shows thick coatings on
the polymer nanofiber (Fig. 1b). This could be the result of GA
molecules attached covalently to the surface of the polymer that
had reacted with the ␣NH2 group of N-terminal, the imidazole
ring of histidine and the phenolic group of tyrosine, as zein lacks
the ␧-amino group of lysine [24]. When GA activated ZP nanofiber
was treated with the laccase, a uniform attachment of enzyme to
the nanofiber is evident via covalent attachment to GA (Fig. 1c).
Further, crosslinking with 5% GA solution of the other unbound
enzyme molecules adsorbed onto the bound protein causes the
formation of aggregates, wherein GA reacts with ␧-amino group of
lysine present in the enzyme to form covalent linkages between the
enzyme molecules via a conjugated Schiffs base [25]. This is evident
from the fully-packed inter-fiber pores of ZP by crosslinked enzyme
aggregates (Fig. 1c). Enzyme activity was inversely proportional to
the concentration of glutaraldehyde, because extensive cross link-
ing may result in deformation of the enzyme structure. Thus it may
lead to the less accessibility of the substrate to the active site of
the enzyme, so reducing its biological activity [26]. To avoid this
zein polyurethane nanofiber membrane was first treated with glu-
taraldehyde and washed extensively with distilled H2O. This forms
a cross linking of glutaraldehyde molecules with free overhangs.
2.10.3. LC–ESI-MS analysis
After degradation, the reaction mix contains buffer and
chloroxuron metabolities were subjected to ethyl acetate extrac-
tion to remove impurities. Then it was vacuum dried and the
chloroxuron metabolites were dissolved in acetronitrile for liquid
chromatography-mass spectrometry analysis for the determina-
tion of non-volatile metabolites. For LC–MS analysis, 5 ␮L aliquots
were injected into an HPLC system (Acquity Ultra Performance LC,
Waters), comprising a Waters ACQUITY UPLC®BEH C18 column
(2.1 mm × 100 mm, 1.7 ␮m). LC–MS was controlled by MassLynx
software (version 4.1). The mobile phase consisted of 0.1% formic
acid in distilled water (DW) and 0.1% formic acid in acetroni-
trile. The flow rate was 0.5 mL/min and employed a gradient from
10% to 100% buffer B for 10 min. The mass spectrometer was
equipped with an ESI interface, operating at an ionization voltage
of +3000 V and a source temperature of 380 ◦C. The capillary volt-
age, con voltage, and source offset were set at 3 kV, 30 kV and 30 V,
respectively. Tandem MS analysis was performed using the multi-
reaction-monitoring (MRM) mode. Collision energy, the gas flow of
desolvation, and cone spray nebulizer were set at 650 L/h, 150 L/h
and 7 bar, respectively.

T. Palvannan et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 156–162
159
Fig. 2. FTIR spectrum of laccase immobilized on 10% zein–PU nano fiber activated
with 25% glutaraldehyde followed by 5% crosslinking. (a) Zein–PU nanofiber, (b)
laccase immobilized on zein–PU nanofiber after 25% glutaraldehyde activation and
(c) laccase crosslinked with 5% glutaraldehyde.
was shown by spectrum peak range between 3329–3290 cm−1
(H
peak at 2951–2926 cm−1 and spectrum peak at 1423 cm−1 shows
H aliphatic stretch and CH bending respectively, and indicates
O
H stretch) and 1109–1107 cm−1 (C O stretch). Spectrum
C
the existence of polyol soft-segment in the chain of polyurethane. In
the ZP nanofiber spectra, bands observed in the region correspond
to C O stretching (1750 cm−1) and C H stretching (2820 cm−1
)
vibrations. This can be attributed to the characteristic absorption of
aldehyde groups, which act as a binding site for the primary amino
groups of enzyme molecules [29,30]. Though the ZP nanofiber is
having aldehyde groups the treatment of ZP nanofiber with 25% of
glutaraldehyde vastly improves the binding of the laccase to the
membrane which is evident from the enzyme activity assay, pro-
tein content. The spectrum of laccase-immobilized ZP nanofiber
(Fig. 2b) has pronounced bands corresponding to N H bending
(1560 cm−1), which may be due to primary amino groups of the
immobilized enzyme and N H bending (1650–1580 cm−1). This
Fig. 1. (a) SEM image of 10% zein–PU nano fiber, (b) FE-SEM image of the electrospun
zein–PU nanofiber surface topography activated by 25% glutaraldehyde, and (c) FE-
SEM image of laccase immobilized on 25% glutaraldehyde activated 10% zein–PU
fiber followed by crosslinking with 5% glutaraldehyde.
