A Novel esterase from Pseudochrobactrum asaccharolyticum WZZ003: Enzymatic properties toward model substrate and catalytic performance in chiral fungicide intermediate synthesis

Article abstract of DOI:10.1016/j.procbio.2018.03.011

Only a few esterases have been used for the synthesis of optically pure fungicide. For example, (R)-metalaxyl synthesized using esterase-involved bioreaction displays fungicide activity, whereas (S)-enantiomer is redundant. However, the biosynthesis of (R

Full text of DOI:10.1016/j.procbio.2018.03.011

ProcessBiochemistryxxx(xxxx)xxx–xxx
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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
A Novel esterase from Pseudochrobactrum asaccharolyticum WZZ003:
Enzymatic properties toward model substrate and catalytic performance in
chiral fungicide intermediate synthesis
Feng Chenga,1, Feifei Chenga,1, Jianyong Zhenga, Guanzhong Wub, Yinjun Zhanga,⁎, Zhao Wanga
a
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China
Yifan Biotechnology Group Co. Ltd., Wenzhou, 32500, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Esterase
(R,S)-DMPM
Metalaxyl
High activity
High enantioselectivity
Only a few esterases have been used for the synthesis of optically pure fungicide. For example, (R)-metalaxyl
synthesized using esterase-involved bioreaction displays fungicide activity, whereas (S)-enantiomer is re-
dundant. However, the biosynthesis of (R)-metalaxyl is currently hampered by the lower activity, selectivity and
thermostability of esterase. Therefore, to obtain a better biocatalyst, several esterase genes were cloned from
Pseudochrobactrum asaccharolyticum WZZ003. The esterase PAE07, among eight enzymes, was selected because it
exhibited the highest hydrolysis activity toward (R,S)-DMPM. The DNA and amino acid sequence analysis
suggested that PAE07 is a new member of lipolytic enzyme family V. The enzymatic properties of PAE07 toward
(R,S)-DMPM and model substrate (p-nitrophenyl acetate) were investigated. PAE07 was found to be a highly
active esterase with excellent enantioselectivity. The reaction conditions including temperatureand pH were
optimized, and the effects of metal ions, organic solvents and detergents were also investigated. Results indicated
that PAE07 is a competitive candidate for (R)-metalaxyl manufacturing.
1. Introduction
derivatives requires toxic or chiral catalysts [11], which are not en-
vironmentally friendly and not suitable for scale-up. The enzymatic
Esterases (EC 3.1.1.X), which catalyze esterification, ester hydro-
lysis, transesterification and acylation, are one of the most widely ap-
plied enzymes in white biotechnology [1–4]. These esterase-catalyzed
reactions have many attractive properties, such as high chemo-, regio-,
and stereo- selectivity, and do not require cofactors. Thus, esterases as
potential biocatalysts have been used to produce enantiopure com-
pounds in food, pharmaceutical and agrochemical industries [5–7].
Optically pure fungicides exhibit higher safety than the racemic
ones (different enantiomers usually demonstrate distinct biological
activities). For instance, (R)-enantiomer exhibits fungicide activity,
while (S)-enantiomer is redundant or even harmful [8,9]. Mefenoxam,
an efficient chiral-fungicide produced by Novartis has been widely used
in seed treatment, foliar spray and broadcast soil applications. This
fungicide is the (R)-enantiomer of metalaxyl fungicide (full name:
methyl N-(2,6-dimethylphenyl)-N-(methoxyacethyl)-D-alaninate). Me-
thyl(R)-N-(2,6-dimethylphenyl)-alaninate ((R)-DMPM) is the key in-
termediate to (R)-metalaxyl; therefore, o(R)-DMPM synthesis is the
research hotspot for chiral fungicide synthesis [10].
kinetic resolution process of (R,S)-DMPM is high stereoselective and
environmentally friendly but still at the laboratory research stage. A
study isolated and characterized stereoselective lipases from Bur-
kholderia sp. and identified five lipases possessing different enantios-
electivities for racemic DMPM hydrolysis [12]. Among them, lipase MC
16-3 yielded 91.8% enantiomeric excess of the product (e.e.p) of (R)-
DMPM at 25% conversion and lipase 99-2-1 yielded 91.0% e.e.p of (R)-
DMPM at 30% conversion. In another study, immobilization of lipase
PS was employed and yielded a high conversion (> 80%) and a sa-
tisfactory enantiomeric excess (96% e.e.p) [13]. However, for the large
scale-up of this bioprocess, the currently used lipase exhibits several
shortcomings such as insufficient activity, selectivity, thermostability,
and organic solvent tolerance. Therefore, a novel, highly active, highly
enantioselective, and robust esterase is greatly desired.
In a previous study, a Pseudochrobactrum asaccharolyticum WZZ003
was isolated from soil sample and identified as a candidate for (R)-
DMPM synthesis (see the route for (R)-DMPM synthesis in Fig. 1). In the
current study, the gene of esterase PAE07 was mined and cloned from P.
asaccharolyticum WZZ003, and successfully heterogeneously expressed
Traditionally, the chemical synthesis of (R)-DMPM and its
⁎
Corresponding author at: College of Biotechnology and Bioengineering, Zhejiang University of Technology, Chaowang Road 18, Hangzhou, 310014, China.
