Asymmetric hydrolysis of styrene oxide by PvEH2, a novel Phaseolus vulgaris epoxide hydrolase with extremely high enantioselectivity and regioselectivity

Article abstract of DOI:10.1016/j.catcom.2017.08.026

A novel EH from Phaseolus vulgaris, PvEH2, was discovered based on the computer-aided analysis, and its encoding gene was cloned and expressed in E. coli Rossetta (DE3). The substrate spectrum of recombinant (re) PvEH2 was assayed, among which the enantiomeric ratio of rePvEH2 towards racemic styrene oxide (rac-1a) was > 200, while its regioselectivity coefficients, α_S and β_R, towards (S)- and (R)-1a were 99.1 and 69.8%, respectively. The asymmetric hydrolysis of 20 mM rac-1a by rePvEH2-expressing whole cells was performed at 25 °C, retaining (R)-1a with > 99.5% ee_s and 49.4% yield and producing (R)-phenyl-1,2-ethanediol (1b) with 96.2% ee_p and 49.7% yield in 40 min.

Full text of DOI:10.1016/j.catcom.2017.08.026

Accepted Manuscript
Asymmetric hydrolysis of styrene oxide by PvEH2, a novel
Phaseolus vulgaris epoxide hydrolase with extremely high
enantioselectivity and regioselectivity
Chuang Li, Die Hu, Xun-Cheng Zong, Chao Deng, Lei Feng,
Min-Chen Wu, Jian-Fang Li
PII:
DOI:
Reference:
S1566-7367(17)30366-7
doi: 10.1016/j.catcom.2017.08.026
CATCOM 5175
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Catalysis Communications
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Accepted date:
8 June 2017
19 August 2017
21 August 2017
Please cite this article as: Chuang Li, Die Hu, Xun-Cheng Zong, Chao Deng, Lei Feng,
Min-Chen Wu, Jian-Fang Li , Asymmetric hydrolysis of styrene oxide by PvEH2, a
novel Phaseolus vulgaris epoxide hydrolase with extremely high enantioselectivity and
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all authors. Please check if appropriate. Catcom(2017), doi: 10.1016/j.catcom.2017.08.026
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ACCEPTED MANUSCRIPT
Asymmetric hydrolysis of styrene oxide by PvEH2, a novel Phaseolus vulgaris epoxide
hydrolase with extremely high enantioselectivity and regioselectivity
Chuang Li a,1, Die Hu a,1, Xun-Cheng Zong a, Chao Deng b, Lei Feng b, Min-Chen Wu b,*, Jian-Fang Li c,*
1 Chuang Li and Die Hu, the two first authors,contributed equally to this work.
a Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,School of
Biotechnology,Jiangnan University,Wuxi 214122, PR China
b Wuxi Medical School,Jiangnan University,Wuxi 214122,PR China
c School of Food Science and Technology,Jiangnan University,Wuxi 214122,PR China
* Corresponding authors.
E-mail addresses: biowmc@126.com (M.-C. Wu), lijf@163.com (J.-F. Li).
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ABSTRACT
A novel EH from Phaseolusvulgaris, PvEH2, was discovered based on the computer-aided analysis, and its
encoding gene was cloned and expressed in E. coli Rossetta(DE3). The substrate spectrumof recombinant (re)
PvEH2 was assayed,among which the enantiomeric ratio of rePvEH2 towards racemic styrene oxide (rac-1a)
was more than 200, while its regioselectivity coefficients, αS and βR, towards (S)- and (R)-1a were 99.1 and
69.8%, respectively. The asymmetric hydrolysis of 20 mM rac-1a by rePvEH2-expressing whole cells was
performed at 25 °C, retaining (R)-1a with > 99.5% ees and 49.4% yield and producing (R)-phenyl-1,2-ethanediol
(1b) with 96.2% eep and 49.7% yield in 40 min.
Keywords: Epoxide hydrolase; Phaseolusvulgaris; Styrene oxide; Enantioselectivity; Regioselectivity;
Asymmetric hydrolysis
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1. Introduction
Chiral epoxides and their corresponding vicinal diols are extensively used in the synthesis offine chemicals,
pharmaceuticals and agrochemicals [1]. Among these versatile building blocks, chiral 1a and its hydrolysate,
(R)- or (S)-1b, are important synthons forproducing anticancer agents,adrenergic receptor agonists and kinase
inhibitors [2]. Recently, several chemical asymmetric syntheses ofchiral 1a and 1b have been reported, such as
