Biotransformation of the diphenyl ether herbicide lactofen and purification of a lactofen esterase from brevundimonas sp. LY-2

Article abstract of DOI:10.1021/jf101974y

The diphenyl ether herbicide lactofen is commonly used to control broadleaf weeds. Once released into the environment, this herbicide is subject to microbial reactions. This study describes the biotransformation of lactofen by Brevundimonas sp. LY-2 isolated from enrichment cultures inoculated with soil sample. This strain degraded about 80% of 50 mg L^-1 lactofen in 5 days of incubation in flasks. The metabolic behaviors of the herbicide in the media are described. The results show a transformation pathway of lactofen by the bacterium leading to the formation of 1-(carboxy)ethyl-5-(2-chloro-4- (trifluoromethyl)phenoxy)-2-nitrobenzoate and ethanol. An esterase, which could cleave the right ester bond of the alkanoic side chain of lactofen, was purified 113.3-fold to homogeneity with 6.83% recovery. The current results suggested that Brevundimonas sp. LY-2 degraded lactofen via the ester bond cleavage catalyzed by esterase.

Full text of DOI:10.1021/jf101974y

J. Agric. Food Chem. 2010, 58, 9711–9715 9711
DOI:10.1021/jf101974y
Biotransformation of the Diphenyl Ether Herbicide Lactofen
and Purification of a Lactofen Esterase from
Brevundimonas sp. LY-2
B
O
L
IANG, Y
U-
KUN
Z
HAO, PENG
L
U
, SHUN
-
PENG
L
I
,
AND
X
ING
HUANG
*
Key Laboratory for Microbiological Engineering of Agricultural Environment, Ministry of Agriculture,
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China
The diphenyl ether herbicide lactofen is commonly used to control broadleaf weeds. Once released
into the environment, this herbicide is subject to microbial reactions. This study describes the
biotransformation of lactofen by Brevundimonas sp. LY-2 isolated from enrichment cultures inocu-
lated with soil sample. This strain degraded about 80% of 50 mg L-1 lactofen in 5 days of incubation
in flasks. The metabolic behaviors of the herbicide in the media are described. The results show a
transformation pathway of lactofen by the bacterium leading to the formation of 1-(carboxy)ethyl-
5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoate and ethanol. An esterase, which could cleave
the right ester bond of the alkanoic side chain of lactofen, was purified 113.3-fold to homogeneity
with 6.83% recovery. The current results suggested that Brevundimonas sp. LY-2 degraded lactofen
via the ester bond cleavage catalyzed by esterase.
KEYWORDS: Biotransformation; lactofen; intermediate metabolites; Brevundimonas sp.; esterase
INTRODUCTION
poron beigelii SBUG 752 was able to transform diphenyl ether,
and several oxidation products were identified (13). Sphingomo-
nas paucimobilis could metabolize diclofop-methyl to diclofop
acid and degraded diclofop acid to 4-(2,4-dichlorophenoxy)-
phenol and 2,4-dichlorophenol and/or phenol (14). Keum et al.
(12) found that nitrodiphenyl ethers could be degraded by
S. wittichii RW1 through the initial reduction and N-acetylation
of nitro groups, followed by ether bond cleavage. Lysinibacillus
sp. ZB-1 could degrade fomesafen by reduction, acetylation of
nitro groups, and dechlorination (15).
Up to now, little is known about the biotransformation of
lactofen by pure culture. In this paper, a potent lactofen degrader
was isolated from soils through repetitive enrichment and succes-
sive subculture. We also describe the transformation of lactofen
by a newly selected bacterium together with identification of the
corresponding metabolites and the purification of a lactofen
esterase from cellfree extracts of this strain.
Lactofen is a member of the diphenylether herbicides. The major
target of this class of herbicides is protoporphyrinogen oxidase in
the porphyrin biosynthetic pathway (1, 2). Lactofen has high
activity and is commonly used to control broadleaf weeds in
soybeans, cereal crops, potatoes, and peanuts (3). As a postemergent
herbicide, it is directly released to the environment, and the
environmental fate of lactofen has been studied (4, 5). Hydrolysis
is expected to be an important process under alkaline conditions
based on an estimated hydrolysis half-life of 37 days at pH 8.
