Elucidation of the enantioselective enzymatic hydrolysis of chiral herbicide dichlorprop methyl by chemical modification

Article abstract of DOI:10.1021/jf104500h

Up to 25% of the current pesticides are chiral, the molecules have chiral centers, but most of them are used as racemates. In most cases, enantiomers of chiral pesticides have different fates in the environment. Knowledge of the function of amino acids of

Full text of DOI:10.1021/jf104500h

ARTICLE
pubs.acs.org/JAFC
Elucidation of the Enantioselective Enzymatic Hydrolysis of Chiral
Herbicide Dichlorprop Methyl by Chemical Modification
†
,‡
‡
‡
,‡
†,‡
Yuezhong Wen, Chandan Li, Zhaohua Fang, Shulin Zhuang,* and Weiping Liu
†
MOE Key Laboratory of Environmental Remediation & Ecosystem Health, College of Environmental and Resource Sciences,
Zhejiang University, Hangzhou 310058, China
‡
Institute of Environmental Science, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
ABSTRACT: Up to 25% of the current pesticides are chiral, the molecules have chiral centers, but most of them are used as
racemates. In most cases, enantiomers of chiral pesticides have different fates in the environment. Knowledge of the function of
amino acids of enzymes involved in enantioselective behaviors contributes to the understanding of the enantioselectivity of chiral
pesticides. In this work, Aspergillus niger lipase (ANL, EC3.1.1.3) was chemically modified using bromoacetic acid (BrAc), 2,3-
butanedione (BD), N-bromosuccinimide (NBS), and methanal. The enantioselectivity of the enzymatic hydrolysis of 2,4-
dichlorprop-methyl (DCPPM) was investigated by chiral GC. The results have suggested that histidine, arginine, and tryptophan
are essential for lipase activity and might be involved in the catalytic site of ANL. In addition, histidine and lysine play an important
role in determining the observed enantioselective hydrolysis of chiral herbicide dichlorprop methyl. The molecular modeling study
revealed that the essential hydrogen bonds formed between DCPPM and catalytic residues of ANL might be responsible for the
enantioselectivity of DCPPM. The loss of enantioselectivity can also arise from the fact that the modification of the amino acids may
cause changes in both the nature of the ANL enzyme conformation and the binding pattern of DCPPM. Our study provides basic
information for the exploration of the enantioselective interaction mechanism of enzymes with chiral pesticides.
KEYWORDS: Chemical modification, lipase, enantioselectivity, hydrolysis, pesticide
’
INTRODUCTION
lipases). Because of their central role in the soil environment,
soil lipase activities are attractive as indicators for monitoring
various impacts on soils. It should be noticed that lipases are a
Up to 25% of the current pesticides are chiral, the molecules
15
have a chiral center and nonsuperimposable mirror images called
enantiomers, but most of them are used as racemates.
Although the enantiomers have identical physical-chemical
properties, in most cases, enantiomers behave different from
biochemical processes, the length of lag phases as well as the
degradation rates. It is also found that environmental factors can
affect the chiral preference of pesticides. Therefore, the effects
and the environmental fate of the enantiomers of chiral pesticides
need to be investigated separately.
Nowadays, most environmental research on chiral pesticides
has thus far been limited to investigating the enantioselectivity of
group of hydrolytic enzymes that couple a wide specificity to a
1
,2
16,17
high regio- and enantioselectivity and specificity.
Thus, when
pesticides are released into environments, they can interact with
lipases. However, although lipase is an intriguing enzyme with
diverse roles, our knowledge on its progress of stereoselective
transformation is still rather limited.
As a means of identifying the functional roles of critical
residues of enzymes, chemical modification of amino acids has
3
,4
5
been exploited for many years, and the chemistry is well
18,19
established.
However, there are few reports to provide
unequivocal information on the mechanism of the chiral inter-
actions between lipase and pesticide by using chemical modifica-
tion. Therefore, in the present work, the amino acid residues
chosen for study were histidine, arginine, tryptophan, and lysine,
which are known to be often catalytically essential. We per-
formed chemical modification on Aspergillus niger lipase (ANL,
EC3.1.1.3) and investigated the effects of chemical modifiers on
ANL-catalyzed enantioselective hydrolysis of 2,4-dichlorprop-
methyl (DCPPM). DCPPM is a highly effective, broad-spectrum
chiral herbicide with low toxicity. Its racemate of DCPPM
contains a pair of (R)- and (S)-DCPPM isomers (Figure 1), with
only the (R)-form being herbicide active. The enantioselective
6
-10
degradation and toxicity.