N
H stretching and bending becomes more pronounced in the FTIR
spectrum of ZP nanofiber (Fig. 2c), where laccase is crosslinked with
5% GA which indicates that ZP nanofiber binds laccase effectively
and efficiently via GA activation.
Then this pretreated membrane was incubated laccase solution.
This constitutes a complete cross linking of laccase with all glu-
taraldehyde molecules bonded with nanofiber membrane. Lower
concentration of glutaraldehyde was added to this solution for a
complete cross linking of laccase with zein polyurethane mem-
brane. Minimal amount of glutaraldehyde was used to avert the
loss of enzyme activity [27].
3.3. Properties of immobilized laccase
The amount of laccase attached to the ZP nanofiber was 0.25 mg
(5 U) to 5 mg of ZP nanofiber was estimated according to manu-
facturer’s instructions of Thermo® Scientific Pierce Coomassie Plus
(Bradford) Protein Assay Kit. The relative activity of immobilized
laccase was 85 4% that of free laccase. After immobilization, a
minor decrease in relative activity was observed. The loss in the
immobilized system may be due to changes in the enzyme diffusion
rate, non-biospecific interactions, and variations in the microenvi-
ronment [31].
The pH and temperature profiles of the free and immobilized
enzyme are shown in Fig. S1a and b. Both free and immobilized
laccase showed optimum activity at pH 4.5 and at 60 ◦C. These
results suggest that there were no significant changes in the elec-
trostatic state of the amino acids at the active sites of the laccase
after immobilization [29]. The charge density of the nanofibrous
membrane surface does not change the enzyme microenviron-
ment as is the case with other polycations [30]. Compared to the
free enzyme, temperature stability was pronounced in the case
of immobilized laccase (Fig. S1c). The presence of proline of zein
3.2. FTIR spectrum of immobilized laccase
The FTIR spectrum of ZP nanofiber (Fig. 2a) exhibited a broad, but
not so intense band around 3300 cm−1, which shows the stretching
of protons. Absorptions in the region 3650–3250 cm−1 correspond
to hydroxyl or amino groups, which mainly dominate this region,
both giving rise to very characteristic band profiles. The presence
or absence of hydrogen bonding is well-delineated in this region
[28]. The main absorption band in the region 3600–3000 cm−1 was
broad and hence the compound probably possesses a hydroxyl
or amino group. Since zein contains prolamin and polyurethane
have a hydroxyl and an amide linkage, the presence of amino
group and hydroxyl groups overlapping in the spectrum cannot
be ruled out. According to the FTIR spectrum, urethane linkage

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T. Palvannan et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 156–162
polymer may influence this observed effect. Proline substitution
enhancing enzyme stability is reported elsewhere [32,33].
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2013.10.022.
A kinetic study was performed to obtain Km and Vmax, for the free
and ZP immobilized laccase. These kinetic parameters were deter-
mined by analysis of the Eddie–Hofstee plot (Fig. S2). The Km for
the immobilized laccase was 0.67 mM, which varies significantly
from the free laccase (0.087 mM), and the Vmax of the immobi-
lized laccase decreased to 1.9 from 2.1 U mg−1 for the free laccase.