Photolysis is thought to be minimal, and the photodegradation half-
life of lactofen was reported as 24 days (6). This herbicide is
degraded in soil mainly by microbial activity, and aerobic conditions
speed the rate of microbial breakdown of lactofen (7). The biode-
gradation half-life of lactofen applied to a clay loam at an applica-
tion rate of 5 mg kg-1 and maintained under anaerobic conditions
was 30 days. The biodegradation products of lactofen in soil had
been reported as acifluorfen and 1-(carboxy)ethyl 5-(2-chloro-
4-(trifluoromethyl)phenoxy)-2-nitrobenzoate (8). The enantioselec-
tivity of the transformation of lactofen also had been studied (9).
Currently, few reports on the microbial degradation of diphenyl
ethers have been published. Sphingomonas sp. strain SS3 could
utilize diphenyl ether and its 4-fluoro, 4-chloro, and 4-bromo
derivatives as sole sources of carbon and energy (10). Azotobacter
chroococcum could utilize herbicide oxyfluorfen as the sole carbon
source for growth (11). Keum et al. (12) reported Sphingomonas
wittichii RW1 could degrade chlomethoxyfen, nitrofen, and
oxyfluorfen. The metabolism pathways of the microbial degrada-
tion of several diphenyl ethers also have been studied. Trichos-
MATERIALS AND METHODS
Soil, Chemicals, and Media. Soil samples were collected from an
agricultural field, which had been exposed to lactofen for many years, in
the city of Qiqihaer, Heilongjiang Province, China. Lactofen (99% purity)
was purchased from Sigma-Aldrich Chemical Co. (Shanghai, China). All
other chemicals used in this study were of analytical grade. The Luria-
Bertani medium contained yeast extract (5.0 g L-1), tryptone (10.0 g L-1),
and NaCl (10.0 g L-1), pH 7.0. Mineral salts medium (MM) contained
NH4NO3 (1.0 g L-1), K2HPO4 (1.5 g L-1), KH2PO4 (0.5 g L-1), NaCl
(0.5 g L-1), MgSO4 (0.2 g L-1), and 10 mL of a trace element solution, pH
7.0 (16). Solid medium plates were prepared by adding 1.5 wt %/vol agar
into the above liquid medium. Lactofen dissolved in acetone was added to
the media at a final concentration of 50 mg L-1
.
Isolation and Identification of the Strain. Soil samples (10.0 g) were
added to 100 mL of MM with 50 mg L-1 lactofen and incubated at 30 °C in
*Corresponding author (phone 86-25-84396685; fax 86-25-84396314;
e-mail huangxing@njau.edu.cn.).
© 2010 American Chemical Society
Published on Web 08/16/2010
pubs.acs.org/JAFC

9712 J. Agric. Food Chem., Vol. 58, No. 17, 2010
Liang et al.
Figure 1. Phylogenetic tree for strain LY-2 and related species based on the 16S rRNA gene sequences.
a rotary shaker at 160 rpm for about 7 days. About 5 mL of enrichment
culture was subcultured into 100 mL of fresh enrichment medium weekly.
After six rounds of transfer, the loss of lactofen was measured by HPLC
(as described below). Dilutions of the sequential enrichment were plated
onto MM agar plates containing 50 mg L-1 lactofen. After incubation at
30 °C for 2 days, the colonies were purified and selected to verify their
degrading capabilities. One pure isolate with lactofen degradation effici-
ency was isolated (designated LY-2) and selected for further investigation.
The identification of the strain was carried out by standard laboratory
procedures and those described in Bergey’s Manual of Determinative
Bacteriology (17). The 16S rRNA gene was amplified by PCR using the
following primers: 50-AGAGTTTGATCCTGGCTCAG-30 as forward
and 50-TACGGTTACCTTGTTACGACTT-30 as reverse (18). This se-
quence was compared to known sequences found in the GenBank
database using BLAST. Phylogenesis was analyzed with MEGA version
3.0 software, and its distance was calculated using the Kimura two-
Figure 2. Biodegradation of lactofen by Brevundimonas sp. LY-2: (2)
autoclaved LY-2; (9) active LY-2. Error bars represent the standard error
of three replicates.
parameter distance model (19). Unrooted trees were built using the
neighbor-joining method. Each data set was bootstrapped 1000 times.