Little is known about the mecha-
nism of enantioselectivity of chiral pesticides in the environment.
As we know, enantioselective degradation implies that one or
more enzyme reactions involved in different degradation steps
must be able to differentiate between the enantiomers, and only
subtle differences in the enzymatic active sites determine their
enantioselectivities. Thus, for a better understanding of enantio-
selective degradation and toxicity, more detailed studies of
enzymes involved in enantioselective behaviors need to be
2
,11-13
conducted.
Hydrolysis is an important degradation approach of pesticides
in environments, and the presence of hydrolytic enzymes largely
14
promotes the degradation of pesticides. Among the hydrolytic
enzymes, the lipases (EC 3.1.1.3) are a group of important
hydrolytic enzymes in water and soils and are widely distributed
throughout the organisms of different nature (some microorgan-
isms are known to have the ability to produce extracellular
Received: November 23, 2010
Accepted: January 24, 2011
Revised:
January 17, 2011
Published: February 11, 2011
r 2011 American Chemical Society
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Journal of Agricultural and Food Chemistry
ARTICLE
Figure 1. Enantiomers of dichlorprop methyl ester.
Table 1. Modification of Lipase by Chemical Modifiers
modifier
modified amino acid
modified group
ref
bromoacetic acid
histidine
arginine
tryptophan
lysine
imidazole
guanidyl
indolyl
22
23
24
25
2,3-butanedione
N-bromosuccinimide
methanal
amido
Figure 2. Tertirary structure of ANL generated by homology modeling.
8
,20
The catalytic triad was composed of Ser173 (purple color), Asp228
degradation and toxicity of DCPPM are observed.
On the
(orange color), and His285 (green color). This canonical Ser/Glu/His
basis of the chemical modification, the molecular modeling
method was applied to explore the binding mode of (R)- and
S)-DCPPM isomers to the lipase. Combining the chemical
catalytic triad was located in a cavity with a lid composed of seven
residues spanning from Ile113 to Asp119.
(
modification and molecular modeling, we have identified the
important role of four amino acids in the enantioselectivity of
ANL. This study provides basic information for the exploration
of the enantioselective hydrolysis mechanism of ANL with the
chiral herbicide.
In the second method, DCPPM was used as the hydrolytic substrate
to assay the enantioselectivity of lipase to DCPPM by slightly modifying
2
6
the literature procedure. The lipase-catalyzed hydrolysis of DCPPM
was carried out in the dark. Ten milliliters of sodium phosphate buffer
(pH 7.0) was added to 0.2 mL of substrate (1.0 mg/mL) and 0.5 mg of
lipase. The mixture was incubated at 25 ( 1 °C in a rotary shaker at 120
rpm for 5 h. After the completion of the reaction, aliquots (0.2 mL) of
the reaction mixture were withdrawn and extracted with 2 mL of n-
hexane, followed by drying over anhydrous Na SO . The samples were
’
MATERIALS AND METHODS
Materials and Reagents. ANL was supplied by Shenzhen
2
4
Leveiking biological engineer Co. Ltd., with a reported activity of 10
analyzed using chiral gas chromatography. Each treatment was sampled
in triplicate, and the enzyme-free samples were included as controls for
the analytical background of enzyme activity.
-
1
KLUg . Bromoacetic acid (BrAc), 2,3-butanedione (BD),and N-
bromosuccinimide (NBS) were supplied by Sigma Co. Ltd. as modifiers
of lipase. (R)-Dichlorprop methyl ester, (S)-dichlorprop methyl ester,
and (Rac)-dichlorprop methyl ester with 99% purity were synthesized
Enantioselectivity Gas Chromatography Separation.
