Exploring the Biocatalytic Scope of a Novel Enantioselective Halohydrin Dehalogenase from an Alphaproteobacterium

Article abstract of DOI:10.1007/s10562-019-02659-0

A gene encoding halohydrin dehalogenase from an alphaproteobacterium (AbHHDH) was identified, cloned and over-expressed in Escherichia coli. AbHHDH was able to catalyze the stereoselective dehalogenation of prochiral and racemic halohydrins. It showed the highest enantioselectivity in the dehalogenation of 20?mM (R,S)-2-bromo-1-phenylethanol, which yielded (S)-2-bromo-1-phenylethanol with 99% ee and 34.5% yield. Moreover, AbHHDH catalyzed the azidolysis of epoxides with low to moderate (S)-enantioselectivity. The highest enantioselectivity (E = 18.6) was observed when (R,S)-benzyl glycidyl ether was used as the substrate. A sequential kinetic resolution catalyzed by HHDH was employed for the synthesis of chiral 1-chloro-3-phenoxy-2-propanol. We prepared enantiopure (S)-isomer with a high enantiopurity of ee > 99% and a yield of 30.7% (E-value: 21.3) by kinetic resolution of 20?mM substrate. The (S)-isomer with 99% ee readily obtained from 40 to 150?mM (R,S)-1-chloro-3-phenoxy-2-propanol. Taken together, the results of this study demonstrate the applicability of this HHDH for the production of optically active compounds. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s10562-019-02659-0

Catalysis Letters
https://doi.org/10.1007/s10562-019-02659-0
Exploring the Biocatalytic Scope of a Novel Enantioselective
Halohydrin Dehalogenase from an Alphaproteobacterium
Feng Xue1,2 · Xiangju Ya1,3 · Yuansong Xiu1 · Qi Tong1 · Yuqi Wang1 · Xinhai Zhu1 · He Huang2
Received: 10 November 2018 / Accepted: 6 January 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A gene encoding halohydrin dehalogenase from an alphaproteobacterium (AbHHDH) was identified, cloned and over-
expressed in Escherichia coli. AbHHDH was able to catalyze the stereoselective dehalogenation of prochiral and racemic
halohydrins. It showed the highest enantioselectivity in the dehalogenation of 20 mM (R,S)-2-bromo-1-phenylethanol, which
yielded (S)-2-bromo-1-phenylethanol with 99% ee and 34.5% yield. Moreover, AbHHDH catalyzed the azidolysis of epox-
ides with low to moderate (S)-enantioselectivity. The highest enantioselectivity (E =18.6) was observed when (R,S)-benzyl
glycidyl ether was used as the substrate. A sequential kinetic resolution catalyzed by HHDH was employed for the synthesis
of chiral 1-chloro-3-phenoxy-2-propanol. We prepared enantiopure (S)-isomer with a high enantiopurity of ee>99% and a
yield of 30.7% (E-value: 21.3) by kinetic resolution of 20 mM substrate. The (S)-isomer with 99% ee readily obtained from
40 to 150 mM (R,S)-1-chloro-3-phenoxy-2-propanol. Taken together, the results of this study demonstrate the applicability
of this HHDH for the production of optically active compounds.
O
OH
O
O
Cl
Tris-buffer pH8.0
HHDH
(R,S)-phenyl glycidyl ether
(R,S)-1-chloro-3-phenoxy-2-propanol
HHDH
HHDH
Tris-buffer
Tris-buffer
pH 7.5
NaN3
pH 7.5
NaN3
O
OH
HHDH
O
O
Cl
ee>99%
(R)-phenyl glycidyl ether
ee>99%
(S)-1-chloro-3-phenoxy-2-propanol
Keywords Halohydrin dehalogenase · Epoxides · Halohydrins · Enantioselectivity · Biocatalysis
1 Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10562-019-02659-0) contains
Halohydrin dehalogenases (HHDHs) are employed by
supplementary material, which is available to authorized users.
microbes for the biodegradation of halogenated organic
compounds [1]. Biochemical and structural studies have
shown that HHDHs and the enzymes of the short-chain
dehydrogenases/reductases family (SDR) are not only evo-
lutionarily but also mechanistically related, catalyzing the
dehalogenation of ortho-halogenated alcohols into their
* Feng Xue
coldsun_xf@163.com
* He Huang
biotech@njtech.edu.cn
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

