Purification and characterization of a novel pyrethroid hydrolase from Aspergillus niger ZD11

Article abstract of DOI:10.1021/jf051460k

The pyrethroid pesticides residues on foods and environmental contamination are a public safety concern. Pretreatment with pyrethroid hydrolase has the potential to alleviate the conditions. For this purpose, a fungus capable of using pyrethroid pesticides as a sole carbon source was isolated from the soil and characterized as Aspergillus niger ZD11. A novel pyrethroid hydrolase from cell extract was purified 41.5-fold to apparent homogeneity with 12.6% overall recovery. It is a monomeric structure with a molecular mass of 56 kDa, a pl of 5.4, and the enzyme activity was optimal at 45°C and pH 6.5. The activities were strongly inhibited by Hg²⁺, Ag⁺, and p-chloromercuribenzoate, whereas less pronounced effects (5-10% inhibition) were observed in the presence of the remaining divalent cations, the chelating agent EDTA and phenanthroline. The purified enzyme hydrolyzed various insecticides with similar carboxylester. trans-Permethrin is the preferred substrate.

Full text of DOI:10.1021/jf051460k

J. Agric. Food Chem. 2005, 53, 7415−7420 7415
Purification and Characterization of a Novel Pyrethroid
Hydrolase from Aspergillus niger ZD11
WEI Q. LIANG,† ZHUO Y. WANG,† HE LI,† PEI C. WU, JI M. HU, NA LUO,
LI X. CAO, AND YU H. LIU*
State Key Laboratory of Biocontrol, School of Life Science, Zhongshan University, Guangzhou,
510275, People’s Republic of China
The pyrethroid pesticides residues on foods and environmental contamination are a public safety
concern. Pretreatment with pyrethroid hydrolase has the potential to alleviate the conditions. For this
purpose, a fungus capable of using pyrethroid pesticides as a sole carbon source was isolated from
the soil and characterized as Aspergillus niger ZD11. A novel pyrethroid hydrolase from cell extract
was purified 41.5-fold to apparent homogeneity with 12.6% overall recovery. It is a monomeric structure
with a molecular mass of 56 kDa, a pI of 5.4, and the enzyme activity was optimal at 45 °C and pH
6.5. The activities were strongly inhibited by Hg2+, Ag+, and F-chloromercuribenzoate, whereas less
pronounced effects (5-10% inhibition) were observed in the presence of the remaining divalent
cations, the chelating agent EDTA and phenanthroline. The purified enzyme hydrolyzed various
insecticides with similar carboxylester. trans-Permethrin is the preferred substrate.
KEYWORDS: Pyrethroid insecticides; pyrethroid hydrolase; purification; Aspergillus niger ZD11;
degradation
INTRODUCTION
lation, and microbial transformation (6). Microbial degradation
has been deemed the most influential and significant cause of
pesticides removal. Therefore, biodegradation is considered to
be a reliable cost-effective technique for pesticides abatement
and a major factor determining the fate of pyrethroid pesticides
in the environment (7). Some pyrethroid carboxylesterases from
pyrethroid-resistant insects have been purified and characterized
(8). A few pyrethroid-degrading bacteria including Bacillus
cereus SM3 (9), Pseudomonas Fluorescens (10), Vibrio hollisae,
Burkholderia picketti, and Erwinia carotoVora have been
isolated from soils and rivers (11). The only pyrethroid-
degrading enzyme from Bacillus cereus SM3 was purified and
characterized. These studies have indicated that the first step
in the microbial degradation and detoxification of pyrethroid
compounds is the hydrosis of carboxylester linkage. There is,
however, little information on pyrethroid pesticides biotrans-
formation by fungi (9, 10). Fungus is an important member of
microorganisms that are critical to the biogeochemical cycle
and are responsible for the bulk of the degradation of xenobiotics
in the biosphere. To understand thoroughly the role of pyrethroid
pesticides biodegradation by microbes, it is necessary to carry
out studies on isolation of fungi, metabolic mechanism, and
molecular biology, and evaluate objectively their role. Taking
into consideration that many synthetic organic compounds in
many cases, although biodegradable, may persist in nature for
a long time, the populations and microorganisms bringing about
their destruction are not large or active enough (12, 13). A low
biodegradation rate resulting from low microbial growth or
insufficient quantities of organism is one of the common
Synthetic pyrethroid insecticides have been widely used for
controlling many agricultural and urban pests in agriculture,
forestry, horticulture, animal and public health, and homes all
around the world (1). They are likely to become more widely
used as organophosphate insecticides including diazinon and
chlorpyrifos are phased out due to the concerns regarding their
safety (2). Extensive applications not only result in pest
resistance to these insecticides, but also may lead to environ-
mental issues and human exposure. A variety of personnel are
exposed to pyrethroids during manufacture and application, diet,
and drinking water. Although these compounds are widely
considered safe for humans (3), numerous studies have shown
that very high exposure to pyrethroids might cause potential
problems to man (4). Such effects include suppressive effects
on the immune system, endocrine disruption, lymph node and
splenic damage, and carcinogenesis (1). In addition, most
synthetic pyrethroids possess apparent toxicity to fish and other
aquatic organisms, including aquatic invertebrates, often at
concentrations less than 1 µg L-1 (5). Therefore, it is important
to develop a rapid and efficient disposal process to eliminate
or minimize contamination of surface water, groundwater, and
agricultural products by pyrethroid insecticides.