DCPPM was analyzed by gas chromatography (GC-2010 Shimadzu),
equipped with a chiral glass capillary column (β-cyclodextrin BGB-176,
30 m length, outer diameter 0.35 mm, inner diameter 0.25 mm, and film
thickness 0.25 μm; BGB Analytik AG). The oven temperature of GC
was 140 °C for 50 min, whereas that of the injection port and the
detector were 200 and 230 °C, respectively. The analytes were mon-
itored by electric capture detection. Peaks were identified by comparison
of the retention times of (R)-DCPPM and (S)-DCPPM, respectively.
The enantiomeric ratio (ER) was expressed as the peak area of the (R)-
isomer divided by that of the (S)-isomer.
21
according to Camps’s method. n-Hexane, NaH
2 4 2 4
PO , Na HPO , and
Na SO were analytically pure and used without further purification.
2
4
Chemical Modification of Lipase. Histidine (His), arginine
(Arg), tryptophan (Try), and lysine (Lys) residues in lipase were
modified separately by different modifiers according to the slightly
modified literature procedures listed in Table 1. All modification of
amino acid residues were carried out in the dark in 5 mL total reaction
volume (25 °C) and in 50 mM sodium phosphate buffer, pH 7.00. First,
the 4 mg lipase dry powder was added into the buffer solution. Then,
different concentrations of chemical modifiers were added, and the
solutions were incubated. After 20 min, aliquots (1 mL) were removed
and assayed for residual enzyme activity.
Enzymatical Hydrolysis. Two methods were used to carry out
the lipase-catalyzed hydrolysis reactions. In the first method, fluoresce-
indiacetate (FDA) was used as the lipase substrate to assay the hydrolytic
activity of lipase. First, phosphate buffer solution (pH 7.0) was added to
the tubes to make a constant volume of 5 mL and 0.5 mM FDA, and then
Spectrometry Experiments. The spectra of lipase were mea-
2
sured as reports. Fluorescence emission spectra were recorded using a
Hitachi F2500 spectrofluorimeter. UV spectra were measured using a
Shimadzu UV-2401PC spectrophotometer.
Homology Modeling. The amino acid sequence of ANL (GenBank
accession number ABG37906.1) was used for the structural construction.
The homology modeling for the tertiary structure of ANL was performed
2
7,28
with the I-TASSER server.
The initial template was identified using
0
.8 mg lipase was added to start the reaction. For all determinations of
the LOMETS threading and the following structure assembly, model
selection, and refinement, and structure-based functional annotation
FDA hydrolytic activity, fluorescence emission spectra of fluorescein
released during the assay was used. The scanning parameters were as
follows: scanning time of 3 min, excitation wavelength of 492 nm,
emission wavelength of 514 nm, and an experimental temperature of
29
were performed with the reported protocols. The quality of the obtained
3
0
model was further evaluated by the Prosa web server. The knowledge-
based potentials of mean force were used to evaluate the model accuracy
3
1
2
5 °C. The fluorescence intensity at 200 s was recorded. Each treatment
following the reported protocol.
was sampled in triplicate, and the enzyme-free samples were included as
controls for the analytical background of enzyme activity.
Molecular Docking. To build the structures for the complex of
ANL with with (R)- and (S)-DCPPM isomers, automated molecular
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Journal of Agricultural and Food Chemistry
ARTICLE
Figure 4. Remaining activity of lipase incubated with different con-
centrations of BD.
reactions. The modeled tertirary structure of ANL showed that
the catalytic His285 locates at the active site, which is in line with
the experimental study of the modification of lipase by dye-
33
sensitized photooxidation. Considering BrAc is a specific
modifier for protein histidine residues and is being used widely
22,34
in enzyme modification,
by modification of histidine residue
with BrAc, we can understand whether the amino acids at (or
near) the active center of the enzyme take part in the progress of
stereoselective transformation of the chiral herbicide DCPPM.
As shown in Figure 3a, the enzyme activity remained constant
at low concentration (0-0.288 mM). This result is consistent
35
with Liu’s result, since it was impossible to label histidine
residues in the low range of modifier concentration. At high
concentration of BrAc (0.288-0.864 mM), the loss of enzyme
activity increased quickly with an increase of modifier concentra-
tion and the enzyme activity remained 77.59% in the 0.864 mM
BrAc. These data indicated the possible involvement of histidine
residues in the catalytic mechanism of action of the enzyme.
