Molecular modification of a halohydrin dehalogenase for kinetic regulation to synthesize optically pure (S)-epichlorohydrin

Article abstract of DOI:10.1016/j.biortech.2018.12.103

Asymmetric synthesis of chiral epichlorohydrin (ECH) from 1,3-dichloro-2-propanol (1,3-DCP) using halohydrin dehalogenases (HHDHs) is of great value due to the 100% theoretical yield and high enantioselectivity. The vital problem in the asymmetric synthesis is to prepare optically pure ECH. In this study, key amino acid residues located at halide ion channels of HheC (P175S/W249P) (HheC_PS) were modified to regulate the kinetic parameters. HheC_PS I81W, F86N and V94R were constructed with the corresponding halide ion channels destroyed. The catalytically efficiencies (k_cat/K_m) of the three mutants exhibited 0.38-, 0.23- and 0.23-fold decrease toward (S)-ECH and the reverse reaction was significantly inhibited. As the results, (S)-ECH was synthesized with >99% enantiomeric excess (e.e.) and 63.42%, 67.08% and 57.01% yields, respectively, under 20 mM 1,3-DCP as substrate. To our knowledge, this is the first investigation of the molecule kinetic modification of HHDHs and also the first report for the biosynthesis of optically pure (S)-ECH from 1,3-DCP using HHDHs.

Full text of DOI:10.1016/j.biortech.2018.12.103

Bioresource Technology 276 (2019) 154–160
Contents lists available at ScienceDirect
Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Molecular modification of a halohydrin dehalogenase for kinetic regulation
to synthesize optically pure (S)-epichlorohydrin
⁎
Xiao-Jian Zhang, Han-Zhong Deng, Nan Liu, Yi-Chuan Gong, Zhi-Qiang Liu , Yu-Guo Zheng
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China
G R A P H I C A L A B S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords:
Asymmetric synthesis of chiral epichlorohydrin (ECH) from 1,3-dichloro-2-propanol (1,3-DCP) using halohydrin
dehalogenases (HHDHs) is of great value due to the 100% theoretical yield and high enantioselectivity. The vital
problem in the asymmetric synthesis is to prepare optically pure ECH. In this study, key amino acid residues
located at halide ion channels of HheC (P175S/W249P) (HheCPS) were modified to regulate the kinetic para-
meters. HheCPS I81W, F86N and V94R were constructed with the corresponding halide ion channels destroyed.
Halohydrin dehalogenase
Optically pure chiral epichlorohydrin
Kinetic regulation
(
S)-Epichlorohydrin
Halide ion channel
The catalytically efficiencies (kcat/K
m
) of the three mutants exhibited 0.38-, 0.23- and 0.23-fold decrease toward
(
S)-ECH and the reverse reaction was significantly inhibited. As the results, (S)-ECH was synthesized with > 99%
enantiomeric excess (e.e.) and 63.42%, 67.08% and 57.01% yields, respectively, under 20 mM 1,3-DCP as
substrate. To our knowledge, this is the first investigation of the molecule kinetic modification of HHDHs and
also the first report for the biosynthesis of optically pure (S)-ECH from 1,3-DCP using HHDHs.
1. Introduction
building block for various transformations in pharmaceutical and or-
ganic synthesis, such as β-adrenergic blocking agents, atorvastatin, L-
Biodiesel is a biological alternative energy and boasts a huge market
carnitine, and trehalostatin etc. (Liu et al., 2011; Elenkov et al., 2018;
Wu et al., 2010). Asymmetric synthesis of chiral ECH using HHDHs has
attracted much attention because of the low catalyst cost, mild reaction
conditions and 100% theoretical yield (Schallmey et al., 2013, 2014;
Shen et al., 2018; Zheng et al., 2017).
needs, but the huge demand leads to the accumulation of by-product
glycerol with an inefficient use (Duarte et al., 2015; Shibasaki-Kitakawa
et al., 2013). It is reported that 1,3-DCP which is from glycerol can be
transformed into chiral ECH by a biological enzymatic method in an
effective way (Xue et al., 2015a). Chiral ECH is an important versatile
HHDHs (EC 4.5.1.X) are enzymes to catalyze the reversible
⁎
Corresponding author at: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of
Technology, Hangzhou 310014, China.
E-mail address: microliu@zjut.edu.cn (Z.-Q. Liu).
https://doi.org/10.1016/j.biortech.2018.12.103
Received 16 November 2018; Received in revised form 24 December 2018; Accepted 26 December 2018
Available online 27 December 2018
0960-8524/ © 2018 Elsevier Ltd. All rights reserved.

X.-J. Zhang et al.
Bioresource Technology 276 (2019) 154–160
dehalogenation of vicinal halohydrins to form epoxides and the corre-
sponding halide ions and H+ through a molecular kernel substitution
mechanism (Hu et al., 2015; Tang et al., 2003; Xue et al., 2015b). In the
2.3. Homology modeling and molecular docking simulation
The three-dimension (3-D) structures of the HHDHs were con-
structed using Modeller 9.19 program (http://www.salilab.rog) and
based on the crystal structure of HheC from Agrobacterium radiobacter
AD1 (PDB ID: 1ZO8). The quality of the final structure models were
evaluated using the Procheck program (http://www.ebi.ac.uk/
thornton-srv/databases/pdbsum/Generate.html). The halide ion chan-
nels were predicted by MOLE 2.0 (https://webchem.ncbr.muni.cz/
Platform/App/Mole). The sequence alignment was carried out by
ESPript 3.0 (http://espript.ibcp.fr/ESPript/ESPript/). The molecular
docking procedure was performed according to the program of
AutoDock 4.2.1 (Scripps Res. Inst., La Jolla, CA). ChemDraw 8.1
(CambridgeSoft, Cambridge, UK) was used to draw the 3D structures of
1,3-DCP and (S)-ECH. All the structures were visual analyzed using the
PyMOL program (http://www.pymol.org).