E-mail address: zhangyj@zjut.edu.cn (Y. Zhang).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.procbio.2018.03.011
Received 25 December 2017; Received in revised form 6 March 2018; Accepted 15 March 2018
1359-5113/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Cheng,F.,ProcessBiochemistry(2018),https://doi.org/10.1016/j.procbio.2018.03.011

F. Cheng et al.
ProcessBiochemistryxxx(xxxx)xxx–xxx
Fig. 1. Enzymatic resolution of (R,S)-DMPM.
in Escherichia coli BL21(DE3). This gene encoding enzyme (PAE07)
exhibited high hydrolysis activity toward (R,S)-DMPM with excellent
enantioselectivity. The enzymatic properties of PAE07 including op-
timal temperature and pH of its enzymatic reaction, and the effects of
metal ions, organic solvents and detergents were investigated. The ki-
netic parameters (kcat value and KM value) and specific activity of
PAE07 toward (R,S)-DMPM and model substrate p-nitrophenyl acetate
(p-NPA) were detected and compared with those of other excited li-
pases.
isolated from the harvested cells was transformed into competent E. coli
BL21(DE3). The E. coli BL21(DE3) harboring pEASY-blunt-pae was
grown in LB media supplemented with ampicillin at 37 °C at 200 rpm.
When the OD600 reached 0.6–0.8, isopropyl-β-D-thiogalactopyranoside
(IPTG) was added to a final concentration of 0.2 mM to induce the gene
expression at 22 °C for 8 h.
The harvested cells were resuspended in 50 mM of Tris-HCl (pH 8.0)
and disrupted by sonication and the cell lysate was centrifuged for
5 min. The resulting crude enzyme solution was loaded onto a Ni-NTA
column (1 ml, Bio Basic Inc), and the retained protein was gradually
eluted with an increasing gradient of imidazole from 10 mM to 250 mM
at a flow rate of 1 ml/min. The resulting enzyme fractions were ana-
lyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis
2. Materials and methods
2.1. Chemicals, enzyme and kits
(SDS-PAGE).
A Thermo Scientific Pierce BCA protein assay kit
(Shanghai, China) was used to quantify the proteins as previously re-
ported [15]. The purified solution was stored at −20 °C for further
characterization.
All chemicals were of analytical-reagent grade or higher quality and
were purchased from Sigma-Aldrich or Aladdin Co., Ltd. (Shanghai,
China) unless stated otherwise. Racemic methyl-(R,S)-N-(2,6-di-
methylphenyl)-alaninate and (R,S)-N-(2,6-dimethylphenyl)alanine was
purchased from Zhejiang Heben Chemical Co., Ltd (Zhejiang, China).
DEAE Sepharose™ Fast Flow and Phenyl Sepharose™ 6 Fast Flow were
purchased from GE Healthcare Co., Ltd. (Shanghai,China). TransStart™
Taq DNA Polymerase was purchased from TransGen Biotech Co., Ltd.
(Beijing, China).
2.4. Enzyme assay
The recombinant enzyme activity was measured. A total of 1 mM p-
NPA was hydrolyzed to p-nitrophenol (p-NP) at 40 °C in 50 mM of Tris-
HCl (pH 8.0) and was assayed by spectrophotometry at 405 nm. One
unit of enzyme activity (U) was defined as the amount of enzyme re-
leasing 1.0 μmol of p-NP per minute [16].
2.2. Strains and plasmids
Pseudochrobactrum asaccharolyticum WZZ003 strain was isolated
from soil sample and deposited in China Center for Type Culture
Collection (CCTCC M2014209). pEASY-blunt vector (TransGen Biotech
Co., Ltd, Beijing, China) was used for pae gene cloning and expression.
E. coli DH5α and BL21(DE3) chemically competent cells purchased
from TransGen Biotech Co., Ltd (Beijing, China) were applied to the
plasmid transformation experiment as a cloning host and an expression
host, respectively.
2.5. Substrate specificity
The substrate specificity was evaluated using p-NP esters with dif-
ferent chain lengths (C2–C16) dissolved in isopropanol to a final con-
centration of 0.33 mM. The reaction mixtures consisted of purified PAE,
0.33 mM p-NP ester and 50 mM Tris-HCl (pH 8.0). The reactions were
conducted at 35 °C for 5 min and terminated of the reactions was by
adding acetonitrile [17,18]. The enzyme activity was determined by
measuring the absorbance at 405 nm [19]. Denatured PAE07 was added
to the reaction as a negative control.