The fate of pyrethroid pesticides in the environment is
associated with both abiotic and biotic process, including
evaporation, photooxidation, chemical oxidation, bioaccumu-
* Author to whom correspondence should be addressed [telephone 86-
20-84113712; fax 86-20-84036215; e-mail ls112@zsu.edu.cn].
† These authors contributed equally to this work.
10.1021/jf051460k CCC: $30.25
© 2005 American Chemical Society
Published on Web 08/19/2005

7416 J. Agric. Food Chem., Vol. 53, No. 19, 2005
Liang et al.
Chemicals and Reagents. Sephadex G-100, DEAE-Sepharose CL-
6B, and DEAE-Sephacel were purchased from Amersham Pharmacia
Biotech Co. (UK). Cypermethrin (98%), trans-permethrin and cis-
permethrin (99%), fenvalerate (98%), malathion (98%), and delta-
methrin (98%) were kindly provided by Zhong shan pesticide factory
(Guang dong, China). All F-nitrophenyl esters were purchased from
Sigma. All other chemicals and reagents were of analytical grade and
were purchased from commercial sources, unless otherwise stated.
Enzyme Purification. All of the experiments described below were
carried out between 0 and 4 °C unless otherwise specified.
(a) Preparation of Crude Extract. For enzyme production, the
medium was inoculated with Aspergillus niger ZD11 viable spores.
The culture was incubated at 30 °C for 5 days in 500-mL Erlenmer
flasks containing 200 mL of medium on a rotary shaker at 150 rpm,
harvested by centrifugation at 10 000g for 20 min at 4 °C, washed
twice with cold 50 mM Tris-HCl buffer (pH 6.8), and stored at -20
°C until used later.
Next, 12.6 g of washed mycelia was resuspended in 50 mM Tris-
HCl buffer (pH 6.8) and disrupted in a vibration homogenizer (Vibrogen
vi4, Edmund tubingen, German) with glass beads (0.1 mm in diameter).
After standing at 4 °C overnight, the suspension was centrifuged
(12 000g for 20 min at 4 °C) to remove the unbroken cells and cellular
debris. The suspension was centrifuged at 20 000g for 30 min, the
supernatant was subjected to ultracentrifugation at 100 000g for 1 h,
and the resulting supernatant was used as an enzyme source for
subsequent enzyme purification.
obstacles that must be overcome in developing bioremediation
process for chemical pollutants; this limitation may be overcome
by the following two ways. One way involves the introduction
of appropriate plasmid-borne catabolic genes into well-
established and competitive indigenous populations; an alterna-
tive approach is bioaugmentation by inoculation of a habitat
with microorganisms that survive adverse condition (14-16).
The ability of fungi to produce spores is an attractive feature
for improving efficiency of remediation of contaminated
environment; there is a great deal of interest in fungus with the
broad-spectrum substrate specificity and high catalytic turnover
rate for various pyrethroid pesticides, making it a promising
candidate for managing exposure to pyrethroid pesticides. This
paper describes the purification and characterization of a novel
pyrethroid hydrolase from Aspergillus niger ZD11, previously
isolated from the pesticides contaminated soil, in an attempt to
better understand its specific role on pyrethroid pesticides
degradation. To our knowledge, this is the first broad-spectrum
pyrethroid hydrolase purified to homogeneity from fungi, which
degrades various pesticides containing similar carboxylester
linkage effectively, and further genetic studies may lead to the
discovery of novel genes involved in the future.