–
–
–
–
–
−
2 3
presence of negative ions such as NO , N , OCN , HCOO , CN , SCN ,
−
−
Br and Cl , the reverse reaction is occurred to convert the epoxide
rings into the corresponding β-substituted alcohols (Lin et al., 2011;
Spelberg et al., 2001). Different approaches have been tried to prepare
chiral ECH with high optical purity. It is reported that (R)-ECH was
prepared with the e.e. > 92.3% by HheB8 from Bradyrhizobium ery-
throphlei (Xue et al., 2018). In our previous work, a mutant HheCPS was
constructed with high enantioselectivity and the e.e. of (S)-ECH
changed greatly from 5% to 95.3%, compared with the parent HheC
(
Xue et al., 2015b). Recently, HheCPS was immobilized and employed in
non-aqueous bioconversion system to obtain (S)-ECH with 98% e.e. and
2.34% conversion (Zhang et al., 2018). However, the optical purity of
5
chiral ECH was not high enough (> 99%) for industrial application,
and as the reaction prolonged, the chiral ECH became racemic.
Due to the difference in the affinity of a HHDH to (R)- or (S)-ECH,
the reverse reaction of asymmetric conversion from 1,3-DCP to chiral
ECH is responsible for the conversion of (S)-ECH to 1,3-DCP and (R)-
ECH, which is considered as the key factor for the low optical purity of
chiral ECH products (Tang et al., 2010; Wan et al., 2018; Xue et al.,
2.4. Construction of the mutagenesis libraries
The HheCPS plasmid was isolated using the AxyPrep Plasmid
Miniprep Kit and was used as the template. Key amino acid residues
which were predicted to be located at halide ion channels were selected
to be replaced by random amino acids with the target codon of NNS
(N = A/T/C/G in a 1:1:1:1 ratio, S = C/G in a 1:1 ratio) by polymerase
chain reaction (PCR). The PCR was performed with Phanta Max Super-
Fidelity DNA polymerase using the following program: 95 °C for 3 min,
15 s at 95 °C, annealing at 55 °C for 15 s, 3 min 30 s at 72 °C, step 2 to
step 4 were repeated for 30 cycles, and followed by a final extension at
72 °C for 10 min (Cheng et al., 2018; Wang et al., 2018). The PCR
products were determined using gel electrophoresis. Subsequently, PCR
cleanup kit was used to purify the PCR products (Liu et al., 2017c,
2018b). After digestion with DpnI at 37 °C for 3 h, the products were
transformed into E. coli BL21(DE3) competent cells and cultivated on
agar/kanamycin LB plates, respectively.
2015a). The modification of the kinetic parameters of HHDHs for the
forward and reverse reactions would affect the synthesis of (S)- or (R)-
ECH and thus define the optical purity of chiral ECH. Currently, most
mutagenesis focused on the active sites to improve the enantioselec-
tivity, and for preparing optically pure ECH, no report has been pub-
lished about the molecular modification to alter the kinetic parameters
of a HHDH for both forward and reverse reactions.
In the present study, the amino acid residues located at the halide
ion channels of HheCPS were predicted by specialized toolkit MOLE 2.0
and site-directed saturation mutagenesis was carried out to modify the
kinetic characters of the enzyme. The positive mutants were employed
for the synthesis of (S)-ECH with > 99% e.e. To our knowledge, this is
the first investigation of the molecule kinetic modification of a HHDH
and also the first report for the biosynthesis of optically pure (S)-ECH
from 1,3-DCP using HHDH as catalyst.
2.5. Screening of mutagenesis libraries
The colonies of the mutant libraries and controls were inoculated
into a 96-deep-well plate containing 1 mL LB medium with 50 µg/mL
Kan and cultivated at 37 °C for 8 h. The seed solutions were transferred
into another new 96-deep-well plate containing 1 mL LB medium with
2
. Materials and methods
.1. Materials
,3-DCP, (S)-ECH and (R)-ECH were purchased from J & K Scientific
2
50 µg/mL Kan and 0.1 mM IPTG (final concentration) to induce protein
expression at 28 °C for 12 h. Cells were collected by centrifugation and
suspended in 200 mM sodium phosphate buffer at pH 8.0 with 20 mM
1
Ltd. (Shanghai, China). Phanta Max Super-Fidelity DNA Polymerase
and DpnI methylation-sensitive restriction enzyme were purchased from
Vazyme Biotech Co., Ltd. (Nanjing, China). AxyPrep Plasmid Miniprep
Kit was purchased from Axygene Biotech Ltd. (Hangzhou, China).
Kanamycin (Kan) and the inducer isopropyl β-D-thiogalactoside (IPTG)
were purchased from Sango Biotech Co., Ltd. (Shanghai, China). PCR
cleanup kit was purchased from Axygen Scientific, Inc. (Jiangsu,
China). Yeast extract, tryptone and other reagents were of analytical
grade purity and were commercially available.