2.3. Gene cloning, heterogeneous expression, and purification of PAE07
Sequence alignment was performed using a multiple-alignment
program (ClustalW) [14]. The primer was designed according to the N-
terminal and C-terminal amino acid sequences of the predicated α/β
hydrolases from Pseudochrobactrum sp. AO18b and Pseudochrobactrum
sp. B5. PCR was conducted out as follows: denaturation of DNA tem-
plate at 94 °C for 5 min; 30 cycles at 94 °C for 30 s, 54 °C for 30 s, and
72 °C for 1 min; and final extension at 72 °C for 10 min. The PCR pro-
ducts were purified using the Genomic DNA Fragment Rapid Purifica-
tion Kit (BioDev-Tech, Beijing, China) and were inserted into a pEASY-
blunt vector. The ligated product pEASY-blunt-pae was then trans-
formed into E. coli DH5α competent cells. The plasmid pEASY-blunt-pae
2.6. Effect of pH and temperature on the activity and stability of PAE07
To investigate the effects of pH and temperature on the enzyme
activity, the initial hydrolysis rate of p-NPA was measured under var-
ious reaction conditions. The optimum pH was determined in the fol-
lowing buffers: 50 mM sodium citrate buffer (pH 3.0–5.0), 50 mM so-
dium phosphate buffer (pH 6.0–8.0), 50 mM Tris-HCl buffer (pH 9.0),
and Na2CO3-NaHCO3 (pH 10.0–11.0). The reaction mixture was agi-
tated for 2 h at 40 °C. The optimal temperature of the enzymatic reac-
tion was investigated by measuring the residual activities at 20–70 °C
for 30 min. The stability of PAE07 was measured at different
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temperature (30, 40, 50, 60 and 70 °C) for 2 h. The residual activities
were measured every 30 min using the standard enzyme assay.
retrieved from the PDB database [23]. The sequence identity of PAE07
toward 4G9E, 4G8D, and other selected esterase was determined using
ClustalW. Docking was performed using AutoDock Vina to predict the
binding energy and fine tune the ligand placement in the binding site
under the default docking parameters, in which point charges were
initially assigned according to the AutoDock semi-empirical force field
[24,25].
2.7. Kinetic study of PAE07 for the determination of KM, Vmax, and kcat
values
The reaction velocities of hydrolyzing p-NPA at different substrate
concentrations (0.01–1 mM) were determined for 5 min at 40 °C and pH
8.0. The kinetic parameters of PAE07 was calculated by adding
1.66 mg/mL of pure enzyme to each assay. The relationship between
initial velocity and substrate concentration was fitted to the
Lineweaver-Burk plot, and the Michaelis-Menten constant (KM), max-
imum velocity (Vmax), and catalytic constant (kcat) were also calculated.
3. Results and discussion
3.1. Hunting for esterase gene from the genome of P. asaccharolyticum
WZZ003
In our previous work, the WZZ003 strain that can enantioselectively
hydrolyze (R,S)-DMPM to (R)-MAP-acid was isolated from 425 soil
samples, and identified as P. asaccharolyticum based on a 16S rDNA
phylogenetic tree analysis and physiological-biochemical characteriza-
tions. Although the genome sequence of P. asaccharolyticum was not
available in the NCBI database, two genomic sequences of closely re-
lated species (Pseudochrobactrum sp. B5 and Pseudochrobactrum sp.
AO18b) were used as references for the analysis. The functional pre-
diction identified 16 (in the genomic B5) and 13 (in the genomic
AO18b) candidate genes encoding lipases/esterases, respectively.
Twenty-nine pairs of primers were designed for the amplification of the
lipase/esterase genes from the genome DNA of P. asaccharolyticum
WZZ03. Eight genes were successfully cloned from P. asaccharolyticum
WZZ003 and expressed in E. coli BL21(DE3) (data not shown). Among
them, the enzyme PAE07 displayed the highest conversion and ex-
cellent enantioselectivity (e.e. > 99%) toward (R,S)-DMPM (Fig. 1 and
Table S1). Consequently, this enzyme was selected as a potential bio-
catalyst for further studies.
2.8. Effect of metal ions, organic solvents, and surfactants on the enzyme
activity of PAE07
A concentration of metal ions (Na+, K+, Ca2+, Fe2+, Zn2+, Mg2+
,
Ba2+, Cu2+, Mn2+, Co2+, Ni2+, and Al3+) and ethylenediaminete-
traacetic acid (EDTA) were added to a final concentration of 1 and
5 mM. The reaction mixtures were incubated at 40 °C for 30 min, and
the residual enzyme activity was measured at the end of the reaction.
The solution without metal ions was regarded as a control.
The PAE07 was incubated in the presence of various organic sol-
vents (glycerol, DMSO, DMF, methanol, acetonitrile, ethanol, acetone,
isopropanol, tetrahydrofuran, n-butanol, chloroform, toluene, and n-
hexane) at 10% and 30% (v/v) in a water bath at 40 °C for 30 min, and
then the residual enzyme activity was measured.
A solution was incubated in the presence of detergents (namely,
EDTA, SDS, Tween 20, Tween 80, Triton X-100, and Urea at 1% and 5%
(v/v) for 30 min. The residual enzyme activities were assayed by the
standard enzyme assay.