Jacobsen epoxidation and Sharpless dihydroxylation [3]. However, catalysts having potential pollution were
required in these methods, and the enantiomeric excess (ee) values and/oryields of products were unsatisfactory
in some cases [4].
Epoxide hydrolases (EHs, EC 3.3.2.3) stereoselectively catalyze the addition of water to epoxide ring. With
the green wave of global industries, the biotransformation by EHs, an environmental-friendly way, has received
much attention [5]. To date, EHs from plants,microorganisms, mammals and invertebrates have been discovered
and characterized, but few EHs exhibited desirable enantioselectivity and regioselectivity [6,7]. For example,
AuEH2 from Aspergillususamii displayed a modest preference for (R)-1a in the resolution of rac-1a, retaining
(S)-1a with 99.2% ees, but only 38% yield, lower than its theoretical yield of 50% [8]. As to plant EHs, only one
from Solanumtuberosum, StEH, had high enantiomeric ratio (E) value of 30. Thus, StEH highly preferentially
hydrolyzed (S)-1a, retaining (R)-1a with nearly 100% ees. Additionally, owing to its high and complementary
regioselectivity towards (S)- and (R)-1a, an eep of (R)-1b reached 93% in 90 min. However, its enantiopurity
decreased to 89% eep when the reaction time was prolonged to 120 min [9,10].
In this work, using the Phaseolusvulgaris EH1 (PvEH1, GenBank: AKJ75509) [11] as the template, a novel
EH also from P. vulgaris, PvEH2, was selected based on the homology sequence search and multiple sequence
alignment. A PvEH2-encoding gene pveh2 was amplified by RT-PCR from P. vulgaris total RNA, and expressed
in Escherichia coli Rosetta(DE3). The E values of rePvEH2 towards ten rac-epoxides (Fig. 1) and its αS and βR
values towards (S)- and (R)-epoxides were determined, respectively. Then, the asymmetric hydrolysis of rac-1a
was conducted by the whole cells expressing rePvEH2 to simultaneously prepare (R)-1a and (R)-1b.
2. Experimental
2.1. Materials
P. vulgaris was purchased from the local supermarket for total RNA extraction. All enzymes and kits used for
gene cloning were from TaKaRa (Dalian, China), while pET-28a(+) and E. coli Rosetta(DE3) (Novagen,
Madison,WI) were used for expression of pveh2. E. coli Rosetta(DE3) and its recombinants were cultured in the
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LB medium and induced by IPTG. Rac-1a, 7a, (S)-1a, (R)-1a, (S)-1b and (R)-1b were products ofTCI (Tokyo,
Japan), while rac-6a was purchased from Energy (Shanghai, China). Rac-2a5a and 8a10a were synthesized in
our lab.
2.2. Database search and sequence analysis
Sequences of hypotheticalproteins with unknown functions,sharing identities more than 75% with that of
PvEH1 (AKJ75509), were searched at NCBI (http://www.ncbi.nlm.nih.gov/) by BLAST server. Among them, a
hypotheticalone (XP_007147001) also from P. vulgaris, PvEH2, was selected. Based on the multiple sequence
alignment with five plant EHs by ClustalW2 program (http://www.ebi.ac.uk/Tools/msa/clustalw2/), PvEH2 was
confirmed as the study object in this work.
2.3. Cloning and expression of pveh2
The first-strand cDNA was obtained from P. vulgaris total RNA by reverse transcription using Oligo
dT-Adaptorprimer in an RNA PCR kit. Then, the PvEH2-encoding gene (pveh2), flanked by Nde I and Xho I
sites,was PCR-amplified using a pair of primers Pv2-F and Pv2-R (Table S1), and ligated with pET-28a(+),
followed by DNA sequencing. The correct recombinant expression plasmid pET-28a-pveh2 was transformed into
E. coli Rosetta(DE3), forming a recombinant E. coli (E. coli/pveh2). E. coli Rosetta(DE3) transformed with
pET-28a(+) (E. coli/pET-28a) was used as the negative control.
A single colony of E. coli/pveh2 or E. coli/pET-28a was inoculated into LB medium containing 100 g/mL
kanamycin, and cultured at 37 °C overnight as the seed culture. Then, fresh LB medium was inoculated with 2%