1
,3-DCP (final concentration). At the sampling time points, the reac-
tions were terminated by sulfuric acid immediately. The products were
extracted by ethyl acetate and prepared for chiral GC analysis. After
screening, positive mutants with high enantioselectivity and activity
were selected for DNA sequencing and further experimental researches.
2.6. Expression and purification of enzyme
The recombined E. coli BL21(DE3) cells containing positive mutant
genes were cultured and all the HheCPS proteins were induced and
purified according to our previous reports (Xue et al., 2015b; Zhang
et al., 2018). One unit (U) of activity was defined as the amount of
enzyme necessary for producing 1 μmol of ECH from 1,3-DCP in 1 min
under the standard assay conditions (Liu et al., 2014). The secondary
structures of HheCPS and HheCPS mutants were determined using a
circular dichorism (CD) equipment (Applied Photophysics Ltd.,
Britain).
2
.2. Strains and growth condition
Escherichia coli BL21(DE3) (Invitrogen, Carlsbad, CA, USA) was used
as the host cell. The pET-28b(+) plasmid (Novagen, Darmstadt,
Germany) was used for gene expression. Recombinant E. coli
BL21(DE3)/pET28b-HheCPS containing a 6*His tag tail was constructed
for the expression of halohydrin dehalogenase gene HheCPS (Xue et al.,
2
1
5
015b). Lysogeny broth (LB) medium containing 5 g/L of yeast extract,
0 g/L of tryptone, and 10 g/L of NaCl with the final concentration of
0 μg/L Kan was employed for the recombinant E. coli cells cultivation
2.7. Kinetic characteristics of HheC mutants
(
Huang et al., 2018; Liu et al., 2017a, 2018a). IPTG was used as the
To study the kinetic parameters of HheCPS and the HheCPS mutants,
inducer for protein expression (Liu et al., 2017b).
reactions were performed in 200 mM sodium phosphate buffer at pH
155

X.-J. Zhang et al.
Bioresource Technology 276 (2019) 154–160
8
.0 containing purified enzymes at 37 °C respectively. Forward and
analysis and halide ion channel prediction.
.2. Screening of the mutagenesis libraries
Site-saturation mutagenesis of residues Ile81, Phe82, Glu85, Phe86,
reverse reactions with different concentrations of 1,3-DCP or (S)-ECH at
different time points were analyzed by chiral GC. The Vmax and K
calculated using the Lineweaver-Burk plot and the kcat and kcat/K
obtained from Vmax and enzyme concentration [E].
m
were
were
3
m
Val94, Leu178 and Phe186 were performed. Mutagenesis libraries with
over 40,000 colonies were generated and were screened for beneficial
mutants with high enantioselectivity and activity.
Km
Vmax [S]
1
1
Vmax
v =
+
where [S] was the concentration of 1,3-DCP for the forward reaction or
the concentration of (S)-ECH for the reverse reaction.
Based on DNA sequencing and chiral GC analysis, three mutants
(
HheCPS I81W, F86N and V94R) of HheCPS were identified and dis-
played the highest enantioselectivity (> 99% e.e. towards (S)-ECH),
while 95.3% e.e. of (S)-ECH was observed using HheCPS. Compared
with the parent enzyme (relative activity was defined as 100%), the
relative activity of three mutants decreased from 100% to 37.65%,
2
.8. Asymmetric synthesis of (S)-ECH
The asymmetric reactions were performed in 10 mL of 200 mM so-
dium phosphate buffer at pH 8.0 with different HHDH enzymes and
different concentration of 1,3-DCP at 37 °C. Reaction solution was taken
out at different time points and mixed immediately with sulfuric acid to
terminate the reaction. Ethyl acetate was added to extract the products.
The extracted samples were detected using chiral GC.
41.66% and 27.03%, respectively, in a 5-min reaction. Then further
investigations of the combined mutagenesis of HheCPS I81W/F86N,
HheCPS I81W/V94R, HheCPS F86N/V94R and HheCPS I81W/F86N/
V94R were performed. No mutant enzyme with high enantioselectivity
or activity was detected. The secondary structures of HheCPS and
HheCPS mutants were also analyzed by CD. Results showed that there
was no significant difference among them in secondary structures.
2.9. Chiral GC analysis
1,3-DCP, (R)-ECH and (S)-ECH were determined using a chiral ca-
3.3. Stability assay of (S)-ECH
pillary BGB-175 column (0.25 mm × 30 m × 0.25 µL film thickness)
and an Agilent-7890 gas chromatography equipped with an FID de-
tector. The analytical method was carried out with the following pro-
gram: the initial column temperature was set at 60 °C and the inlet and
FID detector temperatures were both 220 °C. The retention times were
as follows: 6.5 min for (S)-ECH, 6.6 min for (R)-ECH, and 9.5 min for
To explore the effects of three HheCPS mutants on the reverse re-
action, the stability of (S)-ECH was investigated in the present of dif-
−
ferent HheCPS enzymes. Reaction systems with and without Cl were
used as the controls. As shown in Fig. 1, (S)-ECH in all solutions in-
cubated with HheCPS and HheCPS mutant enzymes displayed downward
trends. The control sample also showed slight spontaneous hydrolysis
and racemization. At the end of the 6-hour incubation, the remaining
concentration of (S)-ECH with HheCPS I81W and HheCPS F86N and
HheCPS V94R showed 1.91-, 1.90- and 1.25-fold higher respectively,
than that with the parent protein. The trends were similar to the
1,3-DCP. The e.e. was derived from the two enantiomers [e.e.