3.2. DNA and amino acid sequence analysis of PAE07
2.9. Enantioselectivity toward methyl(R,S)-N-(2, 6-dimethylphenyl)-
alaninate
The gene of PAE07 contained an of 813 bp ORF encoding a 270-
amino acid protein. The nucleotide sequence of PAE07 has been de-
posited in the GenBank database (accession no. MF591571). The online
tools BlastN and BlastP were used to obtain similar sequences of this
protein. The BlastN analysis could not find any sequences with sig-
nificant similarity to the pae07 gene. The BlastP results showed that
only two amino acid sequences of proteins shared high homology
The purified PAE07 was used in catalyzing a target substrate and
compared with commercial enzymes. Enzymatic reactions were per-
formed in the mixture, which consisted of optical methyl(R,S)-N-(2,6-
dimethylphenyl)-alaninate and an enzyme solution dissolved in 50 mM
Tris-HCl (pH 8.0), at 40 °C. The hydrolytic activity and enantiomeric
excesses (e.e.) were measured using HPLC with a chiral Ultimate®
Cellud-Y column (0.5 ml/min, 4.6 × 250 mm, n-hexane/isoPrOH/
Trifluoroacetic acid = 98:2:0.1) at 220 nm. The retention times for the
enantiomers of DMPM were tS(S) = 10.406 min and tS(R) = 9.909 min,
and the resulting retention times of product were tP(S) = 32.178 min
and tP(R) = 24.283 min. One unit of enzyme activity was defined as the
amount of enzyme that produces 1 μmol/min of MAP acid.
(> 50%)
with
PAE07,
i.e.,
alpha/beta
hydrolase
from
Pseudochrobactrum sp. AO18b (WP_022712336.1) and alpha/beta hy-
drolase from Pseudochrobactrum sp. B5 (WP_075655351.1) at 92% and
91% sequence identities, respectively. The phylogenetic analysis of
esterase PAE07 was conducted based on the amino acid sequences of
different lipolytic enzymes belonging to families I–VIII obtained from
the GenBank database. As shown in Fig. 2, esterase PAE07 is in close
proximity to the lipolytic enzymes which belong to family V (lipolytic
enzymes from Streptomyces sp. and Sulfolobus acidocaldarius). The
multiple sequence alignment (Fig. S1) revealed the conserved catalytic
triad of Asp220, His249 and Ser99 and the GxHxG motif. These results
indicated that esterase PAE07 is a new member of lipolytic enzyme
family V.
2.10. Effect of substrate concentration on the hydrolysis of (R,S)-DMPM
The reactions, including 1.65 mg ml−1 of purified PAE07, 50 mM
sodium phosphate (pH = 8.0), and a series of concentration gradients
(19–200 mM), were conducted at 40 °C for 10 min. Initial reaction rate
was determined by the conversion of substrate. At a low substrate
concentration, the kinetic constants of PAE were calculated from the
Lineweaver-Burk plot.
3.3. Expression and purification of PAE07
The E. coli BL21 (DE3) cells harboring pEASY-blunt-pae07 were used
to express active PAE07. The esterase activity determination indicated
that the recombined PAE07 was successfully expressed in E. coli and the
expression level of PAE07 was estimated using SDS-PAGE analysis (Fig.
S2a). On the basis of C-terminal 6 × His affinity tag, esterase PAE07
was purified using the Ni-NTA Sepharose column. After purification,
esterase PAE07 was loaded on SDS-PAGE and was separated as a single
protein consistent with the predicted molecular weight (29.7 kDa) (Fig.
S2b).
2.11. Homology modelling and docking
A
maximum-likelihood tree was constructed using Molecular
Evolutionary Genetics Analysis software (MEGA 7) by analyzing the
relationship between PAE07 and other lipolytic enzymes [20,21]. The
homology model of PAE07 was generated using the HHpred Server and
MODELLER [22]. The crystal structures of N-acyl homoserine lactonase
with N-butanoyl homoserine (chain A of 4G9E and 4G8D) were
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Fig. 2. Maximum-likelihood phylogenetic tree. The phylogenetic analysis was conducted using the MEGA 7 software, and the esterase PAE07 is shown in red color.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Substrate specificity of PAE07 toward p-NP esters
of PAE07 was measured after incubation at 30, 40, 50, 60, and 70 °C for
at different periods. The residual activity remained approximately 90%
after 2 h of incubation at 30, 40 and 50 °C. When the incubation tem-
perature increased to 60 °C, the activity remained above 60% after 2 h
(Fig. 3d). Thus, PAE07 has good thermostability.
The substrate specificity of PAE07 was investigated using a broad
range of p-NP esters with different acyl chain lengths (C2–C16) as
substrates. PAE07 displayed the highest activity toward p-nitrophenyl
caproate (C6, 100%, set as reference) and p-nitrophenyl caprylate (C8,
83%) (Fig. S3). PAE07 exhibited 64% and 65% relative activity on C2
and C4 esters, respectively, compared with p-nitrophenyl caproate (C6
substrate) (Fig. S3). However, the hydrolytic activity of PAE07 de-
creased dramatically with the elongation of carbon chains. The relative
activity of PAE07 toward the C10 to C16 substrates were only < 30%
compared with the C6 substrate. These results indicated that PAE07 is a
“true” esterase that preferentially catalyzes the hydrolysis of short acyl
chains.
3.6. Effect of metal ions, organic solvents, and detergents on PAE07 activity
Metal ions are key factors that influence enzyme activity by inter-
acting with the disulfide bond of enzyme structure, or binding to the
substrate to form a salt [28–30]. The effects of metal ions on PAE07
were determined at 1 and 5 mM concentrations of a series of metal ions.
The activity of PAE07 was affected by many of the metal ions and was
strongly inhibited by Cu2+, Co2+, Zn2+, Fe2+, Al3+, Ag+ and Hg2+
(Table 1). Given that Ag+ and Hg2+ strongly bind to sulfhydryl re-
agents, the thiol group was essential for PAE07’s catalytic activity.