MATERIALS AND METHODS
(b) Ammonium Sulfate Precipitation. The 100 000g supernatant was
brought to 40% ammonium sulfate saturation and stirred for 30 min,
the cloudy suspension was centrifuged at 20 000g for 30 min, and
supernatant was brought to 90% ammonium sulfate saturation; after
being stirred for 30 min, the pellet obtained by centrifugation at 20 000g
for 30 min was dissolved in the smallest possible volume 50 mM Tris-
HCl buffer (pH 6.8) and dialyzed 1000-fold against 50 mM Tris-HCl
buffer (pH 6.8), and the supernatant was filtered through a 0.22 µm
membrane (Millipore) and concentrated.
(c) Gel Filtration through Sephadex G-100. The concentrated enzyme
solution was loaded onto Sephadex G-100 column (1.8 × 100 cm)
preequilibrated with 50 mM Tris-HCl buffer (pH 6.8). The column
was washed at a flow rate of 24 mL/h with 400 mL of the same buffer,
and 5-mL fractions were collected. Proteins were eluted in fractions
15-76, whereas the enzyme activity was confined to fractions 26-
34. The fractions with high specific activity were then pooled and
concentrated for further purification.
(d) Ion-Exchange Chromatography. The concentrated enzyme was
loaded onto a TSK-DEAE column (30 × 2.5 cm) that had been
equilibrated with 50 mM Tris-HCl buffer (pH 6.8). The column was
washed with 150 mL of the same buffer, and proteins were eluted with
a linear gradient of NaCl from 0 to 1 M. Fractions (2.5 mL) were
collected every 6 min and screened for enzyme activity; fractions that
eluted with the running buffer and exhibited enzyme activity were
pooled, dialyzed overnight, lyophilized, dissolved in 50 mM Tris-HCl
buffer (pH 6.8), and placed on a DEAE-Sepharose CL-6B column (30
× 2 cm) previously equilibrated with 50 mM Tris-HCl buffer (pH 6.8).
The column was eluted with a linear gradient of NaCl from 0 to 0.5 M
at a flow rate of 24 mL/h, 2-mL fractions were collected and tested for
enzyme activity, and active fractions that eluted with the NaCl gradient
were pooled, dialyzed 1000-fold against 50 mM Tris-HCl buffer (pH
6.8) and lyophilized, and loaded on a Sephacryl S-200 column (90 ×
1 cm). They were then equilibrated and washed with 50 mM Tris-HCl
buffer (pH 6.8), and 1-mL fractions were collected. Active fractions
were pooled for subsequent analysis.
Microorganism Isolation and Culture Conditions. Aspergillus
niger ZD11, which was used in all experiments, was isolated from the
soils where pesticides were heavily used. The minimal medium (MM
medium) consisted of 0.2% NaNO3, 0.1% KH2PO4, 0.1% K2HPO4,
0.05% MgSO4‚7H2O, 0.02% NaCl, 0.001% MnSO4, and 0.005% CaCl2
(pH 6.8). After being autoclaved, the pyrethroid pesticide was prepared
by dissolving it into 10 times its volume of ethanol and added at the
concentration (200 mg/L). The soil sample was inoculated into MM
medium, and then pyrethroid-degrading fungi were enriched by
repeating subcultivations. Appropriate dilutions of the enrichment
culture were placed on MM agar plates and incubated at 30 °C for 3
days. The colonies were selected from the plates, and the positive colony
was isolated and purified. The pyrethroid hydrolysis was further
confirmed by pyrethroid-degrading experiments, quantification, and
identification of pyrethroids, and transformation products were based
on retention times and pear areas of pure standards through GC-MS
(4, 17). The pesticide assay was conducted using a Hewlett-Packard
(Palo Alto, CA) 6890 gas chromatograph equipped with a 5973 mass
spectrometer, and the MS chemstation with window system (1701AA
series) software was used for analysis. A 30 m × 0.25 mm × 0.25 µm
HP-5MS fuse-silica capillary column, coated with 5% biphenyl and
95% dimethylpolysiloxane (from HP Inc.), was employed. The column
temperature program was set as follows: 90 °C (hold for 5 min) to
265 °C at 30 °C/min, 265 °C to 300 °C at 4 °C/min, hold for 4 min.