Protein UV absorption is mainly due to the electronic excita-
tion of aromatic amino acids such as tryptophan and tyrosine,
followed by phenylalanine and histidine. The absorption spectra
of these chromophores changes with varying solution conditions.
In the UV differential spectrometry experiments, the absorption
peak of lipase was approximately 210 nm with an absorption
intensity of 0.27. Differential spectra of lipase with different
concentrations of BrAc were shown in Figure 3b. As illustrated, it
was found that the absorption spectrum of lipase underwent
changes when BrAc was added. It can be seen that the maximum
absorption of lipase decreases regularly with a red shift with
increasing concentrations of BrAc (from 0 to 1.152 mM). This
indicates that lipase forms complexes with BrAc and that this
complex produces observable changes in the absorption spectra.
The decrease in the UV-vis differential spectral intensity and red
shift in the absorption maximum may be attributed to the
perturbation of the microenvironment of histidine residues at
higher concentrations of BrAc. These results further indicated
the involvement of histidine residues in the catalytic mechanism
of action of the enzyme.
Figure 3. (a) Remaining activity of lipase incubated with different
concentrations of BrAc. (b) The UV absorption spectra of lipase in the
presence of BrAc. c(BrAc)/mM: 1, 0; 2, 0.144; 3, 0.288; 4, 0.432; 5,
0.576; 6, 0.72; 7, 0.864; 8, 1.008; 9, 1.152.
32
docking soft Gold 5.0 was used. The binding pocket was defined as a
sphere with a radius of 5 Å to cover the ligands. Twenty times of genetic
algorithm runs were carried out during the molecular docking process.
The energetic evaluations were performed using the GoldScore fitness
function. Ten poses of each isomer were finally generated, and the best
pose with the highest docking score was finally chosen.
’
RESULTS AND DISCUSSION
To better characterize the biological functions of ANL, its
tertiary structural details are essential. Considering the lack of an
experimentally determined structure of ANL, the tertiary struc-
ture of ANL was modeled with the established I-TASSER server,
and the obtained model was verified by the Prosa web server.
The structure of ARL shares a canonical topology with the
lipase family (Figure 2). Its catalytic triad was composed of
Ser173 (purple color), Asp228 (orange color), and His285
(
green color). This canonical Ser/Glu/His catalytic triad was
To further reveal the role of histidine in enantioselectivity of
the enzyme, the enzymatical hydrolysis of racemate DCPPM was
investigated. It was found that ER value decreased from 2.93 to
1.77 after histidine residues were modified by BrAc and was only
60.4% that of native lipase. This result showed that the histidine
residue of lipase was essential for its enantioselective catalytic
located in a bowl-shaped cavity with a lid (pink color) composed
by seven residues spanning from Ile113 to Asp119.
Modification of Histidines. Histidine, an essential amino
acid, has as a positively charged imidazole functional group. The
imidazole makes it a common participant in enzyme catalyzed
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Journal of Agricultural and Food Chemistry
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Figure 7. (a) Changes in the UV spectra of lipase induced by NBS. (b)
Changes in the fluorescence emission spectra of lipase induced by NBS.
(a) c(NBS/mM: 1, 0; 2, 0.045; 3, 0.90; 4, 0.13; 5, 0.18; 6, 0.22; 7, 0.27; 8,
0
0
.32; 9, 0.36. (b) c(NBS)/mM: 1, 0; 2, 0.11; 3, 0.22; 4, 0.33; 5, 0.44; 6,
.55; 7, 0.66; 8, 0.77; 9, 0.88.
Figure 5. (a) Changes in the UV spectra of lipase induced by BD. (b)
Changes in the fluorescence emission spectra of lipase induced by BD.
(a) c(BD)/mM: 1, 0; 2, 0.14; 3, 0.28; 4, 0.41; 5, 0.55; 6, 0.70. (b) c(BD)
/
9
mM: 1, 0; 2, 0.35; 3, 0.69; 4, 1.04; 5, 1.38; 6, 1.73; 7, 2.07; 8, 2.42;
, 2.76; 10, 3.11.