Na2+ and Ca2+ slightly increased the activity of PAE07. Nevertheless,
no inhibition was observed in the presence of chelating agent EDTA
suggesting that PAE07 have no metal ion requirements for catalysis.
Esterase an industrial biocatalyst, commonly used in non-aqueous
media such as organic solvents [31,32]. Thus, the effects of various
organic solvents with different log P values on the enzyme activity were
investigated at two different concentrations (10% and 30%, Table 2).
Most of organic solvents exhibited a certain inhibition at low con-
centration (10%), but some exhibited a positive promotion. For in-
stance, glycerol, DMSO and n-hexane weakly enhanced the enzyme
activity at high concentration (30%). The other organic solvents
showed evident negative influence.
3.5. Effect of pH and temperature on the activity and stability of PAE07
toward the model substrate
p-NPA is commonly used as a model substrate for characterization of
lipases/esterases [16,26]. The influences of pH and temperature on
PAE07 activity were investigated on p-NPA. Given that pH may affect
the protein’s conformation, the investigation of the effect of pH on the
stability of enzyme is indispensable [27]. The changes in PAE07 activity
along with a broad pH range (3.0–11.0) were shown in Fig. 3a. The
enzyme exhibited the highest activity at pH 7.0–9.0, and maximal ac-
tivity at pH 8.0. PAE07 demonstrated good pH- stability at pH 5.0–9.0.
The residual activity of PAE07 remained more than 80% after 2 h in-
cubation in this pH range (Fig. 3b).
The effects of different temperatures on the activity and stability of
PAE07 were also investigated. The optimal temperature of PAE07 to-
ward p-NPA was 40 °C, and the enzyme showed comparative activity
between 30 and 50 °C. However, the enzyme activity significantly de-
creased when temperature increased above 50 °C (Fig. 3c). The stability
The enzyme activity of PAE07 was detected with different con-
centrations of various detergents. As shown in Table 3, all surfactants
exerted a certain inhibition on its activity. Urea, Triton X-100, Tween
20, and Tween 80 showed the inconspicuous effects on PAE07 activity
at low level (1%) but exerted significant inhibition after concentration
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Fig. 3. Effect of pH and temperature on the activity and stability of PAE07. (a) Optimal pH for hydrolyzing p-NPA (sodium citrate buffer (pH 3.0–5.0), sodium
phosphate buffer (pH 6.0–8.0), Tris-HCl buffer (pH 9.0), Na2CO3-NaHCO3 (pH 10.0–11.0); (b) Enzyme stability at different pH (■ pH 3; ● pH 5; ▲ pH 7; ▼ pH 9; ♦
pH 11). (c) Optimal temperature for hydrolyzing p-NPA. (d) Thermostability was determined at different temperatures (■ 30 °C; ● 40 °C; ▲ 50 °C; ▼ 60 °C; ♦ 70 °C)
from 0.5 to 2.0 h.
increase (5%). The residual activity remained only 48% after adding
SDS. This finding revealed SDS exerted an apparent inhibition on
PAE07 activity. DTT exhibited no effect on esterase PAE07 activity.
This result suggested that the recombinant esterase has no disulfide
bonds.
3.7. Specific activity and kinetic parameters of PAE07 toward p-NP esters
The specific activity of purified PAE07 to p-NPA (the model struc-
ture) was 421.0 U/mg protein, which was ∼13-fold higher than the
specific activities of esterases from Aureobasidium pullulans (31.8 U/mg)
Table 1
Table 2
Effect of metal ions on PAE07 activity.
Effect of organic solvents on PAE07 activity.
Metal ion
% Relative activity (SD)
Organic solvents
Log Pow
% Relative activity ( SD)
1 mM
100.0
5 mM
10% (v/v)
100.0
30% (v/v)
Control
Na+
100.0
105.8
85.5
102.8
96.6
12.8
23.2
2.9
46.2
94.7
83.7
7.7
Control
Glycerol
DMSO
DMF
Methanol
Acetonitrile
Ethanol
–
100.0
103.8
110.6
31.2
18.3
8.4
91.2
93.8
98.2
97.6
18.3
75.9
13.3
80.0
95.3
93.1
29.8
71.1
101.9
5.3
8.8
5.1
9.0
4.9
1.4
1.3
5.6
1.6
1.8
1.0
1.5
5.5
5.2
6.7
1.4
3.3
0.3
−1.76
−1.35
−1.0
−0.76
−0.34
−0.3
−0.24
0.38
0.49
0.88
1.97
2.73
111.9
106.6
49.0
42.6
39.0
133.3
99.1
69.3
9.9
3.4
4.8
6.3
2.6
1.5
1.6
4.9
1.5
5.6
4.4
0.3
1.9
0.5
K+
Ca2+
Mg2+
Cu2+
Fe2+
Zn2+
Co2+
Mn2+
Ba2+
Ag+
45.2
33.7
15.6
2.4
1.2
0.7
0.4
0.1
Acetone
0.8
4.7
6.0
0.7
4.1
1.2
Isopropanol
Tetrahydrofuran
n-butanol
Chloroform
Toluene
0.9
2.9
79.3
75.7
36.4
117.1
9.7
10.7
2.3
76.9
19.9
32.2
105.9
7.9
11.7
2.8
Al3+
EDTA
24.9
98.9
0.7
n-hexane
3.5
8.6
12.5
The activity was calculated using the p-NPA assay at 40 °C and pH 8.0. The
activity of PAE07 in the non-metal ion solution was the reference.