The GC injector port was used in the “splitless” mode and held
isothermally at 270 °C for injections for the duration of the run. Helium
was used as the carrier gas at a flow rate of 1 mL min-1 by using
electronic pressure control. The GC/MS interface temperature was
maintained at 280 °C. The MS was operated in the electron impact
(EI) ionization mode with electron energy of 70 eV, and mass-to-charge
ratio scan ranged from 50 to 550 amu to determine appropriate masses
for selected ion monitoring. The MS ion source and mass filter (quad)
temperatures were held at 230 and 150 °C, respectively. Extraction of
the synthetic pyrethroids was performed using solid-phase micro-
extraction (SPME) equipment. The solid-phase microextraction (SPME)
manual fiber holder and poly(dimethylsiloxane) (PDMS) with 100 µm-
thickness coated fibers were from Supelco Inc. (Bellefonte, PA). The
SPME fibers were conditioned under helium for 1-2 h in hot GC
injection port before extraction. The SPME sampling stand and heat/
stir plate used in extraction were also from Supelco. The magnetic
stirring bars (10 × 3 mm) were from Aldrich (St. Louis, MO). The
sampling was performed by immersing a SPME fiber directly into the
3 mL sample solution or at 25 °C under magnetic stirring condition
for an appropriate period for the absorption of analytes onto the fiber
coating.
Determination of Protein. Protein amounts were determined by the
method of Bradford. Bovine serum albumin (Sigma) was used as
standard for calibration (18).
Determination of Enzyme Activity. The esterase activity against
F-nitrophenyl esters was determined by measuring the amount of
F-nitrophenol released by esterase-catalyzed hydrolysis. The hydrolysis
of substrate was performed at 30 °C for 10 min in 50 mM sodium
phosphate buffer (pH 6.5) containing 1% acetonitrile. The production
of F-nitrophenol was monitored at 405 nm by Labsystems Dragon
Wellscan MK3. One unit of enzyme activity was defined as the amount

Purification and Characterization of a Novel Pyrethroid Hydrolase
J. Agric. Food Chem., Vol. 53, No. 19, 2005 7417
Table 1. Summary of Purification of Pyrethroid Hydrolase from
Aspergillus niger ZD11
total
protein
(mg)
total
activity
Sp act
purification
(fold)
yield
(%)
-1
purification step
crude extract
(NH4)2SO4
Sephadex G-100
TSK-DEAE
(units)a
(units mg
)
151
37.6
10.5
2.48
±
±
±
±
±
±
8
1.8
0.51 432
0.12 216
0.06 152
693
613
±
±
±
±
±
±
35
4.6
±
±
±
±
±
±
0.24
0.82
2
1
3.5
8.9
18.9
26.7
41.5
100
88
62.3
31
22
12.6
31 16.3
22
11
7
41
87
123
191
4.6
6
9.6
Figure 1. SDS-PAGE analysis of the purified pyrethroid hydrolase from
Aspergillus niger ZD11 (lane 2) and protein markers (lane 1) stained with
Coomassie blue. Markers from top to bottom are phosphorylase b (97.4
kDa), bovine serum albumin (66.2 kDa), ovalbumin (43 kDa), carbonic
anhydrase (31 kDa), and trypsin inhibitor (20.1 kDa).
DEAE-sephrose CL-6B 1.23
Sephacryl S-200 0.46
0.02
87
4
a Enzyme activity was measured using
F-nitrophenyl acetate as substrate.
of enzyme that produced 1 µmol of F-nitrophenol per minute from
substrate under these conditions (19). Pesticide hydrolase activity assays
were carried out according to the methods of Huang et al., Wheelock
et al., and Stok et al. (20-22). All pesticides were prepared in ethanol
(25 mM). The enzyme specificity was determined by adding 4 µL (25
mM in ethanol) of substrate solution to 2 mL enzyme solution for a
final concentration of 25 µM and incubating for 5 min at 30 °C. After
that, samples were extracted, and the remaining pesticides were
quantified by GC-MS. In every measurement, the effect of nonenzy-
matic hydrolysis of substrates was taken into consideration and
subtracted from the value measured when the enzyme was added.
Determination of Molecular Mass and pI. The molecular mass of
the denatured protein was determined by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE). An SDS-12.5%
polyacrylamide gel was prepared by the method of Laemmli (23).