Figure 8. Remaining activity of lipase in the presence of methanal.
of BD. Similar to the result of the modification of the histidine
residue, the enzyme activity decreased with the increase of
modifier concentration (0-3.11 mM). The chemical modifica-
tion of lipase was executed for 20 min in the presence of BD.
Incubation of lipase with 3.11 mM BD to labeling arginines
decreased the enzyme activity 50%. This result also indicated the
possible involvement of arginine residues in the catalytic me-
chanism of action of the enzyme.
Figure 6. Remaining activity of lipase incubated with different con-
centrations of NBS.
The UV absorption spectra of lipase modified by BD are
shown in Figure 5a. Increasing the BD concentration resulted in a
decrease in the enzymatic absorption intensity of the 280 nm
region, which suggested a change in tyrosine exposure, besides
indicating contributions from the variation of the environment or
the ionization of tryptophan residues, phenylalanine residues,
activity, and the modification of histidine residue by BrAc led to
the loss of enantioselectivity of lipase.
Modification of Arginines. BD has been exploited widely for
23,35
the modification of arginine residues.
Figure 4 shows the
36
remaining activity oflipase incubatedwithdifferentconcentrations
and probably also disulfide groups. It suggested that the
1
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Journal of Agricultural and Food Chemistry
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Figure 9. Binding mode of (R)- and (S)-DCPPM isomers to ANL. The dashed line indicates the hydrogen bond formed between DCPPM isomers and
ANL. The DCPPM isomers are show, in stick and the residues of ANL in licorice representation.
arginine modified by BD may interact with tyrosines or trypto-
phans of lipase.
decreased in the presence of increasing concentrations of NBS.
These results showed that the tryptophan residue was located at
or near the substrate binding sites of the enzyme and that
oxidation of these moieties by NBS led to conformational
changes in the protein, either at the active site or at some other
To further reveal the interactions between lipase and modifier
BD, the fluorescence of lipase was investigated, and the fluores-
cence quenching spectra are shown in Figure 5b. It can be seen
that increasing the concentration of BD resulted in a decrease in
the fluorescence emission intensity of lipase. Since arginine was
not a fluorescent amino acid, chemical modification of arginines
had an effect on the microenvironment of tyrosines or trypto-
phans residues which were fluorescent residues of lipase.
To understant the role of arginine in the enantioselectivity of
enzyme, the enzymatical hydrolysis of racemate DCPPM was
also investigated. It was found that ER value remained unchanged
after histidine residues were modified by BD. This result showed
that the arginine residue of lipase was not essential for its
enantioselective catalytic activity. But because the modification
of the arginine residue by BD led to the loss of enzyme activity,
these results indicated that there is no inevitable link between
enzyme activity and enantioselectivity of lipase.
24
location which, in turn, can affect the active site. The study of
enzymatical hydrolysis of racemate DCPPM found that the ER
value also remained unchanged after tryptophan residues were
modified by NBS. This result also indicated that the tryptophan
residue of lipase was not essential for its enantioselective catalytic
activity. In other words, the enantioselectivity and catalytic
activity of lipase are two different processes, and the amino acids
between two processes are not completely the same nor identical.
Modification of Lysines. Chemical modification of lysine
residues has been reported to cause the inactivation of several
2
5
enzymes, and methanal is widely used as lysine modifier.
Figure 8 shows the remaining activity of lipase in the presence
of methanal. It can be found that the enzyme activity toward FDA
remained fully active for 20 min in the presence of methanal (0-
6.8 mM). This finding indicated that no lysine residues appeared
to be essential for lipase activity. In addition, methanal did not
have effects of the UV absorption spectra and fluorescence
spectra of lipase. These results indicated that the modification
of lysine residues by methanal also did not lead to conformational
changes in the protein, either at the active site or at some other
location which, in turn, can affect the active site.
Modification of Tryptophans. NBS has been widely used for
the specific modification of tryptophan residues, and changes in
catalytic activity and loss of tryptophan residues (i.e., conversion
of the indole ring into an oxindole derivative) were followed as a
24
function of the molar ratio of NBS to enzyme. In the present
study, NBS was used for labeling tryptophan residues of lipase.