Degradation of Lactofen by Strain LY-2 in Liquid Culture. Strain
LY-2 was grown in LB medium. Cell culture was harvested in the late-
exponential growth phase by centrifugation at 5000g for 5 min and washed
removed by centrifugation (10000g, 15 min). The resulting supernatant was
subjected to further precipitation by ammonium sulfate at saturations of
20-40, 40-60, and 60-100%. All fractions were desalted by dialysis and
then concentrated. The esterase was purified using anion exchange column
chromatography. The sample was applied to a DEAE Sepharose Fast Flow
column (1.6 cm ꢀ 8 cm, GE Healthcare Bio-Sciences AB), which was pre-
equilibrated with 20 mM Tris-HCl, pH 7.2. The column was initially eluted
with 2 bed volumes of the buffer to wash out unabsorbed proteins. The
adsorbed proteins were then eluted with a linear gradient of 0-250 mM NaCl
in Tris-HCl, pH 7.2, buffer at a flow rate of 0.5 mL min-1. The gradient eluate
was collected in 2.5 mL fractions. Protein fractions were collected and
monitored for esterase activity as described above. Those fractions containing
esterase activity were combined and stored at -20 °C. The concentrated
enzyme solution was subjected to native polyacrylamide gel electrophoresis
(PAGE) as described by Waterborg and Matthews (21). When the PAGE
completed, the polyacrylamide gel was transferred onto a 200 mg L-1 lactofen
agar plate and incubated for 60 min at 30 °C. Then a clear transparent band
appeared on the plate due to the degradation of the lactofen. The poly-
acrylamide gel slice corresponding to the transparent band was excised with a
scalpel. The separation of the lactofen-degrading enzyme from the gel was
achieved using a D-tube Electroelution Accessory Kit (Novagen).
twice with fresh MM medium. After the optical density at 600 nm (OD600
)
had been adjusted to 1.0, an inoculum (2%, vol/vol) was inoculated into
50 mL of MM medium with the lactofen (50 mg L-1). The cultures were
incubated at 30 °C and 160 rpm. At regular time intervals, aliquot samples
were collected, and the concentration of the herbicide was determined by
HPLC. During the degradation, two products appeared and were ana-
lyzed by LC-MS and GC, respectively. Each treatment was performed in
three replicates, and the control experiment with autoclaved microorgan-
ism was carried out under the same conditions.
Preparation of Cellfree Extracts. Cells in LB liquid medium were
harvested by centrifugation (5000g, 5 min) at 4 °C, washed twice with Tris-
HCl buffer (pH 7.2), and resuspended in the same buffer (20 mL) at a
concentration equivalent to an OD600 of 5.0. The cells were ruptured by 3 s
pulsed sonication for 10 min. The disrupted cell suspension was centrifuged at
12000g for 20 min at 4 °C to remove cell debris and undisrupted cells. The
supernatant was passed through a cellulose acetate filter with a pore size of
0.2 mm. The microbial extract was suspended in Tris-HCl buffer, pH 7.2.
Protein concentration was quantified according to the method of Bradford
(20). Bovine serum albumin (Sigma) was used as standard for calibration.
Protein Purification. All of the following experiments were carried
out at 4 °C unless otherwise specified. The cellfree extract of strain LY-2
produced a transparent halo of lactofen degradation on the phosphate
buffered saline solid plates containing 200 mg L-1 lactofen within 1 h of
incubation at 30 °C, which was utilized as an indicator to monitor the
lactofen-degrading activities. Solid ammonium sulfate was added, with
continuous stirring, to the enzyme suspension at 0-20% saturation and
the resulting suspension settled for 20 min. The precipitate formed was
Determination of Esterase Activity. Esterase activity was measured
by adding 20 μL of enzyme preparation to 2 mL of 20 mM Tris-HCl, pH
7.2, containing 50 mg L-1 lactofen and incubating for 30 min at 30 °C.
Samples were extracted, and the lactofen residues were measured by HPLC.