F. Xue et al.
corresponding epoxides [1, 2]. In the reverse reaction, they
can accept a variety of alternative anionic nucleophiles
such as CN−, NO2−, and N3−, to open the epoxide ring,
which provides a novel biocatalytic strategy for the prepa-
ration of fine chemicals and pharmaceutic intermediates
[2, 3].
further characterization of their biochemical and structural
properties and for further applications in organic synthesis.
Herein, a series of predicted putative HHDHs were
selected as the candidates on the basis of NCBI data-
base searches with the reported HheC and HheB as query
sequences. Eight genes bearing 36–46% amino acid
sequence identity with the probe HHDHs were cloned and
expressed in Escherichia coli BL21(DE3). After one round
of testing and screening, the HHDH from an alphaproteobac-
terium (isolate 46_93_T64), was identified as the best candi-
date based on its high enantioselectivity toward (R,S)-phenyl
glycidyl ether. The substrate scope of this novel enzyme was
investigated in detail. Furthermore, sequential kinetic resolu-
tion of (R,S)-1-chloro-3-phenoxy-2-propanol catalyzed by
the halohydrin dehalogenase in conjunction with azide was
performed to verify the practical applicability of HHDH.
Recent investigations focus on the biotechnological appli-
cations of HHDHs, especially regarding the stereoselective
biotransformations [1]. At the early stage, HHDHs were
utilized in the kinetic resolution of racemic halohydrins to
obtain an optically active enantiomer [4–7]. Chiral epox-
ides are valuable and extremely important building blocks
for the synthesis of enantiomerically pure pharmaceuticals
and agrochemicals. In addition, the kinetic resolution of
racemic epoxides by HHDHs is another way to obtain the
optically pure isomers [3, 8–10]. The HHDH-catalyzed ste-
reoselective ring opening of epoxides is widely used in the
asymmetric synthesis of enantiopure β-substituted alcohols,
which are important building blocks for a number of phar-
maceuticals and biologically active compounds [11–14].
The most successful example is the synthesis of the statin
side-chain precursor ethyl (R)-4-cyano-3-hydroxybutyrate
by coupling highly engineered variant of HheC with stere-
oselective ketoreductase [15, 16].
2 Materials and Methods
2.1 Materials
The reagents 1,3-dichloro-2-propanol (1), 1,3-dibromo-
2-propanol (2), (R,S)-2,3-dichloro-propanol (3), (R,S)-
2,3-dibromo-2-propanol (4), (R,S)-2-chloro-1-phenylethanol
(5), (R,S)-2-bromo-1-phenylethanol (6), (R,S)-1-chloro-3-
phenoxy-2-propanol (7), (R,S)-epichlorohydrin (8), (R,S)-
styrene oxide (9), (R,S)-phenyl glycidyl ether (10), (R,S)-
benzyl glycidyl ether (16), (R,S)-naphthyl glycidyl ether
(17) were purchased from Sigma-Aldrich Chemical Co. (St.
Louis, MO, USA). All reagents were of analytical grade.
The racemic epoxides 11–15 were prepared according to a
previously described synthesis method [23]. Isopropyl-β-d-
thiogalactopyranoside (IPTG) and kanamycin were obtained
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
Yeast extract, tryptone and agar were purchased from Oxoid
(Basingstoke, England). All other organic compounds were
obtained from local commercial suppliers and used without
further purification (Fig. 1).
Despite these diversity of reported biocatalytic applica-
tions, it is particularly surprising to remember that all the
reactions studied in the past 20 years have involved only a
very small number of HHDHs [2]. Before 2014, only six
different HHDH sequences had been cloned, characterized
and classified into three different phylogenetic subtypes A,
B, and C by sequence homology and substrate specificity
[1]. To explore novel promising and attractive HHDHs,
many new techniques and approaches have been adopted.
Among them, database mining is an effective strategy to
discover new HHDHs. Using a database mining approach
based on the two sequence motifs representing the nucle-
ophile binding pocket (TX4[FY]XG) and catalytic triad
(SX12YX3R), the number of available HHDH sequences
was greatly increased [1, 17]. Four new HHDH family
subtypes, D through G, could be identified, with percent-
age identities below 30% for protein sequence alignments
between them [1, 17]. Of these HHDHs, 21 representative
proteins from six phylogenetic subtypes were characterized
in detail, several of which showed broad substrate spectra
[13, 18–22]. Among all the HHDHs mentioned above, HheC
still seems to be the only HHDH enzyme with relatively high
enantioselectivity in the conversion of various substrates,
whereas most other HHDHs are either non-selective or dis-
play only low to moderate enantioselectivity toward a few
halohydrins and epoxides [1, 18]. Thus, there is still a lack
of enantioselective HHDHs with high enantioselectivity for
various halohydrins and epoxides. Therefore, it is neces-
sary to identify and clone more enantioselective HHDH for
2.2 Strains and Plasmid
Escherichia coli BL21 (DE3) (Novagen, Darmstadt, Ger-
many) was used as host strains for the heterologous expres-
sion of recombinant HHDH. pET28a (+) was used as vector
for the expression of HHDH gene in E. coli BL21 (DE3).
E. coli transformants were grown in Luria–Bertani (LB)
medium.
2.3 Database mining for HHDHs
A toolbox of HHDHs was collected by database mining.
The candidates were further confirmed as putative HHDHs
1 3