Proteins were stained with Coomassie brilliant blue G. The molecular
mass of the native protein by gel filtration on a Superose 12HR 5/30
column, gamma globulin (160 000 Da), bovine serum albumin (67 000
Da), ovalbumin (43 000 Da), carbonic anhydrase (30 000 Da), was used
as the reference protein. Isoelectric point (pI) was estimated by PAGE
with 6.25% Ampholine (pH 3.5-10) in a gel rod (0.5 × 10 cm) using
a kit for Isoelecric Focusing Calibration (Pharmacia LKB) according
to recommendations by the supplier.
Effect of Temperature and pH on Pyrethroid Hydrolase Activity.
For determination of the optimum pH, the activity was determined by
incubating purified enzyme (0.2 µg/mL) with trans-permethrin as a
substrate at 30 °C for 5 min in 50 mM buffer at pH values between 4
and 10. The buffers (at a final concentration of 50 mM) used for the
measurement are described below: citric acid-NaOH (pH 4.0-5.5);
potassium phosphate (pH 5.0-7.0); Tris-HCl buffer (pH 6.5-9.0),
glycine-NaOH buffer (pH 8.5-10.0). Overlapping pH values were used
to verify that there were no buffer effects on substrate hydrolysis. For
the pH stability determination, samples were incubated in 50 mM
buffers from pH 4 to 10 at 30 °C for 2 h. The relative residual activity
was assayed as described above. The optimum temperature was
determined analogously with a constant pH of 6.5 and different
temperatures. For determination of the thermostability, purified enzyme
(0.2 µg/mL) was incubated in reaction buffer for 1 h at temperatures
ranging from 20 to 70 °C, and retaining pyrethroid hydrolase activity
was measured as before.
Effect of Various Chemicals on Pyrethroid Hydrolase Activity.
The effects of various chemicals on the enzyme activity were
investigated by preincubating the enzyme with chemicals in 50 mM
Tris-HCl buffer (pH 6.8) for 30 min at 30 °C and then measuring the
residual activities by using trans-permethrin as a substrate as described
above.
Determination of Substrate Specificity and Kinetic Measurement.
Substrate specificity against F-nitrophenyl esters was determined using
F-nitrophenyl acetate ranging from 0.025 to 1.5 mM, F-nitrophenyl
propionate ranging from 0.02 to 0.8 mM, F-nitrophenyl butyrate ranging
from 0.01 to 0.5 mM, F-nitrophenyl valerate ranging from 0.005 to
0.5 mM, F-nitrophenyl caproate ranging from 0.015 to 0.8 mM, and
F-nitrophenyl caprylate ranging from 0.1 to 3.5 mM as a substrate in
50 mM sodium phosphate buffer (pH 6.8) containing 1% acetonitrile
at 30 °C. An additional 0.04% Triton X-100 was included in the reaction
mixture in the case of F-nitrophenyl caprylate. Substrate specificity
against different pesticides was analogously determined by measuring
enzyme activity over a range of final concentrations from 0.01 to 9
µM according to different pesticides. All initial velocities were
determined at five time points at which no more than 10% of the
substrate had been consumed, and solution content never exceeded 1%
of the total assay volume, so the decrease in substrate concentration
remains linear with time over the period of measurement and the rate
was almost constant throughout the assay. The r-square values ranged
from 0.963 to 0.982 according to different substrates. Initial reaction
velocities measured at various concentrations of substrates were fitted
to the Lineweaver-Burk transformation of the Michaelis-Menten
equation (6). Kinetic analyses by curve fitting were performed with
the SigmaPlot software.
All of the experiments were carried out at least in triplicate, and the
results were expressed as means of those data. Standard deviations were
determined and reported.