The transformation products by esterase were identified by LC-MS and
GC, respectively. Control samples containing boiled extract were treated
and analyzed in the same way. All experiments were carried out in

Article
J. Agric. Food Chem., Vol. 58, No. 17, 2010 9713
Figure 3. Mass spectrum of metabolite product A.
triplicate. One unit of enzyme activity was defined as the amount of
enzyme that liberated 1 μmol of ethanol from the substrate with concen-
tration of 50 mg L-1 per minute at 30 °C. The molecular mass of the
denatured protein was determined by SDS-PAGE (22). The molecular
mass markers used were rabbit muscle phosphorylase b (97.2 kDa), bovine
serum albumin (66.4 kDa), hen egg white ovalbumin (44.2 kDa), bovine
carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20.1 kDa), and
hen egg lysozyme (14.3 kDa). To determine the optimum pH, all activities
were measured using 20 mM citric acid-NaOH buffer at pH 4.0-5.5,
20 mM phosphate buffer at pH 5.0-8.0, 20 mM Tris-HCl buffer at pH
7.2-9.0, and 20 mM glycine-NaOH buffer at pH 8.5-10.0. The optimum
temperature was determined analogously with a constant pH of 7.0 and
different temperatures from 10 to 80 °C. The kinetic parameter study was
measured at the same conditions used for esterase activity determination
by adding 20 μL of enzyme preparation to 2 mL of 20 mM Tris-HCl, pH
7.2, containing 0.1-100 μM lactofen and incubating for 30 min at 30 °C.
Chemical Analysis. Lactofen in liquid cultures were extracted with
the same volume of dichloromethane and dried over anhydrous sodium
sulfate. The mixture was warmed in a hot water bath to evaporate the
dichloromethane. The residues were dissolved in acetonitrile. All samples
were analyzed by HPLC equipped with a Zorbax C-18 ODS Spherex
column (250 mm ꢀ 4.6 mm). The mobile phase was acetonitrile/water
(75:25, v/v), and the flow rate was 0.8 mL min-1. Detection of lactofen was
recorded at 230 nm. The injection volume was 20 μL.
The MS apparatus was an LC-MSD-Trap-SL equipped with an electro-
spray ionization source and operated in the negative polarity mode. The ES-
MS interface was operated under the condition of a gas temperature of
350 °C and a drying gas flow of 9.0 L min-1. The nebulizer nitrogen gas
pressure was 45 psi. Full scan signals were recorded within the m/z range
from 50 to 1000. For LC-MS, the source parameter was spray voltage =
7.0 kV. The sheath and auxiliary gases were supplied with nitrogen.
For metabolic product ethanol from liquid culture, 1 mL of culture was
collected from the medium and centrifuged at 12000g for 5 min and filtered
througha hydrophilic polyether sulfone(PES) syringe filter. The analysis of
ethanol was conducted by gas chromatography coupled with a flame
ionization detector (FID) (Shimadzu GC-2010, Japan), a 30 m Rtx-Wax
capillary column (0.25 mm id, film thickness = 0.25 μm), and a flame
ionizing detector (H2, 30 mL min-l; air, 250 mL min-l). Nitrogen was used
as the carrier gas (20 mL min-l). The temperatures at the injector, detector,
and oven were set at 210, 250, and 280 °C, respectively. The injection
volume was 0.5 μL. Standard solutions of ethanol (in 0.1% w/v) were mixed
with MM medium as following concentrations (mg L-1): 0, 0.5, 1, 2, 4, 8.
Recovery Assay. Triplicate analyses were conducted with liquid culture
at levels of 0.1, 1.0, 10.0, and 50 mg L-1 lactofen. Extraction and analysis
were performed as described above. The recoveries of lactofen from liquid
culture at levels of 0.1, 1.0, 10.0, and 50 mg L-1 were determined to be
87.91 ( 6.2, 90.12 ( 4.3, 96.89 ( 2.7, and 99.12 ( 0.8%, which indicated
that the procedure was efficient in extracting lactofen from liquid culture.
Figure 4. Time course of ethanol (product B) produced by Brevundimo-
nas sp. LY-2. Error bars represent the standard error of three replicates.