The decrease in Km and Vmax values indicates shift in affinity and
catalytic ability of the immobilized enzyme compared to the free
enzyme toward the substrate. The increased limitations to diffu-
sion of the substrate to the active site of the immobilized enzymes
might be the cause of decrease in affinity of immobilized laccase
when compared to the free enzyme [34].
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2013.10.022.
3.4. Removal of phenyl urea herbicide chloroxuron
UV–vis spectral analysis of chloroxuron showed a decrease
in absorbance at 240 nm indicating the effective degradation of
chloroxuron by using the immobilized laccase (Fig. 3). The reduc-
tion is more pronounced with increase in time (data not shown).
The action of redox mediators on degradation of chloroxuron
Fig. 4. HPLC chromatogram of 50 mg/L chloroxuron degradation. (a) Chloroxuron
control, (b) chloroxuron treated by immobilized laccase and (c) chloroxuron treated
by immobilized laccase presence of 1 mM HoBt.
cannot be verified, as these mediators have absorption maximum
in the UV region, which makes it difficult to interpret the results.
The interference of various buffers on chloroxuron detection in UV
range was checked (Fig. 3a). It was found that 0.1 M sodium phos-
phate buffer does not show any interference when compared to
citrate or sodium acetate buffers; hence for detection of cholorox-
uron under UV range 0.1 M sodium phosphate buffer pH4.5 was
employed. The degradation of chloroxuron by immobilized laccase
was further corroborated with HPLC studies.
HPLC chromatogram of chloroxuron showed a sharp peak with
retention time (RT) at 19 min, and a minor peak at RT 20 min
(Fig. 4a). Treatment of chloroxuron with immobilized laccase dras-
tically reduces the peak at RT 19 min within 30 min of reaction time
(Fig. 4b). Addition of redox mediator HoBt to chloroxuron in pres-
ence of immobilized laccase (5 U) resulted in the complete removal
of peak at RT 19 min with the formation of new peak with RT 17 min
(Fig. 4c). Other redox mediators via syringaldehyde and 3-hydroxy
anthranilic acid do not show any effect on chloroxuron removal
(data not shown). HPLC results clearly showed that laccase has the
ability to decompose chloroxuron to other species, leading to its
degradation. This result was further corroborated with LC–ESI-MS
analysis.
Fig. 5a shows the decrease in the peak of chloroxuron in LC
spectrum at RT 6.30 min when treated with immobilized laccase
in presence of 1 mM HoBt. This again clearly demonstrates the
ability of laccase to effectively degrade chloroxuron. Fig. 5b (i
and ii) shows the ESI-MS analysis of chloroxuron control and
chloroxuron treated with immobilized laccase. Analysis of chlorox-
uron control chromatogram (Fig. 5b (i)) reveals that the peak
at m/z 293 as chloroxuron, whereas minor peak at m/z 278
Fig. 3. UV–vis spectral scanning. (a) Selection of buffer with less interference for
identifying the chloroxuron degradation by UV–vis scanning and (b) UV–vis spectral
scanning of 0.175 mM of chloroxuron degradation by immobilized laccase using
sodium acetate buffer pH 4.5.

T. Palvannan et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 156–162
161
Fig. 6. Toxicity assessment of solution containing 0.175 mM chloroxuron treated
with and without immobilized laccase in presence of 1 mM HoBt. (CX: chloroxuron
control; ILCX: immobilized laccase treated chloroxuron and DW: distilled water
control).
Fig. 5. LC–ESI-MS analysis. (a) TIC of LC–MS analysis of ethyl acetate extract of sam-
ples. (i) Chloroxuron control, peak corresponds to retention time (RT) 6.30 min; (ii)
chloroxuron treated with immobilized laccase in presence of 1 mM HoBt, peak at RT
6.30 min disappears. (b) ESI-MS of chloroxuron degradation. (i) Chloroxuron control;
(ii) chloroxuron degraded by immobilized laccase in presence of 1 mM HoBt.