(v/v) seed culture, and cultured until OD600 reached 0.60.8. After induction by 0.2 mM IPTG at 20 °C for 9 h,
the cells were collected and resuspended in Na2HPO4NaH2PO4 buffer (50 mM, pH 7.2). The resulting cell
suspension (100 mg wet cell/mL) was used as the catalyst.
2.4. Protein and enzyme activity assay
The expression of rePvEH2 was analyzed by SDS-PAGE. Its apparent molecular weight was estimated by
Quantity One software based on the standard proteins. EH activity was measured by high-performance liquid
chromatography (HPLC) as described previously [11] using an e2695 apparatus (Waters, Milford, MA) equipped
with a C18 column. One unit (U) of EH activity was defined as the amount of rePvEH2 generating 1 mol 1b
per min under the assay conditions.
2.5. Stereoselectivity assay of rePvEH2
The hydrolytic reactions of rac-1a–10a, (S)- and (R)-1a were performed as follows: 1.0 mL cell suspension
(100 mg/mL) was separately mixed with 1.0 mL rac-1a–10a (10 mM), (S)- and (R)-1a (5 mM) in 50 mM
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Na2HPO4–NaH2PO4 buffer (pH 7.2), and then incubated at 25 °C. During the hydrolytic reaction, 50-L samples
were withdrawn at different time points,extracted with 400 L ethyl acetate, and assayed by chiral gas
chromatography (GC) using a GC-2010 apparatus (Shimadzu, Tokyo, Japan) or HPLC to calculate the ees of
substrate, eep of product and conversion ratio (c).
The enantiomeric ratio (E) represents a degree of preferential hydrolysis of an enantiomer over the other one,
that is, enantioselectivity [12]. The ees of retaining epoxide was calculated: ees  [(R  S)/(R  S)]  100%, while
E value: E  ln [(1  c)  (1  ees)]/ln [(1  c)  (1  ees)]. R and S are the concentrations of (R)- and (S)-epoxide,
respectively. The regioselectivity coefficients, αS and βR,were related to attacks at Cα (a hindered benzylic
carbon in an epoxide ring) of (S)-epoxide and at Cβ (an unhindered terminal carbon) of (R)-epoxide, respectively
[13]. In this work, the αS and βR of rePvEH2 towards (S)- and (R)-1a were calculated based on the concentrations
of (R)- and (S)-1b, using (S)- and (R)-1a as substrates,respectively [14], while the coefficients towards (S)- and
(R)-2a10a were derived from the equation: eep = αS + βR  1 + [(βR  αS) × ees × (1 c)]/c, using their racemic
forms [15]. The absolute configurations of enantiomers of 2a–10a and 2b–10b were established by comparing
their retention times with those reported previously [13,15,16].
2.6. Asymmetric hydrolysisof rac-1a by whole cells
Both 5.0 mL cell suspension (100 mg/mL) and 4.0 mL Na2HPO4NaH2PO4 buffer were preheated at 25 °C
for 5 min, and then 1.0 mL rac-1a (200 mM) was added, giving the final cell and substrate concentrations of50
mg/mL and 20 mM, respectively. The reaction was carried out, and aliquots of 100 L sample were drawn out at
different time points in 60 min. The eep of (R)-1b was calculated: eep = [(Rp  Sp)/(Rp + Sp)] × 100%, while yields
of (R)-1a and (R)-1b: Ys = [R/(R0 + S0)] × 100% and Yp = [Rp/(R0 + S0)] × 100%. Rp and Sp are the concentrations
of (R)- and (S)-1b, while R0 and S0 are the initial concentrations of (R)- and (S)-1a [7].
3. Results and discussion
3.1. Selection and analysis of PvEH2 sequence
Using PvEH1 sequence as the template, those of six hypotheticalproteins sharing over 75% identities were
searched by BLAST, among which a hypothetical PvEH2 shared 80.1% identity with PvEH1. It was speculated
that PvEH2 may have EH activity owing to higher primary structure homology. Thus, this work selected PvEH2
as the study object.The multiple sequence alignment of PvEH2 with five plant EHs displayed that it has typical
conserved motifs, identical to those of the α/β fold EH family [8,14]: HGXP, GXSmXS/T and SmXNuXSmSm,
in which X, Sm and Nu represent any, small and nucleophilic residues,respectively (Fig. 2). The catalytic triad,
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consisting of nucleophile Asp,general base His and charge-relay Asp or Glu, was predicted to be Asp101-His295