The activity was determined using p-NPA at 40 °C and pH 8.0. The activity of
PAE07 in non-organic solvents was defined as 100%.
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Table 3
reaction was conducted under the following optimization: phosphate
buffer (pH 8.0), temperature 40 °C, 10 mM PAE07 and 50–500 mM
(R,S)-DMPM. When the substrate concentration was 50 mM, the yield of
(R)-MAP-acid could reach 49.5% only after 70 min with e.e. value of
99.9%. Even when the concentration increased to 500 mM, the con-
version could reach 49% after 10 h with e.e.p value of 99.1%. The re-
action times for conversion > 49% toward 100 mM, 250 mM and
350 mM (R,S)-DMPM were 120, 240, and 380 min, respectively, and the
e.e.p values were all more than 99%.
Effect of detergents on PAE07 activity.
Detergents
% Relative activity (SD)
1% (v/v)
100
5% (v/v)
100
None
Urea
SDS
Trtion-X100
Tween20
Tween80
DTT
87
48
88
94
95
99
7.3
4.5
6.9
4.5
6.6
3.6
65
37
57
82
87
97
4.3
5.7
5.7
3.4
4.5
6.9
3.10. Homology modelling and docking study
The activity was determined using p-NPA at 40 °C and pH 8.0. The activity of
PAE07 in non- detergents solvents was defined as 100%.
To gain the molecular understanding of PAE07 of the excellent ac-
tivity and selectivity of PAE07, a homology model of PAE07 was built
based on the crystal structures of N-acyl homoserine lactonase from
Ochrobactrum sp. T63 (PDB: 4G9E and 4G8D). The overall structure of
the PAE07 model has a two-domain architecture that contained a core
domain in the α/β-hydrolase fold and a cap domain (Fig. 4a). The core
domain consisted of an eight-stranded β-sheet surrounded by seven α-
helices and the cap domain was composed of five α-helices. The two
domains were linked by loop 122–131 and loop 196–199. The classic
catalytic triad Ser99/His249/Asp220 (Fig. 4b) of PAE07 was located at
the interface between the core and cap domains. The whole active site
is covered by the cap domain. The substrates of (R)-DMPM and (S)-
DMPM were docked in the substrate bind area (8 Å surrounding the
catalytic triad) using Autodock vina. The catalytic nucleophile residue
Ser99 in the conserved motif GxSxG was nucleophilically attacked on
the substrate ((R)-DMPM). Asp220 and His249 served as acidic and
basic members of the catalytic triad, respectively. The distances from
−OCH3 to Ser99 and from −OCH3 to His249 were 2.4 Å and 3.3 Å,
respectively. The binding energy was −7.2 kcal/mol. The docking of
(S)-DMPM into the substrate binding pocket was restricted due to the
steric hindrance by the surrounding aromatic amino acids of Tyr159,
Phe190, Phe137 and Phe222. Overall, the docking confirmation was in
good agreement withthe detected strict R-selectivity of PAE07 toward
(R,S)-DMPM.
In conclusion, a novel esterase (PAE07) gene was identified from the
genomic DNA of P. asaccharolyticum WZZ003. The PAE07 enzyme ex-
hibited high activity on C2 and C4 esters and excellent kinetic para-
meters toward p-NPA (the model substrate). The optimal temperature
and pH of PAE07 were 40 °C and 8.0, respectively for the bio-
transformation on (R,S)-DMPM to (R)-MAP acid. Furthermore, PAE07
exhibited good thermostability. The enzyme activity remains > 60%
after 2 h incubation at 60 °C. PAE07 demonstrated high catalytic effi-
ciency and specific activity toward (R,S)-DMPM and high enantios-
electivity (e.e.p > 99%). Therefore, PAE07 is a competitive candidate
for (R)-metalaxyl manufacturing.
[33] and Bacillus pumilus PS213 (32 U/mg) [34]. The KM and kcat values
of PAE07 toward p-NPA were determined as 0.66
70.2
0.03 mM and
4.3 s−1, respectively.
3.8. The specific activity and kinetic parameters of PAE07 and
commercialized enzymes toward (R,S)-DMPM
To date, only five esterases, including PAE07, have been identified
to enantioselectively hydrolyze racemic DMPM [10,13]. The specific
activity and enantioselectivity of PAE07 and other four commercialized
enzymes were evaluated on 38 mM racemic DMPM. Three out of the
five enzymes, i.e., PAE07, lipase PS, and Amano lipase AK, showed
excellent e.e.p values (99.9%), and others displayed low e.e.p values
(89.6% for Alcalase and 26.0% for Novozym 435, Table S2). PAE07
exhibited the highest specific activity (764 U/mg), which was 54.6-fold
and 127.3-fold higher than that of lipase PS (14 U/mg) and Amano li-
pase AK (6 U/mg), respectively (Table S2). The kinetic parameters of
PAE07 and commercialized lipase PS were also tested. The kcat and KM
values of PAE07 to (R,S)-DMPM were 1.08 × 107 s−1 and 34.3 mM,
respectively, and the kcat and KM values of lipase PS were
1.15 × 107 s−1 and 87.5 mM, respectively. The catalytic efficiency kcat
/
KM value of PAE07 was 315 μM−1s−1, which was ∼2.4-fold higher
than that of lipase PS (130 μM−1 s−1). To the best of our knowledge,
PAE07 had the highest activity and catalytic efficiency toward (R,S)-
DMPM among all enzymes. This result suggested that PAE07 is a
competitive biocatalyst candidate for the large-scale production of (R)-
DMPM and (R)-metalaxyl in a large scale.