RESULTS
Enzyme Production and Purification. Aspergillus niger
ZD11 was aerobically cultivated at 30 °C in MM with trans-
premethrin as the main carbon sources. Pyrethroid hydrolase
activity was detected at the late log phase and reached the
maximum level 5 days after the start of cultivation. However,
when premethrin in the MM medium was replaced by an equal
amount of glucose, cell extract showed no or only a trace level
of the enzyme activity. These results suggested that the enzyme
was induced by trans-premethrin in the medium. The enzyme
activity was detected at the late log phase and reached the
maximum level 5 days after the start of cultivation. Pyrethroid
hydrolase was purified from the cell-free extract of Aspergillus
niger ZD11 by ammonium sulfate precipitation, gel filtration
on SephadexG-100, TSK-DEAE, DEAE-sephrose CL-6B, and
Sephacryl 200 HR chromatography. The data on the purification
are summarized in Table 1. The enzyme was purified 41.5-
fold to a specific activity of 191 U/mg protein from the cell
with a yield of 12.6%. The purified enzyme gave a single band
in SDS-PAGE. This indicated that the purified sample was
electrophoretically homogeneous under the dissociating condi-
tions. The molecular mass of the purified enzyme was estimated
by SDS-PAGE analysis and was approximately 56 kDa (Figure
1). The relative molecular mass of native enzyme estimated by
gel filtration on a calibrated column of Sephacryl 200 HR was
around 55 kDa. Hence, it is assumed that the native pyrethroid-
degrading enzyme is a monomer. The pI value was estimated
to be 5.4. The purified enzyme catalyzed the hydrolysis of
permethrin to equimolar amounts of 3-phenoxybenzyl alcohol
and 2,2-dimethyl-3-(2,2-dichlorovinyl)-cyclopropanecarboxylic
acid (Figure 2); transformation products were identified and
confirmed by GC-MS. The same hydrolysis was reported by
permethrinase from Bacillus cereus SM3 (9).
Effects of pH and Temperature on Activity and Stability
of the Purified Enzyme. The effect of pH and temperature on

7418 J. Agric. Food Chem., Vol. 53, No. 19, 2005
Liang et al.
Table 2. Effect of the Various Substances on Relative Activity of
Pyrethroid Hydrolase from Aspergillus niger ZD11
relative activity
(%)
substances
none
100
1 mM MgCl2
1 mM CuSO4
1 mM ZnSO4
1 mM MnCl2
1 mM AgNO3
1 mM HgCl2
10 mM EDTA
10 mM 1,10-phenanthroline
1 mM PCMB
94
92
93
92
8
6
96
91
±
±
±
±
±
±
±
±
4.2
4.1
4.1
4.3
0.3
0.2
4.5
4.1
Figure 2. Hydrolysis of permethrin of pyrethroid hydrolase from Aspergillus
niger ZD11.
0
Table 3. Kinetic Constants for Hydrolysis of Various
Esters
F-Nitrophenyl
specific activity
(U/mg)
Km
M)
kcat
(s
kcat/Km
-
1
-
-1
substrate
(
µ
)
(s 1 µ
M
)
F
F
F
F
F
F
F
-nitrophenyl acetate
-nitrophenyl propionate
-nitrophenyl butyrate
-nitrophenyl valerate
-nitrophenyl caproate
-nitrophenyl caprylate
-nitrophenyl laurate
187
243
386
473
331
82
±
±
±
±
±
±
9
211
152
93
62
125
651
±
±
±
±
±
±
9
7
4
3
6
33
152
193
306
375
263
65
±
6
9
15
18
13
3
0.72
1.27
3.3
6.05
2.1
0.1
0
12
19
22
16
4
±
±
±
±
±
Figure 3. Effect of pH on activity (
hydrolase from Aspergillus niger ZD11.
0
) and stability (
b) of pyrethroid
0
0
0
Table 4. Kinetic Constants for Hydrolysis of Various Pesticides
specific activity
(U/mg)
Km
kcat
(s
kcat/Km
-
1
-
-1
substrate
(
µ
M)
)
(s 1 µ
M
)
cis-permethrin
trans-permethrin
cypermethrin
fenvalerate
deltamethrin
malathion
29.3
31.1
8.4
3.1
0.47
2.3
±
±
±
±
±
±
1.5
1.5
0.4
0.016
0.024
0.11
0.18
±
±
±
±
±
±
0.008
0.006
0.012
0.06
0.082 0.015
0.061 0.072
0.91
0.97
0.26
±
±
±
±
±
±
0.05
5.05
6.06
1.13
0.086
0.009
0.055
0.16
0.23
1.12
1.67
1.31
0.045
0.014
0.0043
0.0007
0.0034
0.096
Figure 4. Effect of temperature on activity (0) and stability (b) of
pyrethroid hydrolase from Aspergillus niger ZD11.