Asp260 in PvEH2. In addition, Tyr150 and Tyr230 in PvEH2 are also conserved. There is evidence that the Tyr
residues play key roles in substrate binding and epoxide ring opening via formation of hydrogen bonds [17].
3.2. Cloning and expression of pveh2
-
An about 1.0-kb band of pveh2 was cloned from P. vulgaris total RNA by RT-PCR. DNA sequencing result
verified that pveh2 is exactly 963 bp in length (containing Nde I and Xho I sites), encoding PvEH2 of 316 amino
acids. After E. coli/pveh2 was induced by 0.2 mM IPTG at 20 °C for 9 h, the activity of rePvEH2-expressing
whole cells was assayed to be 8.0 U/g wet cell, while no EH activity was detected in E. coli/pET-28a under the
same conditions. Very low EH activity was detected when the temperature during induction exceeded 25 °C,
resulting in that most rePvEH2 molecules were transformed into insoluble inclusion body [8]. SDS-PAGE
analysis displayed that the apparent molecular weight of rePvEH2 was 38.1 kDa (Fig. 3), which well matched
with its theoretical one (37,898 Da).
3.3. Enantioselectivity (E value) of rePvEH2
To assay the substrate spectrumof rePvEH2, ten racemic epoxides were subjected to biotransformation by E.
coli/pveh2 (Table 1). Except for rac-2a and 4a, rePvEH2 preferentially hydrolyzed (S)-enantiomers and
exhibited E values ranging from 1.6 to > 200, indicating that the type of epoxides and position of substituents
obviously affected its activity and enantioselectivity. In detail, rePvEH2 exhibited the moderate
enantioselectivity towards 6a10a and poorenantioselectivity towards 3a and 5a. Unexpectedly, the E value of
rePvEH2 towards 1a was determined to be > 200, displaying the extremely high preference for (S)-1a. That, to
some extent, implied that the great majority of (R)-1a will be retained, and that the complete hydrolysis of
rac-1a needs a quite long time [4]. To our knowledge, rePvEH2 displayed the highest enantioselectivity (E value)
towards rac-1a among all EHs hitherto reported, such as StEH1 (E = 30) [9], Kau2 (E = 65) [15] and CcEH (E =
8.8) [18].
3.4. Regioselectivity (αS and βR) of rePvEH2
The carbon (Cα or Cβ) that is mainly subjected to nucleophilic attack by Asp in a catalytic triad was highly
dependent on the EH-substrate pair [9]. To make a better understanding ofthe catalytic mechanism of rePvEH2,
its regioselectivity coefficients were determined (Table 1). As a result, rePvEH2 attacked (S)-1a, 3a and 5a
mainly at Cα (αS > 90%), giving (R)-diols via an inversion of configuration. Simultaneously, the Cβ atoms of
(R)-3a and 5a were primarily attacked (βR > 95%), also affording (R)-diols via a retention of configuration. The
high and complementary regioselectivity as well as low enantioselectivity towards 3a (E = 1.6) and 5a (E = 1.9)
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indicated that rePvEH2 has a potential to catalyze their enantioconvergent hydrolysis [11,13]. In the cases of
6a10a, rePvEH2 displays the poor regiocomplementarity. Considering the higher E values towards 7a (E = 18.9)
and 10a (E = 23.0), rePvEH2 can be used for the kinetic resolution of rac-7a and 10a.
3.5. Preparation of both (R)-1a and (R)-1b from rac-1a by whole cells
The asymmetric hydrolysis of 20 mM rac-1a was conducted by E. coli/pveh2 whole cells at 25 °C in 60 min,
and monitored at given time intervals (Fig. 4). During the reaction, the concentration of (S)-1a decreased rapidly
while that of (R)-1a was nearly invariable, which resulted in the rapid increase of ees of (R)-1a with high yield.
Additionally, owing to the high and complementary regioselectivity of rePvEH2 towards two enantiomers of 1a,
the eep of (R)-1b kept more than 95%. As the reaction proceeded to 40 min (c = 51.0%) (Fig. S1), (R)-1a with >
99.5% ees and 49.4% Ys as well as (R)-1b with 96.2% eep and 49.7% Yp was obtained,simultaneously. However,