RESULTS AND DISCUSSION
Strain Isolation and Identification. Strain LY-2, capable of
degrading lactofen, was isolated and selected for further study
due to its degrading efficiency. This strain forms smooth, circular,
yellow pigmented colonies on LB plates. Strain LY-2 is Gram-
negative with rod-shaped morphology. It tested positive for
starch hydrolysis, catalase, and oxidase, but negative for glutin
hydrolysis, Vogese Proskauer, and nitrate reduction. The se-
quence of the 16S rRNA gene of LY-2 was deposited at GenBank
under accession number GU003879. The phylogenetic tree of the
16S rRNA sequence is shown in Figure 1, and this sequence was
99% identical to that of the 16S rRNA gene of Brevundimonas
aurantiaca DSM 4731(23). According to its physiological char-
acteristics and 16S rRNA phylogenetic analysis, strain LY-2
could be identified as a Brevundimonas sp.
Few strains of this genus with the ability to metabolize com-
pounds have been isolated. Deshpande et al. (24) reported that the
organophosphorus insecticide dimethoate could be degraded by
Brevundimonas sp. MCM B-427. Herbicide 4-(2,4-dichlorophenoxy)-
butyric acid and 4-(4-chloro-2-methylphenoxy)butyric acid degrad-
ing communities have been isolated, including Brevundimonas
sp. (25). In our study, strain Brevundimonas sp. LY-2 was capable
of degrading lactofen, which was another documented strain of
Brevundimonas species with the ability to degrade xenobiotics.
Biodegradation of Lactofen by Strain LY-2. The time course
curve oflactofen degradation by strain LY-2 is presented in Figure 2.

9714 J. Agric. Food Chem., Vol. 58, No. 17, 2010
Liang et al.
Figure 5. Proposed metabolic pathway of lactofen degraded by Brevundimonas sp. LY-2.
Table 1. Purification of the Lactofen Esterase
purification step
total activity (U)
total protein (mg)
specific activity (U mg-1
)
purification fold
recovery (%)
cellfree crude enzyme
78.5
52.1
41.2
5.36
183.2 ( 6.7
76.32 ( 1.1
7.3 ( 0.33
0.11 ( 0.005
0.43 ( 0.023
0.68 ( 0.03
5.64 ( 0.21
48.73 ( 0.98
1
100
ammonium sulfate precipitation
ion-exchange chromatography
PAGE gel electronic elution
1.58
13.1
113.3
66.37
52.5
6.83
HPLC analysis showed a substantial reduction in the levels of lacto-
fen. After incubation for 5 days, about 81.8% of 50 mg L-1 lactofen
initially added to the MM medium was degraded by strain LY-2.
This strain was deposited in the China General Microbiological
Culture Collection Center under accession number CGMCC3651.
Up to now, several reports about the biodegradation of diphenyl
ether herbicides have been described. Chakraborty et al. (11)
isolated A. chroococcum, which could utilize oxyfluorfen as the
sole carbon source and degrade >60% of added oxyfluorfen at a
concentration of 250 mg L-1 in 7 days. Adkins (14) reported that
1.5 mg L-1 diclofop-methyl could be degraded by S. paucimobilis
within 54 h. Chlomethoxyfen, oxyfluorfen, and nitrofen were
degraded by S. wittichii RW1 after 7 days of incubation in the
nutrient broth-supplimented M9 medium (12).
Identification of the Metabolites. The metabolites produced by
the degradation of lactofen were extracted and identified by LC-MS
and GC, respectively. One degradation product (designated product
A) appeared during biodegradation of lactofen in MM medium at
12 h, and its concentration reached the highest level at 48 h followed
by a decrease with longer incubation times. At 96 h, product A dis-
appeared. The negative-ion chemical ionization of product A (rete-
ntion time = 4.463) showed a prominent protonated molecular ion
at m/z431.97 [M - H]- and characteristic fragment ion peaks at m/z
359.92 [M - COOH - C2H4 - H]- and 315.92 [M - COOH -
C2H4 - CO2-H]- (Figure 3). On the basis of these results, product
A was preliminarily identified as 1-(carboxy)ethyl 5-(2-chloro-
4-(trifluoromethyl)phenoxy)-2-nitrobenzoate, which was produced
by hydrolysis of the right ester bond of the alkanoic side chain of
lactofen. Product A has been reported as a degradation product of
lactofen in soil (8). In this paper, product A was found during the
degradation of lactofen by pure culture. Accompanying product A,
another product (product B) appeared and has been detected by GC
during degradation of lactofen in MM medium at 12 h. Product B
was confirmed by the same retention time with ethanol authentic
standard chemical in GC analysis, and it could be identified as
ethanol. The time course of the production and disappearance of
ethanol by strain LY-2 are shown in Figure 4. After an initial incu-
bation period of 0-24 h, ethanol was accumulated dramatically,
and the highest concentration of ethanol was observed at 48 h. With
further incubation, its release declined and disappeared after 96 h.