NBS was employed initially at 0.044, 0.066, 0.088, 0.11, 0.22,
0
.33, and 0.44 mM for 20 min of reaction time. From Figure 6, it
It was found that the enantioselectivity decreased by chemical
modification of lysine using methanal. In the presence of 3.4 mM
methanal, the ER value decreased from 1.58 to 1.18. It was
conceivable that lysine residues might be involved in the
enantioselective enzymatic hydrolysis of DCPPM, whereas lysine
was not essential in the hydrolysis of FDA as described above.
Interactions of ANL with DCPPM. Studying the interactions
between DCPPM and ANL at the atomic level is essential to the
elucidation of the mechanism of enantioselective hydrolysis of
DCPPM isomers catalyzed by ANL. Here, we applied the
molecular docking technique to explore the interactions of
ANL with DCPPM isomers. The (R)- and (S)-DCPPM isomers
were separately docked into the active site of ANL using the Gold
5 program, and their binding mode to ANL was compared
(Figure 9).
can be seen that enzyme activities decrease quickly with increase
of the NBS concentration. It was noticed that when the NBS
concentrations increased from 0 to 0.11 mM, the enzyme
activities decreased quickly. Then the enzyme activities de-
creased with the further increase of the NBS concentrations. At
the NBS concentration of 0.33 mM, the modification by NBS
caused over 99% loss of lipase activity.
The UV absorption spectra of lipase modified by NBS are
shown in Figure 7a. Alteration of the concentration of NBS
produced an absorption change in the 210 nm region. Increasing
the NBS concentration resulted in a decrease in the absorption
intensity and a small red-shift of the 210 nm region. The
fluorescent properties of modified ANL by NBS are also pre-
sented in Figure 7b. The fluorescence intensity of lipase also
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Journal of Agricultural and Food Chemistry
ARTICLE
DCPPM isomers were located at the active site of ANL and
were surrounded by several residues: His285, Ser173, Arg108,
Trp116, and Lys114. (S)-DCPPM (Figure 9a) was located at the
active site of ANL and formed three hydrogen bonds with ANL,
including the catalytic hydrogen bonds formed with His285 and
with Ser173. However, (R)-DCPPM (Figure 9b) only has one
hydrogen bond with Arg108, and it did not form hydrogen bonds
with catalytic His285 or Ser173. The hydrolysis experiment
showed that ANL is enantioselective for (S)-DCPPM more than
(9) Liu, H. J.; Xiong, M. Y. Comparative toxicity of racemic meto-
lachlor and S-metolachlor to Chlorella pyrenoidosa. Aquat. Toxicol. 2009,
3, 100–106.
9
(10) Diao, J. L.; Xu, P.; Wang, P.; Lu, D. H.; Lu, Y. L.; Zhou, Z. Q.
Enantioselective degradation in sediment and aquatic toxicity to Daph-
nia magna of the herbicide lactofen enantiomers. J. Agric. Food Chem.
2
010, 58, 2439–2445.
11) Muller, T. A.; Kohler, H. P. E. Chirality of pollutants-effects on
metabolism and fate. Appl. Microbiol. Biotechnol. 2004, 64, 300–316.
12) Zhou, Q. Y.; Liu, W. P.; Zhang, Y. S.; Liu, K. K. Action
(
(
(
R)-DCPPM. This difference in hydrolysis of (R)- and (S)-
mechanisms of acetolactate synthase-inhibiting herbicides. Pestic. Bio-
DCPPM isomers by ANL is possibly due to the lack of hydrogen
bond interactions between (R)-DCPPM and catalytic His285
and Ser173. Therefore, the specific hydrogen bond interactions
between DCPPM and catalystic residues of ANL are essential for
the enantioselective hydrolysis of DCPPM. The modification of
these essential residues may change the binding mode of ANL to
DCPPM and cause conformational changes of the ANL enzyme,
thus, leading to the loss of enantioselectivity.
chem. Phys. 2007, 89, 89–96.
(13) Wen, Y. Z.; Yuan, Y. L.; Chen, H.; Wang, H. L.; Liu, H. J.; Kang,
X. D.; Fu, L. S. Spectroscopic investigations of the chiral interactions of
metolachlor and its (S)-isomer with lipase and phosphatase. J. Environ.