3.9. Time course of enzymatic hydrolysis reaction
The time course of enantioselective hydrolysis of (R,S)-DMPM in
different concentrations by employing PAE07 is shown in Fig. S4. The
Fig. 4. Overall structure of modeled PAE07 (a) and the zoom view of its substrate binding site (b). The structure is shown in cartoon format and the key residues and
(R)-DMPM are represented in stick format.
6

F. Cheng et al.
ProcessBiochemistryxxx(xxxx)xxx–xxx
Conflict of interest
ClustalW, Cold Spring Harb. Protoc. 11 (2016) pdb.prot093088.
[15] F. Cheng, L. Zhu, H. Lue, J. Bernhagen, U. Schwaneberg, Directed arginine deimi-
nase evolution for efficient inhibition of arginine-auxotrophic melanomas, Appl.
Microbiol. Biotechnol. 99 (2015) 1237–1247.
[16] Q.Q. Tian, P. Song, L. Jiang, S. Li, H. Huang, A novel cephalosporin deacetylating
acetyl xylan esterase from Bacillus subtilis with high activity toward cephalosporin C
and 7-aminocephalosporanic acid, Appl. Microbiol. Biotechnol. 98 (2014)
2081–2089.
The authors declare no financial or commercial conflict of interest.
Acknowledgements
This study was funded by National Natural Science Foundation of
China (31600639 and 31660247), Zhejiang Provincial Natural Science
Foundation (LY18B020021), Zhejiang Provincial Qianjiang Talent
project, and Zhejiang Provincial Xinmiao Talents Project
(2017R403090).
[17] I. Navarro-González, S.F. Alvaro, G.C. Francisco, Overexpression purification, and
biochemical characterization of the esterase Est0796 from Lactobacillus plantarum
WCFS1, Mol. Biotechnol. 54 (2013) 651–660.
[18] T.C. Maester, M.R. Pereira, E.G. Machado Sierra, A. Balan, E.G. de Macedo Lemos,
Characterization of EST3: a metagenome-derived esterase with suitable properties
for biotechnological applications, Appl. Microbiol. Biotechnol. 100 (2016)
5815–5827.
[19] G. López, J. Chow, P. Bongen, B. Lauinger, J. Pietruszka, A novel thermoalk-
alostable esterase from Acidicaldus sp. strain USBA-GBX-499 with enantioselectivity
isolated from an acidic hot springs of Colombian Andes, Appl. Microbiol.
Biotechenol. 98 (2014) 8603–8616.
[20] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: molecular evo-
lutionary genetics analysis version 6.0, Mol. Biol. Evol. 30 (2013) 2725–2729.
[21] S. Kumar, G. Stecher, K. Tamura, MEGA7: molecular evolutionary genetics analysis
version 7.0 for bigger datasets, Mol. Biol. Evol. 33 (2016) 1870–1874.
[22] M.A. Marti-Renom, A. Stuart, A. Fiser, R. Sánchez, F. Melo, A. Sali, Comparative
protein structure modeling of genes and genomes, Annu. Rev. Biophys. Biomol.
Struct. 29 (2000) 291–325.
[23] A. Gao, G.Y. Mei, S. Liu, P. Wang, Q. Tang, Y.P. Liu, H. Wen, X.M. An, L.Q. Zhang,
X.X. Yan, D.C. Liang, High-resolution structures of AidH complexes provide insights
into a novel catalytic mechanism for N-acyl homoserine lactonase, Acta Crystallogr.
Sect. D 69 (2013) 82–91.
[24] R. Quiroga, M.A. Villarreal, A scoring function based on Autodock Vina improves
scoring, docking, and virtual screening, PLoS One 11 (2016) e0155183.
[25] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading, J. Comput.
Chem. 31 (2010) 455–461.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.procbio.2018.03.011.
References
[1] X.M. Wang, Y.J. Bai, Y.J. Cai, X.H. Zheng, Biochemical characteristics of three
feruloyl esterases with a broad substrate spectrum from Bacillus amyloliquefaciens
H47, Process Biochem. 53 (2016) 109–115.
[2] B.D. Ma, H.L. Yu, J. Pan, J.Y. Liu, X. Ju, J.H. Xu, A thermostable and organic-
solvent tolerant esterase from Pseudomonas putida ECU1011: catalytic properties
and performance in kinetic resolution of α-hydroxy acids, Bioresour. Technol. 133
(2013) 354–360.
[3] H. Dong, F. Secundo, C. Xue, X.Z. Mao, Whole-cell biocatalytic synthesis of cin-
namyl acetate with a novel esterase from the DNA Library of Acinetobacter hemo-
lyticus, J. Agric. Food Chem. 65 (2017) 2120–2128.