From the structures of the identified metabolites, one pathway is pro-
posed for the biotransformation of lactofen by strain LY-2 (Figure 5).
Protein Purification. The lactofen esterase from cellfree extracts
was purified using a procedure that involved ammonium sulfate
precipitation, ion-exchange chromatography, and polyacryla-
mide gel electrophoretic elution (Figure 6). The summary of the
purification is presented in Table 1. The enzyme was purified
113.3-fold to a specific activity of 48.73 U mg-1 of protein from
the cellfree extract with recovery of 6.83% after the final step.
Figure 6. SDS-PAGE analysis of the purified esterase from Brevundimo-
nas sp. LY-2. Lanes: 1, cellfree extract; 2, after ammonium sulfate
precipitation; 3, after anion exchange column chromatography; 4, after
PAGE gel electronic elution; 5, molecular mass markers.
The lactofen-degrading metabolite produced by the purified
esterase was also identified and confirmed by LC-MS and GC.
The purified enzyme catalyzed the hydrolysis of the right ester
bond of the alkanoic side chain of lactofen to 1-(carboxy)ethyl
5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoate and
ethanol. At 60 min, the concentration of ethanol reached the
highest level, 4.75 mg L-1, followed by stability for the rest of the
incubation time. Therefore, the purified enzyme was designated a
lactofen esterase. These degrading products were also found
during the degradation of lactofen by strain LY-2 in liqulid
culture (products A and B). When lactofen was transformed by
the cells of strain LY-2, ethanol disappeared after a longer period
of incubation, which showed that the strain could utilize ethanol
for growth. However, when lactofen was hydrolyed by the
purified esterase, the concentration of ethanol was stable after
reaching the highest level, indicating the purified esterase only
hydrolysis of the ester bond.

Article
The molecular mass of esterase was found to be 25 ( 1 kDa as
J. Agric. Food Chem., Vol. 58, No. 17, 2010 9715
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estimated by SDS-PAGE (Figure 6). The optimum pH and
temperature for the enzyme were 7.0 and 20 °C, respectively.
Measurement of esterase activity using lactofen concentrations
ranging from 0.1 to 100 μM indicated that the Km and Vmax of the
enzyme were 0.81 μM and 1.26 nmol min-1 mg-1, respectively.
At present, many compound-degrading esterases have been
studied. Esterase purified from Gordonia sp. strain MTCC 4818,
which could utilize a number of phthalate esters as sole source of
carbon and energy, is involved in the hydrolysis of butyl benzyl
phthalate (26). An enzyme hydrolyzing ethylene glycol dibenzoate
was purified from Aspergillus nomius HS-1 with a monomeric
structure, of which the molecular mass was about 60000 (27). Three
sulfonylurea herbicide pyrazosulfuron-ethyl degrading bacteria
could produce extracellular carboxyesterase, which catalyzed the
rapid de-esterification of p-nitrophenyl butyrate to p-nitrophe-
nol (28). Pesticide pyrethroid-degrading esterases have been purified
from Bacillus cereus SM3, Aspergillus niger ZD11, and Klebsiella sp.
ZD112 (29-31). However, there is no report on the purification of
esterase from pure strain, which could degrade diphenyl ether
herbicides. In this paper, a lactofen esterase was purified from strain
LY-2. To our knowledge, this is the first lactofen esterase purified to
homogeneity from pure strain, and further genetic studies may lead
to the discovery of novel genes involved in the future.
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Received for review May 24, 2010. Revised manuscript received July 29,
2010. Accepted August 3, 2010. This work was funded by the Chinese
National Natural Science Fund (30900044), the Social Development
Program Fund of Jiangsu Province (BS2007056), and the Fund for the
Doctoral Program of Higher Education (20090097120031).