(
%) = (S − R)/(S + R) × 100] (Zhang et al., 2018). Optical rotation
was measured on an Autopol IV automatic polarimeter (Rubolph Re-
search Analytical, Hackettstown, NJ, USA).
2.10. Statistical analysis
2
0
Unless otherwise noted, all the experiments in this study were
performed in triplicates. An analysis of variance (ANOVA) was carried
out using the SAS program version 8.1 (SAS Institute Inc., Cary, NC,
USA). The least significant difference (LSD) was computed at p < 0.05.
All figures in this study were drawn using origin software version 8.0
18
1
1
6
4
(
Origin Lab Corp., Northampton, MA, USA).
12
1
0
8
6
4
2
0
Treatment 1
Treatment 2
Treatment 3
Treatment 4
Treatment 5
Treatment 6
3
. Results and discussion
3.1. Homology modeling and docking
HheCPS with high enantioselectivity was employed as the parent
enzyme for molecular modification in the present study. The 3-D
structure model for HheCPS was generated using Modeller 9.19 program
according to HheC crystal structure (PDB ID: 1ZO8) and was evaluated
by Procheck (de Jong et al., 2002). The simulated model with the
highest GA341 score and the lowest DOPE score was selected for further
research. Halide ion channels of the protein were predicted by MOLE
1
h
2h 4h 6h
0
min 2min 5min
10min15min30min
Time of Reaction
2
.0, the probe radius was set as 0.5 Å; the shell radius was set as 3.0 Å,
Fig. 1. The comparisons of stability of (S)-ECH under different conditions.
Treatment 1: 20 mM (S)-ECH was incubated in 10 mL 200 mM sodium phos-
phate buffer at pH 8.0. Treatment 2: 20 mM (S)-ECH and 20 mM NaCl were
incubated in 10 mL 200 mM sodium phosphate buffer at pH 8.0. Treatment 3:
and the shell depth was 16.0 Å.
Halide ion channels closed to the catalytic triad (Ser132-Tyr145-
Arg149) were determined and colored as red, blue and green (the
common part colored as yellow), respectively. 7 amino acid residues,
Ile81, Phe82, Glu85, Phe86, Val94, Leu178 and Phe186, located at
three different halide ion channels were considered as the key residues
for the formation the halide ion channels and all the amino acid re-
sidues were far away from the halide-binding site (Asn176, Tyr187 and
Trp249). Although the 7 residues were not highly conserved based on
amino acid sequence alignment, they were still selected for the con-
struction of mutagenesis libraries according to the 3-D structure
0
2
.2 g HheCPS cells, 20 mM (S)-ECH and 20 mM NaCl were incubated in 10 mL
00 mM sodium phosphate buffer at pH 8.0. Treatment 4: 0.2 g HheCPS I81W
cells, 20 mM (S)-ECH and 20 mM NaCl were incubated in 10 mL 200 mM so-
dium phosphate buffer at pH 8.0. Treatment 5: 0.2 g HheCPS F86N cells, 20 mM
(S)-ECH and 20 mM NaCl were incubated in 10 mL 200 mM sodium phosphate
buffer at pH 8.0. Treatment 6: 0.2 g HheCPS V94R cells, 20 mM (S)-ECH and
2
8
0 mM NaCl were incubated in 10 mL 200 mM sodium phosphate buffer at pH
.0.
156

X.-J. Zhang et al.
Bioresource Technology 276 (2019) 154–160
Table 1
Kinetics constants of the HheCPS and HheCPS mutants towards 1,3-DCP and (S)-ECH.
Enzyme
1,3-DCPa
(S)-ECHb
K
m
V
max
k
cat
k
cat/K
m
K
(mM)
m
V
max
k
cat
kcat/K
m
(µmol min−1 mg−1
−
1
)
(s−1)
(mM s−1
−1
)
(µmol min−1 mg−1
−1
)
(s−1)
(mM s−1
)
−1
(mM)
HheCPS
20.8
19.3
16.8
15.8
42.7
34.1
25.9
12.0
17.3
14.2
10.8
5.0
0.83
0.73
0.64
0.32
1.38
4.01
7.31
6.75
0.42
0.49
0.51
0.57
0.18
0.20
0.21
0.24
0.13
0.05
0.03
0.03
HheCPS I81W
HheCPS F86N
HheCPS V94R
a
Reactions were performed in 200 mM sodium phosphate buffer at pH 8.0 and 1,3-DCP concentration was varied in range of 10–100 mM.
Reactions were performed with Cl in 200 mM sodium phosphate buffer at pH 8.0 and (S)-ECH concentration was varied in range of 10–100 mM.
b
−
previously report (Xue et al., 2015a), and mutant enzymes obtained in
this study exhibited excellent capability to preserve (S)-ECH with high
optical purity.