(S)-1a could not be completely hydrolyzed in 6 h (c < 25%) when the concentration of rac-1a was increased to
50 mM. The reasons may be instability of EHs under the operating conditions, inhibition of substrate or product
on EHs and low solubility of epoxides in water [19]. Several methods were used to overcome these problems.
For examples, the directed evolution has been adopted to create new enzymes with enhanced stability and
tolerance. Anotherapproach was to reduce the inhibition via a biphasic system [20].
4. Conclusions
In this work, PvEH2 was selected based on the computer-aided analysis, and its encoding gene pveh2 was
cloned and expressed in E. coli Rossetta(DE3). The substrate spectrumassay indicated that rePvEH2 exhibited
the different catalytic modes towards various epoxides. Owing to the extremely high enantioselectivity (E > 200)
and regioselectivity (αS = 99.1%) of rePvEH2, both (R)-1a and (R)-1b with high ees and eep were simultaneously
prepared from the asymmetric hydrolysis of rac-1a by it. More surprisingly, the Ys of (R)-1a was quite close to
its theoretical value. The obtained PvEH2 not only increased the number of EHs with high enantioselectivity and
regioselectivity, but also established a foundation for the study ofits catalytic mechanism.
Acknowledgment
This work was financially supported by the National Nature Science Foundation of China (No. 21676117)
and the Postgraduate Innovation Training Project of Jiangsu,China (No. KYLX16_0804). We are grateful to
Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.
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Figure captions
Fig. 1. Ten epoxides used for substrate spectrumassay. 1a, styrene oxide; 2a, para-nitrostyrene oxide; 3a,
meta-nitrostyrene oxide; 4a, para-chlorostyrene oxide; 5a, meta-chlorostyrene oxide; 6a, benzyl glycidyl ether;
7a, phenyl glycidyl ether; 8a, para-methylphenyl glycidyl ether; 9a, meta-methylphenyl glycidyl ether; 10a,
ortho-methylphenyl glycidyl ether.
Fig. 2. The multiple sequence alignment of six plant EHs. PvEH2 (ASS33914, in this work); PvEH1 (AKJ75509,
80.1% identity with PvEH2); VrEH1 (ADP68585, 75.5%); GmEH (CAA55294, 75.3%); NbEH (ACE82566,
67.8%); StEH (AAA81891, 57.6%). The conserved motifs of HGXP, GXSmXS/T and SmXNuXSmSm are
boxed, while NC, cap and core loops are underlined. A catalytic triad (Asp101-His295-Asp260)and both Tyr150 and
Tyr230 in PvEH2 are marked with stars.
Fig. 3. SDS-PAGE analysis of the expressed rePvEH2. Lane M, standard proteins; lane 1, E. coli/pET-28a; lane
2, E. coli/pveh2 without induction; lane 3, E. coli/pveh2 induced by 0.2 mM IPTG.
Fig. 4. (a) The process curve of hydrolytic reaction of 20 mM rac-1a by rePvEH2-expressing E. coli/pveh2
whole cells at 25 °C. Symbols: triangle, c of rac-1a; solid square, (S)-1a concentration; hollow square,(R)-1a
concentration; solid circle,eep of (R)-1b; hollow circle, ees of (R)-1a. (b) A schematic diagram of asymmetric
hydrolysis of rac-1a by rePvEH2 into both (R)-1a and (R)-1b.
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Table 1
The enantioselectivity of rePvEH2 towards rac-1a–10a and its regioselectivity towards (S)- and (R)-1a–10a.
Substrate
Activity
E value
αS
βR
(U/g wet cell)
(%)
(%)
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
8.0
NDa
1.3
ND
1.5
2.2
3.0
2.4
6.6
4.1
> 200
99.1

69.8

b
–
1.6

90.3

96.4

1.9
8.7
18.9
6.2
9.1
23.0
93.0
23.3
12.9
10.7
1.6
95.8
87.5
93.7
91.1
98.7
96.2
4.5
a No detectable activity.
b No determination.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Graphical abstract
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Highlights




The newly discovered rePvEH2 shows a very high E value (> 200) towards racemic styrene oxide (SO).
The αS and βR of rePvEH2 towards (S)- and (R)-SO are 99.1 and 69.8%, respectively.
(R)-SO with > 99.5% ees and (R)-phenyl-1,2-ethanediol with 96.2% eep were co-produced.
Besides SO, rePvEH2 also exhibits good catalytic properties towards other epoxides.
16