examined. Both Km and kcat values of purified enzyme decreased
with increases in aliphatic chain length up to F-nitrophenyl
valerate (C5). The comparison of catalytic efficiency values (Kcat/
km) for various substrates indicated that these values were
dependent on the aliphatic chain length of substrate. Short-chain
F-nitrophenyl esters seemed to be preferred substrates, whereas
F-nitrophenyl esters of long-chain fatty acids were poor
substrates, and the purified enzyme had no activity against
F-nitrophenyl laurate. Taking into consideration that lipases
prefer substrates with relatively long aliphatic chains, these
results showed the purified enzyme is an esterase and not a
lipase.
the catalytic activity was studied by using trans-premethrin as
a substrate. The pH-activity profile of the enzyme was bell-
shaped, with maximum values at pH 6.5 (Figure 3), and the
enzyme was found to be stable in the pH range between 5.0
and 9.0. The optimum temperature was determined at the range
of 20-70 °C. The optimal temperature for the enzyme was 45
°C (Figure 4). The enzyme was fairly stable up to 45 °C and
had 62% of its activity at 50 °C. It was completely inactivated
at 65 °C.
Effects of Various Compounds and Metal Ions on Enzyme
Activity. The effects of various chemicals on the enzyme
activity were investigated by addition of the tested compounds
into the reaction mixture at the final concentration. The activity
was then measured with trans-premethrin as substrate and
expressed as a percentage of the activity obtained in the absence
of the added compound (Table 2). The presence of Hg2+ and
Ag+ caused a complete inhibition at 1.0 mM, while less
pronounced effects (5-10% inhibition) were observed in the
presence of the remaining divalent cations. The enzyme activity
was strongly inhibited by 1 mM F-chloromercuribenzoate
(PCMB), whereas the chelating agents EDTA and phenanthro-
line (10 mM) showed little effect on the enzyme activity.
Substrate Specificity. The substrate specificity toward F-ni-
trophenyl esters of various fatty acids was shown in Table 3.
The purified enzyme showed the highest activity with F-nitro-
phenyl valerate (473 U/mg) among the F-nitrophenyl esters
A range of pesticides such as malathion, cis-permethrin, trans-
permethrin, cypermethrin, fenvalerate, and deltamethrin were
tested for substrate specificity of the purified enzyme. Although
the purified enzyme hydrolyzed all pesticides tested at different
hydrolysis rates, the hydrolysis of all pesticides was much lower
than the hydrolysis of some F-nitrophenyl esters of fatty acids.
The trans-permethrin was hydrolyzed most rapidly, while
deltamethrin was the least readily attacked. cis-Permethrin was
hydrolyzed at an approximately equal rate toward trans-
permethrin. The Km and kcat values were calculated by fitting
the data to the Michaelis-Menten equation (Table 4). The
purified enzyme showed comparable affinity against structurally
similar pesticides with Km values ranging from 0.16 to 1.67
µM.

Purification and Characterization of a Novel Pyrethroid Hydrolase
J. Agric. Food Chem., Vol. 53, No. 19, 2005 7419
DISCUSSION
approximately equal activity toward permethrin isomers.
This feature is different from that of carboxylesterase E3
from Nephotettix cincticeps Uhler (21) and carboxylesterase
(BAC36707) (25), indicating that the purified enzyme lacked
stereoselectivity. The comparison of Km and kcat revealed that
the pyrethroid hydrolase has about 10-fold higher affinity toward
trans-permethrin than deltamethrin and can hydrolyze the former
about 65-fold faster than the latter. The catalytic efficiencies
(kcat/Km) are considered as a measurement of the enzyme
specificity; among these, trans-permethrin is clearly the pre-
ferred substrate, due to either steric hindrance of hydrolysis or
stabilization of the est bond. Replacement of the chlorovinyl
group of the pyrethoids with bromovinyl also affected the rate
of hydrolysis by the enzyme. Cypermethrin was more readily
transformed than deltamethrin, in agreement with findings for
the permethrinase from Bacillus cereus (9), pyrethroid-
hydrolyzing carboxylesterase (BAC36707) from mouse liver
microsomes, and the esterases associated with mammalian
microsomal fractions (27), suggesting that the pyrethroid
hydrolase has a specific preference for the properties of the
halogen atoms.
In conclusion, the pyrethroid hydrolase described here differs
from those previously reported in at least one of the following
aspects: molecular mass, pI, pH, and temperature optima. These
differences of the isofunctional enzyme suggest diversity in
evolution and a spread of pyrethroid hydrolase gene among
different microorganisms. Environments contaminated with
pyrethroid are regarded as hazardous because the pesticides are
considered to be an endocrine-disrupting chemical (28). Thus,
a technique for rapid degradation of the compounds is required.