63.3 which is 3-fold higher than the rice seeds treated with chlorox-
uron alone but not closer to the control. This may be due to the
residues or the transformation products of chloroxuron which still
remains in the treated solution. However the suppressive influence
on paddy seedling by the herbicide chloroxuron is relieved to a
greater extent by treating with laccase which is also evident from
the effect of chloroxuron and laccase treated chloroxuron on the
damaged and fatality on rice plant. Untreated chloroxuron showed
100% damage and 50% fatality to the rice plants whereas the laccase
treated chloroxuron did not show any damage or dead plants.
This is the first report on effective degradation and detoxifica-
tion of chloroxuron by laccase.
may be N-(4-chlorophenoxy)-phenyl-N-methylurea (contains one
methyl group). Fig. 5b (ii) shows the ESI-MS spectrum of the ele-
unt at RT 6.30 of chloroxuron treated with immobilized laccase.
The major peak at m/z 121 denotes N-phenylformamide which
is the breakdown product of chloroxuron. The minor peaks at
m/z 272 might be N-(4-hydroxyphenoxy)-phenyl-N-methylurea
(chloroxuron reacts with H2O and the Cl− dissociates to form HCl
along with N-(4-hydroxyphenoxy)-phenyl-N-methylurea which
further hydroxylated at different phenyl rings to form two differ-
ent compounds with M.W. 289). The m/z 263, and 138 denotes
a demethylated product of chloroxuron, 1-[4-(4-chlorophenoxy)
phenyl] urea and N-phenyl urea or phenyl carbamate respectively.
ESI-MS studies reveal the degradation products of chloroxuron by
immobilized laccase.
The advantage of immobilized biocatalysts over the free enzyme
is their ability of recycling which can be used in a continuous
reaction processes. Hence, the operational stability and reusability
of the immobilized enzymes are necessary parameters for major
industrial applications. Fig. S3 shows the operational stability of
immobilized laccase during repeated use for chloroxuron transfor-
mation. Results show that the enzyme activity can be retained up
to 25 cycles without losing much of its activity and then it gradu-
ally decreased with number of recycles. This decrease in enzyme
activity could be due to inactivation or loss of enzyme during each
cycle. Similar results have been reported for other nanobiocatalysts
[35–37].
4. Conclusion
A biocompatible, biodegradable zein polymer-based electro-
spun nanofiber with polyurethane was prepared to adsorb laccase.
The immobilized laccase showed better pH and temperature sta-
bility than the free laccase. Kinetic parameters indicated affinity
shift between immobilized enzyme and the substrate. By employ-
ing the immobilization system along with the redox mediator HoBt,
the PHU chloroxuron was completely degraded within 30 min. The
immobilized system was able to retain 80% of its activity up to
25 recycles. The immobilized laccase treated chloroxuron solution
improved the germination percentage of the paddy seeds and did
not cause any damage or mortality to the paddy seedlings.
Acknowledgements
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2013.10.022.
This research was supported by the Technology Development
Program for Agriculture and Forestry, under the Ministry for Food,
Agriculture, Forestry and Fisheries, Republic of Korea. This research
was also supported in part by research funds of Chonbuk National
University in 2012, and Korea Research Council of Fundamental
Science & Technology (Joint Degree and Research Center for Biore-
finery). We would like to thank the Research Institute of Bioindustry
at Chonbuk National University for kindly providing the facilities
for which to conduct this research.
3.5. Toxicity assessment of the laccase treated chloroxuron
solution
Effect of chloroxuron (control and treated with laccase immo-
bilized on ZP nanofiber) was checked for germination index of
paddy seeds (plant strain used: Sindongjin, a japonica rice culti-
var). Fig. 6 shows the pre-mergence application of herbicide had
negative bearing on rice germination (22.2%), whereas the control
(distilled water) had a germination percentage of 72.2. Chloroxuron
treated with immobilized laccase had a germination percentage of
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