20 mM substrate was used, (S)-ECH was synthesized with > 99% e.e.
and 63.42% yield catalyzed by HheCPS I81W in a 15-min reaction. High
e.e. (> 99%) of (S)-ECH and 67.08% yield were obtained with HheCPS
F86N in a 20-min reaction. For HheCPS V94R as the catalyst, e.e. of (S)-
ECH > 99% and 57.01% yield were obtained in a 25-min reaction. As
the concentration of 1,3-DCP increased to 40 mM, for HheCPS
I81W, > 99% e.e. and 42.20% yield of (S)-ECH were observed; > 99%
e.e. and 41.77% yield were obtained by HheCPS F86N; for HheCPS
V94R, > 99% e.e. and 28.50% yield of (S)-ECH were obtained. When
the concentration of 1,3-DCP was even higher, the e.e. and yield of (S)-
ECH decreased significantly. But for HheCPS F86N, the ability to syn-
thesize optically pure ECH was better than other mutants, when the
reaction was carried out at the 1,3-DCP concentration of
3.4. Kinetic parameters of HheCPS mutants
The kinetic parameters of HheCPS and HheCPS mutants towards 1,3-
DCP and (S)-ECH were determined to evaluate the forward and reverse
reaction performance. Compared with the parent enzyme, the apparent
kinetic constants (Km(1,3-DCP)) for HheCPS I81W, HheCPS F86N and
HheCPS V94R showed 0.93-, 0.81- and 0.76-fold decreased, respec-
tively. The catalytic number (kcat(1,3-DCP)) of HheCPS I81W and HheCPS
V94R were 0.82- and 0.29-fold decreased and the catalytic efficiency
70 mM, > 99% e.e. and 25.13% yield were obtained while no optically
(
k
cat(1,3-DCP)/Km(1,3-DCP)) were 0.88- and 0.39-fold lower, respectively.
For HheCPS F86N, 0.62-fold lower in catalytic number (kcat(1,3-DCP)) and
.77-fold lower in catalytic efficiency (kcat/K were observed
Table 1).
To illustrate the difference in ring-opening reaction between HheCPS
pure (S)-ECH were generated by HheCPS I81W and HheCPS V94R. In all
the reactions with different concentrations of 1,3-DCP catalyzed by
parent HheCPS, (S)-ECH with > 99% e.e. was not observed.
To further investigate the optical purity of (S)-ECH catalyzed by the
mutants, long term reactions were performed under the substrate con-
centration of 20 mM. As shown in Fig. 2, for the first 15 min, > 99% e.e.
of (S)-ECH was observed for the mutant enzyme of HheCPS I81W,
HheCPS F86N and HheCPS V94R with the yields of 62.32%, 66.65% and
0
m
)
(
and HheCPS mutants, kinetic studies of the reverse reaction were per-
formed. As shown in Table 1, the apparent kinetic constants (Km((S)-
ECH)) of HheCPS I81W, HheCPS F86N and HheCPS V94R were 2.91-, 5.30-
and 4.89-fold higher and the catalytic efficiency (kcat((S)-ECH)/Km((S)-
ECH)) were decreased to 0.38-, 0.23- and 0.23-fold respectively, com-
pared with the parent enzyme. HheCPS mutants effectively suppressed
the catalytic ability of the (S)-ECH in reverse reaction.
5
6.69% respectively, while the 58.32% e.e. and 79.89% yield of (S)-
ECH were obtained for the parent enzyme. As the reaction prolonged,
the e.e. decreased slightly for three HheCPS mutants, while for HheCPS
the e.e. and yield decreased rapidly. After 1-hour reaction, the e.e. of
S)-ECH were 82.29%, 86.25% and 77.74% for HheCPS I81W, HheCPS
F86N and HheCPS V94R, respectively, and the yields were 95.13%,
6.24% and 73.94%, while chiral ECH was racemized for the parent
enzyme within 1-hour reaction.
,
For the forward and reverse reactions, HheC displayed different
catalytic capabilities due to the different kinetic parameters towards
(
1,3-DCP and (S)-ECH. The E values which were defined as [kcat(1,3-DCP)
/
9
K
m(1,3-DCP)]/[kcat((S)-ECH)/Km((S)-ECH)] were largely changed in HheCPS
mutant proteins and were 14.6, 21.3 and 10.7 for HheCPS I81W, HheCPS
F86N and HheCPS V94R respectively, while the E value for HheCPS was
6
.38. Because of the regulation of catalytic efficiencies towards 1,3-DCP
3
.6. Molecule mechanism for kinetic regulation
and (S)-ECH, the reverse reaction rates were significantly inhibited in
the three mutants, while the forward reaction rates still kept in the
considerable levels. Thus the ring opening and racemization of (S)-ECH
were largely reduced. The kinetic mechanisms of HHDHs have been
reported regularly recent years (Schallmey et al., 2013; Xue et al.,
To explore the mechanism for the kinetic regulation of HheCPS
mutants, the structure models were constructed using Modeller 9.19
and were evaluated to be with high quality by Procheck. The channels
of HheCPS and HheCPS mutants were predicted by MOLE 2.0. 1,3-DCP
and (S)-ECH were docked into the active sites of the HheCPS, HheCPS
I81W, HheCPS F86N and HheCPS V94R, respectively, using AutoDock
2
015b; Tang et al., 2005). However, no investigation has been pub-
lished about the relationship of halide ion channels and kinetic para-
meters towards 1,3-DCP and chiral ECH. And the present work is the
first report about the molecule modification to regulate the forward and
reverse reactions. The molecular kinetic modified mutants were sub-
sequently employed for asymmetric synthesis of optically pure ECH.
4
.2.1.
In the parent protein, Ile81, Ala83 and Trp185 formed the cavity to
−
promote the Cl releasing or entering in the forward or reverse reaction
Fig. 3(a)). In HheCPS I81W, the existing cavity was closed by the indole
(
group of Trp81 (Fig. 3(b)). Consequently, the channel was destroyed.