[4] L.D. Ye, H.N. Xu, H.W. Yu, Enhanced acylation activity of esterase BioH from
Escherichia coli by directed evolution towards improved hydrolysis activity,
Biochem. Eng. J. 79 (2013) 182–186.
[26] C. Lee, J. Kim, S. Hong, B. Goo, S. Lee, S.H. Jang, Cloning, expression, and char-
acterization of a recombinant esterase from cold-adapted Pseudomonas mandelii,
Appl. Biochem. Biotechnol. 169 (2013) 29–40.
[5] E. Topakas, C. Vafiadi, P. Christakopoulos, Microbial production, characterization
and applications of feruloyl esterases, Process Biochem. 42 (2007) 497–509.
[6] M.C.A. Mukdsi, C. Haro, S.N. González, R.B. Medina, Functional goat milk cheese
with feruloyl esterase activity, J. Funct. Foods 5 (2013) 801–809.
[7] L. Jiang, B. Wang, R.R. Li, S. Shen, H.W. Yu, L.D. Ye, Catalytic promiscuity of
Escherichia coli BioH esterase: application in the synthesis of 3,4-dihydropyran de-
rivatives, Process Biochem. 49 (2014) 1135–1138.
[27] A.J. Nicoll, R.K. Allemann, Nucleophilic and general acid catalysis at physiological
pH by a designed miniature esterase, Org. Biomol. Chem. 2 (2004) 2175–2180.
[28] A. Çolak, D. Şişik, N. Saglam, S. Güner, S. Çanakçi, A.O. Beldzüz, Characterization
of a thermoalkalophilic esterase from a novel thermophilic bacterium,
Anoxybacillusgonensis G2, Bioresour. Technol. 96 (2005) 625–631.
[29] Y. Yang, J. Yang, B. Li, E. Wang, H. Yuan, An esterase from Penicillium decumbens P6
involved in lignite depolymerization, Fuel 15 (2018) 416–422.
[8] B. Chankvetadze, Enantioseparation of chiral drugs and current status of electro-
migration techniques in this field, J. Sep. Sci. 24 (2015) 691–705.
[9] K. Yao, L. Zhu, Z.H. Duan, Z.Z. Chen, Y. Li, Z.S. Zhu, Comparison of R-metalaxyl and
rac-metalaxyl in acute, chronic, and sublethal effect on aquatic organisms: Daphnia
magna, Scenedesmus quadricanda, and Danio rerio, Environ. Toxicol. 24 (2009)
148–156.
[10] O.J. Park, S.H. Lee, T.Y. Park, S.W. Lee, K.H. Cho, Enzyme-catalyzed preparation of
methyl(R)-N-(2,6-dimethylphenyl)alaninate: a key intermediate for (R)-metalaxyl,
Tetrahedron. Asymmetry 16 (2005) 1221–1225.
[11] H.U. Blaser, C. Malan, B. Pugin, F. Spindler, H. Steiner, M. Studer, Selective hy-
drogenation for fine chemicals: recent trends and new developments, Adv. Synth.
Catal. 345 (2003) 103–151.
[12] O.J. Park, S.H. Lee, Stereoselective lipases from Burkholderia sp. cloning and their
application to preparation of methyl(R)-N-(2,6-dimethylphenyl)alaninate, a key
intermediate for (R)-metalaxyl, J. Biotechnol. 120 (2005) 174–182.
[13] O.J. Park, S.H. Lee, T.Y. Park, W.G. Chung, S.W. Lee, Development of a scalable
process for a key intermediate of (R)-metalaxyl by enzymatic kinetic resolution,
Org. Process Res. Dev. 10 (2006) 588–591.
[30] A. Kumar, V. Randhawa, V. Acharya, K. Singh, S. Kumar, Amino acids flanking the
central core of Cu,Zn superoxide dismutase are important in retaining enzyme ac-
tivity after autoclaving, J. Biomol. Struct. Dyn. 34 (2016) 475–485.
[31] J. Kim, S. Kim, S. Yoon, E. Hong, Y. Ryu, Improved enantioselectivity of thermo-
stable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by
directed evolution and characterization of mutant esterases, Appl. Microbiol.
Biotechnol. 99 (2015) 6293–6301.
[32] C. Chen, Zhe Chi, G. Liu, H. Jiang, Z. Hu, Z. Chi, Production, purification, char-
acterization and gene cloning of an esterase produced by Aureobasidium melano-
genum HN6.2, Process Biochem. 53 (2017) 69–79.
[33] K. Rumbold, P. Biely, M. Mastihubová, M. Gudelj, G. Gübitz, K.H. Robra, B.A. Prior,
Purification and properties of a feruloyl esterase involved in lignocellulose de-
gradation by Aureobasidium pullulans, Appl. Environ. Microbiol. 69 (2003)
5622–5626.
[34] G. Degrassi, B.C. Okeke, C.V. Bruschi, V. Venturi, Purification and characterization
of an acetyl xylan esterase from Bacillus pumilus, Appl. Environ. Microbiol. 64
(1998) 789–792.
[14] J.H. Hung, Z. Weng, Sequence alignment and homology search with BLAST and
7