Sci. B 2010, 45, 249–253.
(14) Horvat, C. M.; Wolfenden, R. V. A persistent pesticide residue
and the unusual catalytic proficiency of a dehalogenating enzyme. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 16199–16202.
(15) Margesin, R.; Zimmerbauer, A.; Schinner, F. Soil lipase activity:
a useful indicator of oil biodegradation. Biotechnol. Tech. 1999, 13,
859–863.
’
AUTHOR INFORMATION
(
16) Sonnet, P. E.; Gazzillo, J. A. Evaluation of lipase selectivity of
hydrolysis. J. Am. Oil Chem. Soc. 1991, 68, 11–15.
17) Strohalm, H.; Dold, S.; Pendzialek, K.; Weiher, M.; Engel, K. H.
Corresponding Author
*
Tel: þ86-571-8898-2344. Fax: þ86-571-8898-2344. E-mail:
(
shulin@zju.edu.cn.
Preparation of passion fruit-typical 2-alkyl ester enantiomers via
lipase-catalyzed kinetic resolution. J. Agric. Food Chem. 2010, 58,
Funding Sources
6
328–6333.
18) Lundblad, R. L. Chemical Reagents for Protein Modification; CRC
We acknowledge the financial support from the National Natural
Science Foundation of China (No.20977078), the Fundamental
Research Funds for the Central Universities and Scientific
Research Foundation for Returned Overseas Chinese Scholars,
State Education Ministry, China.
(
press: Boca Raton, FL, 1991; pp 129-215.
(19) Palomo, J. M.; Fernandez-lorente, G.; Guisan, J. M.; Fernandez-
lafuente, R. Modulation of immobilized lipase enantioselectivity via
chemical amination. Adv. Synth. Catal. 2007, 349, 1119–1127.
(20) Ma, Y.; Xu, C.; Wen, Y. Z.; Liu, W. P. Enantioselective
separation and degradation of the herbicide dichlorprop methyl in
sediment. Chirality 2009, 21, 480–483.
’
ABBEVIATIONS USED
DCPPM, 2, 4-dichlorprop-methyl; ANL, Aspergillus niger lipase;
BrAc, bromoacetic acid; BD, 2,3-butanedione; NBS, N-bromo-
succinimide; His, histidine; Arg, arginine; Try, tryptophan; Lys,
lysine; FDA, fluoresceindiacetate.
(21) Camps, P.; Perez, F.; Soldevilla, N. (R)- and (S)-3-hydroxy-4,4-
dimethyl-1-phenyl-2-pyrrolidinone as chiral auxiliaries in the enantio-
selective preparation of alpha-aryloxypropanoic acid herbicides and alpha-
chlorocarboxylic acids. Tetrahedron: Asymmetry 1998, 9, 2065–2079.
(22) Xie, X. L.; Du, J.; Huang, Q. S.; Shi, Y.; Chen, Q. X. Inhibitory
kinetics of bromacetic acid on ss-N-acetyl-D-glucosaminidase from prawn
’
REFERENCES
(
Penaeus vannamei). Int. J. Biol. Macromol. 2007, 41, 308–313.
(
1) Garrison, A. W. Probing the Enantioselectivity of Chiral Pesti-
cides. Environ. Sci. Technol. 2006, 40, 16–23.
2) Wen, Y. Z.; Yuan, Y. L.; Shen, C. S.; Liu, H. J.; Liu, W. P.
(23) Patthy, L.; Smith, E. L. Reversible modification of arginine
residues. Application to sequence studies by restriction of tryptic
hydrolysis to lysine residues. J. Biol. Chem. 1975, 250, 557–564.
(24) Freisheim, J. H.; Huennekens, F. M. Folic acid coenzymes and
one-carbon metabolism. XXV. Effect of N-bromosuccinimide on dihy-
drofolate reductase. Biochemistry 1969, 8, 2271–2276.
(
Spectroscopic investigations of the chiral interactions between lipase
and the herbicide dichlorprop. Chirality 2009, 21, 396–401.
(
3) Lewis, D. L.; Garrison, A. W.; Wommack, K. E.; Whittemore, A;
Steudler, P; Mellilo, J. Influence of environmental changes on degrada-
tion of chiral pollutants in soils. Nature 1999, 401, 898–901.