−
3.5. Asymmetric synthesis of (S)-ECH
Due to the difference of Cl concentration in the substrate pocket and
−
the surrounding solution, Cl could be still easily released though other
HheCPS mutants were employed to prepare (S)-ECH with different
channels for the forward reaction. While for the reverse reaction, the
rate of Cl entering was decreased. The docking results showed that the
distances between the hydroxyl hydrogen atom of Ser132 and the hy-
droxyl oxygen atom of 1,3-DCP, the hydroxyl hydrogen atom of 1,3-
DCP and the phenolic hydroxyl oxygen atom of Tyr145, the oxygen
atom of (S)-ECH and the hydroxyl hydrogen atom of Ser132, the oxygen
−
concentrations of 1,3-DCP. As shown in Table 2, when the 1,3-DCP
concentration was 10 mM, the e.e. of (S)-ECH catalyzed by HheCPS
I81W and HheCPS F86N were all > 99%, and the yields were 74.36%
and 79.12% respectively. For HheCPS V94R, the e.e. of > 99% and the
yield of 60.21% for (S)-ECH were obtained in a 25-min reaction. When
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Bioresource Technology 276 (2019) 154–160
Table 2
Biosynthesis of (S)-ECH using HheCPS and HheCPS mutants at different concentrations 1,3-DCP.
Concentration/mM
HheCPS
HheCPS I81W
HheCPS F86N
HheCPS V94R
e.e./%
Yield/%
e.e./%
Yield/%
e.e./%
Yield/%
e.e./%
Yield/%
1
2
4
5
7
1
0
0
0
0
0
00
92.1 ± 0.36
92.7 ± 0.28
95.3 ± 0.72
92.5 ± 1.01
91.3 ± 0.44
90.4 ± 1.21
94.2 ± 0.31
92.7 ± 0.14
93.7 ± 0.59
92.1 ± 0.92
91.3 ± 0.35
90.4 ± 1.36
> 99
> 99
> 99
> 99
90.43 ± 0.23
87.81 ± 1.36
74.36 ± 0.42
63.42 ± 2.01
42.20 ± 0.23
31.03 ± 0.33
33.15 ± 1.21
25.28 ± 1.01
> 99
> 99
> 99
> 99
> 99
89.05 ± 0.63
79.12 ± 0.45
67.08 ± 0.21
41.77 ± 2.01
36.25 ± 0.42
25.13 ± 0.01
23.66 ± 0.52
> 99
> 99
> 99
> 99
> 99
89.1 ± 0.21
60.21 ± 0.63
57.01 ± 0.31
28.50 ± 2.32
22.80 ± 0.01
6.78 ± 0.32
6.23 ± 0.14
In the site of Phe86, the hydrophobic benzene ring constituted part
of the yellow halide ion channel. The substitution of phenylalanine to
asparagine caused the replacement of benzene ring to a small acyla-
mino group and a gap closed to the catalytic triads was generated. A
new channel passed through the gap and exposed to the medial surface
of the tetramer enzyme. As the result, the efficient ion release me-
chanism became confusing (Fig. 3(d)). For the docking model of the
enzyme and 1,3-DCP, the distance from the hydroxyl hydrogen atom of
1,3-DCP to the phenolic hydroxyl oxygen atom of Tyr145 was extended
from 3.8 Å to 4.5 Å compared with the model of the parent protein,
which led to the decrease in the catalytic efficiency of the forward re-
action. The distance from the hydroxyl hydrogen atom of Ser132 to the
1
00
a
8
6
4
2
0
0
0
0
0
HheC
PS
HheC I81W
PS
HheC F86N
PS
HheC V94R
PS
1,3-DCP hydroxyl oxygen atom was shortened from 3.1 Å to 2.8 Å,
leading to a decreased apparent kinetic constant. For the docking model
of the enzyme and (S)-ECH, the distance between the oxygen atom of
-
20
(
S)-ECH and the phenolic hydroxyl oxygen atom of Tyr145 increased
from 2.7 Å to 2.8 Å, and the distance between the hydroxyl hydrogen
atom of Ser132 and the oxygen atom of (S)-ECH changed from 3.4 Å to
1
h
2h
4h
6h
2
min 5min
1
5min 30min
3
m
.9 Å, which increased the apparent kinetic constant K of reverse re-
Time of Reaction
action. All the results were in agreement with the analysis of kinetics
parameters of HheCPS F86N.
As shown in Fig. 3(e), Val94 was located at the end of the green ion
channel and a gap for ions exiting was formed by Val94, Glu95 and
Arg98. When the amino acid changed from Val to Arg (Fig. 3(f)), the
gap was blocked by the protrusion of Arg, resulting in the disturbing of
efficiency of the halide ion channel. Moreover, Arg is positively
charged, the electric potential between the active site and the protein
surface was altered and the negative charged halide ions could not
enter the active site through the green ion channel. This conjecture was
1
00
b
8
6
4
2
0
0
0
0
0
m
also consistent with the increased in K when the reaction catalysed by
HheC
HheCPS V94R. The substitution of residue at 94 site was far from sub-
strate pocket, and there was no significant effect in the docking of the
enzyme and 1,3-DCP or (S)-ECH.