In view of the resistance to many metals, leakage requirement
for cofactors, relatively broad pH and temperature optimum,
and higher activity against a range of pesticides, the purified
enzyme could conceivably be developed to fulfill the practical
requirements to enable its use in situ for detoxification of
pyrethroids where they cause environmental contamination
problems. Further study is helpful to establish more molecular
knowledge on gene overexpression and regulatory mechanisms.
A few bacterial isolates possessing pyrethroid hydrolase
activity have been isolated, whereas pyrethroid-degrading
enzymatic preparations have not been reported to date on the
fungus. This is the first report to our knowledge on the
production, purification, and properties of a pyrethroid hydrolase
from fungus. The production of pyrethroid hydrolase from
Aspergillus niger ZD11 depended strongly on the carbon
sources. When glucose was added in the minimal medium as
carbon source, cell extract showed no or only a trace level of
the enzyme activity. The pyrethroid was the best carbon source
for the production of intracellular pyrethroid hydrolase. The
specific activity of the purified enzyme under optimal conditions
was 31.1 U mg-1 of protein on trans-permethrin, whereas the
specific activities reported for pyrethroid hydrolase toward trans-
permethrin were from 0.0146 to 0.597 U mg of protein-1 (9,
21, 25). Therefore, the purified enzyme from Aspergillus niger
ZD11 is among the most efficient pyrethroid hydrolase described
so far. As a monomeric 53 kDa protein, the purified enzyme is
also similar to some known pyrethroid hydrolase, whose
molecular masses range from 58.6 to 61 kDa (9, 21, 25). The
optimum pH of the purified enzyme was lower than that
recorded from Bacillus cereus (pH 7.5) (9). The optimal
temperature of 45 °C was higher than that recorded for Bacillus
cereus (37 °C) (9).
The effects observed in the presence of potential inhibitors
or activators of the purified enzyme activity were investigated.
The purified enzyme was significantly affected by sulfhydryl
oxidant metals (Hg2+, Ag+), while other metal ions did not have
a remarkable effect on the activity. This suggested that thiol
may be involved in the active catalytic site. Furthermore, activity
was completely inhibited by thiol-modifying reagents such as
PCMB, therefore, suggesting again that sulfhydryl groups may
be involved in the catalytic center of the enzyme, substrate
binding, and/or recognition (26). EDTA and 1,10-phenanthroline
did not affect activity, indicating that divalent cations are not
required for enzyme activity. This property is similar to that
noted in Bacillus cereus, lacking any requirement for metal ions
at the active site (9).
LITERATURE CITED
The purified enzyme not only hydrolyzed various F-nitro-
phenyl esters of short-medium chain fatty acids, but also
degraded many pesticides with similar carboxylester such as
cypermethrin, permethrin, fenvalerate, deltamethrin, and mala-
thion, which is an organophosphorus pesticide, indicating that
the purified enzyme is an esterase with broader specificity.
However, this observation does not quite agree with data
reported by Motoyama et al., Maloney et al., and Stok et al. (9,
21, 25). The pyrethroid-hydrolyzing carboxylesterase (BAC36707)
from mouse liver microsomes and permethrinase from Bacillus
cereus did not hydrolyze malathion. On the other hand,
Motoyama et al. found that one of five forms of carboxy-
lesterases degraded malathion twice as fast as the fenvalerate,
and three other forms possessed approximately equal activity
toward these two insecticides. Furthermore, the hydrolysis of
cis-permethrin, trans-permethrin, cypermethrin, fenvalerate, and
deltamethrin by the purified pyrethroid hydrolase was much
higher than that of pyrethroid-hydrolyzing carboxylesterase
(BAC36707) from mouse liver microsomes and carboxylesterase
E3 from Nephotettix cincticeps Uhler (21, 25). In a previous
paper, there was a preference in both mammal and insect
carboxylesterases for permethrin to cypermethrin, and trans-
permethrin to cis-permethrin (21, 25). Yet the carboxylesterase
from Nephotettix cincticeps Uhler preferred cis-permethrin to
trans-permethrin. In this study, pyrethroid hydrolase possessed
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Received for review June 21, 2005. Revised manuscript received July
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from the Key Project of Science and Technology plan of Guangdong
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JF051460K