(25) Chen, Q.; Liao, J. H.; Wang, Q.; Chen, Q. X. Studies on the
essential groups of the alkaline phosphatase from Ostrea cucullate. J.
Xiamen Univ. (Nat. Sci.) 2004, 43, 702–705.
(4) Wen, Y. Z.; Yuan, Y. L.; Chen, H.; Xu, D. M.; Lin, K. D.; Liu,
W. P. Effect of chitosan on the enantioselective bioavailability of the
herbicide dichlorprop to Chlorella pyrenoidosa. Environ. Sci. Technol.
(26) Fang, Z. H.; Wen, Y. Z.; Liu, W. P. Influence of kaolinite on
chiral hydrolysis of methyl dichlorprop enantiomers. J. Zhejiang Univ.
SCI. 2005, 6B, 1028–1032.
2
010, 44, 4981–4987.
(
5) Liu, W. P.; Gan, J. Y.; Schlenk, D.; Jury, W. A. Enantioselectivity
(27) Zhang, Y. I-TASSER server for protein 3D structure prediction.
BMC Bioinf. 2008, 9, 40.
(28) I-TASSER. http://zhanglab.ccmb.med.umich.edu/I-TASSER/.
(29) Roy, A.; Kucukural, A.; Zhang, Y. I-TASSER: a unified platform
for automated protein structure and function prediction. Nat Protoc.
2010, 5 (4), 725–738.
in environmental safety of current chiral insecticides. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 701–706.
(6) Buser, H. R.; Muller, M. D.; Poiger, T.; Balmer, M. E. Environ-
mental behavior of the chiral acetamide pesticide metalaxyl: enantio-
selective degradation and chiral stability in soil. Environ. Sci. Technol.
2
002, 36, 221–226.
(30) Prosa web server. https://prosa.services.came.sbg.ac.at/prosa.php.
(31) Wiederstein, M.; Sippl, M. J. ProSA-web: interactive web
service for the recognition of errors in three-dimensional structures of
proteins. Nucleic Acids Res. 2007, 35 (Suppl. 2), W407–410.
(32) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and validation of a genetic algorithm for flexible docking.
J. Mol. Biol. 1997, 267, 727–748.
(
7) Williams, G. M.; Harrison, I.; Carlick, C. A.; Crowley, O.
Changes in enantiomeric fraction as evidence of natural attenuation of
mecoprop in a limestone aquifer. J.Contam. Hydrol. 2003, 64, 253–267.
(8) Li, H; Yuan, Y. L.; Shen, C. S.; Wen, Y. Z.; Liu, H. J. Enantio-
selectivity in toxicity and degradation of dichlorprop-methyl in algal
cultures. J. Environ. Sci. B 2008, 43, 288–292.
1
929
dx.doi.org/10.1021/jf104500h |J. Agric. Food Chem. 2011, 59, 1924–1930

Journal of Agricultural and Food Chemistry
ARTICLE
(
33) Chapus, C.; Semeriva, M. Machanism of pancreatic lipase
action. 2. Catalytic properties of modified lipases. Biochemistry 1976,
5, 4988–4991.
34) Chen, Q. X.; Zhang, Z.; Zhou, X. W.; Zhuang, Z. L. Kinetics of
1
(
inhibition of ss-glucosidase from Ampullarium crossean by bromoacetic
acid. Int. J. Biochem. Cell B 2000, 32, 717–723.
(35) Liu, W.; Zhu, W. M.; Wu, D. Q. Chemical modification of
xylanase from Trichosporon cutaneum shows the presence of carboxyl
groups and cysteine residues essential for enzyme activity. Protein J.
1
999, 18, 677–686.
36) Miggiano, G. A. D.; Mordente, A.; Pischiutta, M. G.; Martorana,
(
G. E.; Castelli, A. Early conformational changes and activity modulation
induced by guanidinium chloride on intestinal alkaline phosphatase.
Biochem. J. 1987, 248, 551–556.
1
930
dx.doi.org/10.1021/jf104500h |J. Agric. Food Chem. 2011, 59, 1924–1930