PS
HheC I81W
PS
HheC F86N
PS
HheC V94R
PS
1,3-DCP could be industrially prepared from glycerol and the
asymmetric synthesis of 1,3-DCP to chiral ECH makes competitive
utilization of glycerol. As the by-product of biodiesel, glycerol from
biodiesel is manufactured abundantly and the researches on the bio-
transfermation of 1,3-DCP into chiral ECH accelerated the efficient use
of glycerol (Duarte et al., 2015; Shibasaki-Kitakawa et al., 2013).
Asymmetric synthesis of chiral ECH using HHDH has been widely in-
vestigated due to high enantioselectivity, 100% theoretical yield, low
production cost and environmental friendly conditions. 89% e.e. of (R)-
ECH was obtained by HHDH from Arthrobacter erithii H10a (Assis et al.,
1
h
2h
4h
6h
2
min 5min
1
5min 30min
Time of Reaction
Fig. 2. The comparisons of e.e. (a) and yield (b) of (S)-ECH catalyzed by
HheCPS, HheCPS I81W, HheCPS F86N and HheCPS V94R.
1
998). HHDHTm catalyzed the preparation of (S)-ECH with over 60%
atom of (S)-ECH and the phenolic hydroxyl oxygen atom of Tyr145
e.e. and 95.2% conversion (Xue et al., 2015a). Molecule modification
and directed evolution technologies were employed to improve the
enantioselectivity of HHDHs. HheCPS was constructed with high en-
antioselectivity in our previous work, and 1,3-DCP was converted to
changed slightly.
−
Besides, for the interaction between (S)-ECH and the enzyme, Cl
was considered as an important factor for the substrate affinity as well
as the stability of the substrate-enzyme complex (Hasnaoui-Dijoux
et al., 2008; Schallmey et al., 2013) and thus effect the K of HheCPS
m
(
S)-ECH with 95.3% e.e. and 93.2% yield (Xue et al., 2015b).
The reverse reaction was proposed as the main reason for the low
I81W towards (S)-ECH. The changes in reaction rates for the forward
and reverse reactions of HheCPS I81W were mainly attributed to the
obstruction of the halide ion channel. All the results were in agreement
with the analysis of kinetic parameters.
optical purity of ECH, which coursed the optical purity of chiral ECH
decreased gradually as the forward reaction going on (Tang et al., 2010;
Xue et al., 2015b). Different approaches on reaction system to inhibit
158

X.-J. Zhang et al.
Bioresource Technology 276 (2019) 154–160
Fig. 3. The halide ion channel prediction of HheCPS (a,c,e), HheCPS I81W (b), HheCPS F86N (d) and HheCPS V94R (f). The amino acids of catalytic triad were shown in
green sticks (a–f). The amino acids changed are shown in yellow sticks (a–f). The amino acids forming special structures were shown in cyan sticks (a, b, e, f).
Different halide ion channels were colored as red, blue and green (the common part colored as yellow). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
the reverse reaction have been investigated. Bi-phase reaction system
was developed to separate chiral ECH from the HHDH catalyst (Jin
et al., 2013). An organic solvent/buffer biphasic system synthesis of
mechanism, the halide ion is first released from the catalytic triad under
the action of halide-binding site (Liu et al., 2014; Tang et al., 2005). The
halide ion channels of HheC were proposed to be the key factor for the
interaction between halide ions and enzyme, and with the guide of the
halide ion channels, the halide ions were released and transported ef-
ficiently (Tao, 2014). In the present study, halide ion channels of
HheCPS were simulated and key amino acid residues were selected for
site saturation mutagenesis. For HheCPS I81W, HheCPS F86N and
HheCPS V94R, one of the main halide ion channels was disrupted re-
spectively. The kinetic parameters for the forward and reverse reactions
(
R)-ECH was also investigated (Zou et al., 2013). However, the optically
pure ECH was not achieved.
−
Cl is the by-product for the forward reaction and one of the sub-
strate for the reverse reaction (Hasnaoui-Dijoux et al., 2008). The in-
−
teraction between Cl and HHDH affected the forward and reverse
reaction rates and thus determined the optical purity of ECH (Jin et al.,
2012; Tang et al., 2003). As the reaction obeys an ordered Uni-Bi
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X.-J. Zhang et al.
Bioresource Technology 276 (2019) 154–160
were regulated. The catalytic efficiencies for the forward reaction were
kept in similar levels as the parent protein, while the catalytic effi-
ciencies for the reverse reaction were significantly decreased. The
changes of the kinetic parameters led to the inhibition of the reverse
reaction. Thus, the optical purity of (S)-ECH increased to > 99% e.e.
and kept in high level during the reaction periods. Our present study is
the first report to regulate the kinetic parameters of a HHDH in protein
molecule level for both forward and reverse reactions, and for the first
time, optically pure (S)-ECH has been synthesized by a HHDH.
epoxide hydrolase in two-phase system. Biochem. Eng. J. 74, 1–7.
Lin, H., Liu, J.Y., Wang, H.B., Ahmed, A.A.Q., Wu, Z.L., 2011. Biocatalysis as an alter-
native for the production of chiral epoxides: a comparative review. J. Mol. Catal. B:
Enzym. 72 (3–4), 77–89.
Liu, Z.Q., Zhang, L.P., Cheng, F., Ruan, L.T., Hu, Z.C., Zheng, Y.G., Shen, Y.C., 2011.
Characterization of a newly synthesized epoxide hydrolase and its application in
racemic resolution of (R, S)-epichlorohydrin. Catal. Commun. 16 (1), 133–139.
Liu, Z.Q., Gao, A.C., Wang, Y.J., Zheng, Y.G., Shen, Y.C., 2014. Expression, character-
ization, and improvement of a newly cloned halohydrin dehalogenase from
Agrobacterium tumefaciens and its application in production of epichlorohydrin. J.
Ind. Microbiol. Biotechnol. 41 (7), 1145–1158.
Liu, Z.Q., Hu, Z.L., Zhang, X.J., Tang, X.L., Cheng, F., Xue, Y.P., Wang, Y.J., Wu, L., Yao,
D.K., Zhou, Y.T., Zheng, Y.G., 2017a. Large-scale synthesis of tert-butyl (3R,5S)-6-
chloro-3,5-dihydroxyhexanoate by a stereoselective carbonyl reductase with high
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Liu, Z.Q., Dong, S.C., Yin, H.H., Xue, Y.P., Tang, X.L., Zhang, X.J., He, J.Y., Zheng, Y.G.,
4. Conclusions
2
017b. Enzymatic synthesis of an ezetimibe intermediate using carbonyl reductase
In our present study, using the molecule kinetic to regulate HheCPS
coupled with glucose dehydrogenase in an aqueous-organic solvent system.
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mutants, optically pure (S)-ECH (e.e. > 99%) was firstly synthesized.
Our study provides a class of HHDH catalysts for the preparation of
chiral ECH with high optical purity and considerable yield. Our present
work makes contribution to the development of green technology for
the synthesis of chiral ECH. However, more works need to be done
including protein engineering, chemical modification of HHDHs and
reaction process control to improve enzyme activity and product yield
in the asymmetric biosynthesis of chiral ECH.
Liu, Z.Q., Wu, L., Zhang, X.J., Xue, Y.P., Zheng, Y.G., 2017c. Directed evolution of car-
bonyl reductase from Rhodosporidium toruloides and its application in stereoselective
synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. J. Agric. Food
Chem. 65 (18), 3721–3729.
Liu, Z.Q., Wu, L., Zheng, L., Wang, W.Z., Zhang, X.J., Jin, L.Q., Zheng, Y.G., 2018a.
Biosynthesis of tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxyhexanoate by carbonyl re-
ductase from Rhodosporidium toruloides in mono and biphasic media. Bioresour.
Technol. 249, 161–167.
Liu, Z.Q., Lu, M.M., Zhang, X.H., Cheng, F., Xu, J.M., Xue, Y.P., Jin, L.Q., Wang, Y.S.,
Zheng, Y.G., 2018b. Significant improvement of the nitrilase activity by semi-rational
protein engineering and its application in the production of iminodiacetic acid. Int. J.
Biol. Macromol. 116, 563–571.
Schallmey, M., Floor, R.J., Hauer, B., Breuer, M., Jekel, P.A., Wijma, H.J., Dijkstra, B.W.,
Janssen, D.B., 2013. Biocatalytic and structural properties of a highly engineered
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Acknowledgments
This work was financially supported by the Public Welfare
Technology Application Research Project of Zhejiang Province (No.
016C31013), the Natural Science Foundation of Zhejiang Province
Schallmey, M., Koopmeiners, J., Wells, E., Wardenga, R., Schallmey, A., 2014. Expanding
the halohydrin dehalogenase enzyme family: identification of novel enzymes by
database mining. Appl. Environ. Microbiol. 80 (23), 7303–7315.
2
Shen, J.W., Qi, J.M., Zhang, X.J., Liu, Z.Q., Zheng, Y.G., 2018. Significantly increased
catalytic activity of Candida antarctica Lipase B for the resolution of cis-( ± )-di-
methyl 1-acetylpiperidine-2,3-dicarboxylate. Catal. Sci. Technol. 8 (18), 4718–4725.
Shibasaki-Kitakawa, N., Kanagawa, K., Nakashima, K., Yonemoto, T., 2013. Simultaneous
production of high quality biodiesel and glycerin from Jatropha oil using ion-ex-
change resins as catalysts and adsorbent. Bioresour. Technol. 142, 732–736.
Spelberg, J.H.L., van Hylckama Vlieg, J.E.T., Tang, L.X., Janssen, D.B., Kellogg, R.M.,
(
No. LQ14B060004), the Project from Department of Education of
Zhejiang Province (No. Y201534805), and the Preferred Fund of
Postdoctoral Scientific Research Project of Zhejiang (No. BSH1602103).
Conflict of interest
2
001. Highly enantioselective and regioselective biocatalytic azidolysis of aromatic
epoxides. Org. Lett. 3 (1), 41–43.
The authors declare that they have no conflict of interest.
Appendix A. Supplementary data
Tang, L.X., Spelberg, J.H.L., Fraaije, M.W., Janssen, D.B., 2003. Kinetic mechanism and
enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter.
Biochemistry 42 (18), 5378–5386.
Tang, L.X., Pazmiño, D.E.T., Fraaije, M.W., de Jong, R.M., Dijkstra, B.W., Janssen, D.B.,
2
005. Improved catalytic properties of halohydrin dehalogenase by modification of
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biortech.2018.12.103.
the halide-binding site. Biochemistry 44 (17), 6609–6618.
Tang, L.X., Li, Y., Wang, X., 2010. A high-throughput colorimetric assay for screening
halohydrin dehalogenase saturation mutagenesis libraries. J. Biotechnol. 147 (3–4),
1
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Tao, Y.W., 2014. Theoretical and computational research on halide ion release channel of
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