Exploring the Biocatalytic Scope of a Novel Enantioselective Halohydrin Dehalogenase from…
OH
based on the presence of the conserved motifs [1]. A total
R
R
OH
of 8 putative HHDHs were selected and confirmed by
multiple sequence alignment with other reported HHDHs
using ClustalW2 program (Table 1). The HHDH nucleotide
sequences were synthesized by Suzhou Hongxun Biotech-
nological Development (China) using the PCR assembly
method after condons optimization for E. coli. The HHDH
genes were cloned individually into the pET28a vector
between the NcoI and XhoI restriction endonuclease sites.
The ligated plasmid pET28a-HHDHs were transformed into
E. coli BL21 (DE3). After heterogeneous overexpression
in E. coli BL21 (DE3), the activity and enantioselectivity
toward (R,S)-phenyl glycidyl ether were determined, as listed
in Table 1.
HO
R
R
R
5: R=Cl
6: R=Br
3: R=Cl
4: R=Br
1: R=Cl
2: R=Br
O
OH
O
O
Cl
Cl
8
9
7
O
O
O
O
O
O
R
10:R=H, 11:R=2-Me
16
17
12:R=3-Me,13:R=4-Me
14:R=2-NO2, 15:R=2-Et:
2.4 Protein Purification
Fig. 1 Substrates used in the dehalogenation and epoxide ring
opening reactions with AbHHDH [1: 1,3-dichloro-2-propanol, 2:
1,3-dibromo-2-propanol, 3: (R,S)-2,3-dichloro-propanol, 4: (R,S)-
2,3-dibromo-2-propanol, 5: (R,S)-2-chloro-1-phenylethanol, 6: (R,S)-
2-bromo-1-phentlethanol, 7: (R,S)-1-chloro-3-phenoxy-2-propanol,
8: (R,S)-epichlorohydrin, 9: (R,S)-styrene oxide, 10: (R,S)-phenyl gly-
cidyl ether, 11: (R, S)-1,2-Epoxy-3-(2-methylphenoxy)propane, 12:
(R, S)-1,2-epoxy-3-(3-methylphenoxy)propane, 13: (R, S)-1,2-epoxy-
3-(4-methylphenoxy)propane, 14: (R, S)-1,2-epoxy-3-(2-nitrophe-
noxy)propane, 15: (R, S)-l-(2′-ethylphenoxy)-2,3-epoxy-propane, 16:
(R,S)-benzyl glycidyl ether, 17: (R,S)-naphthyl glycidyl ether]
Recombinant E. coli cells harboring the pET28a-AbH-
HDH were cultured in 2 L of LB with kanamycin (50 µg/
mL) at 37 °C. When the optical density at 600 nm reached
0.6, the cells were induced by addition of 0.1 mM IPTG
and growth was carried out at 28 °C for extra 10 h. The
cells were harvested by centrifugation at 10,000×g for
10 min. The purification of recombinant HHDH was car-
ried out at 0–4 °C. For preparation of the crude enzyme,
4.0 g of recombinant E. coli (wet cell weight) was resus-
pended in 40 mL Tris–SO4 buffer (20 mM, pH 8.0). After
sonification for 15 min in an ice bath, crude enzyme was
prepared by centrifugation of the lysate at 15,000×g for
Table 1 Evaluation of HHDHs in the toolbox using (R,S)-phenyl glycidyl ether as a model substrate
Entry Accession number Source organism
Amino acid sequence
Activitya
ees(%)/configuration
identify to probe
enzyme
(U/g cww)
1
2
3
4
5
6
WP_092014473.1 Marinobacter daqiaonensis
WP_072897053.1 Hydrocarboniphaga daqingensis
WP_091849011.1 Marinobacter segnicrescens
45% with HheB
227 aa
320 7.8
265 2.5
335 3.4
810 6.6
2.9 0.7
172 2.1
18.5 2.1/(S)
17.7 1.8/(R)
46.0 2.5/(S)
46% with HheB
236 aa
43% with HheB
227 aa
b
OJU34764.1
Alphaproteobacteria incertae sedis isolate 65–37
39% with HheC
-
224 aa
WP_082752032.1 Rhodospirillaceae incertae sedis isolate CCH5-H10
WP_066115330.1 Xylophilus ampelinus
WP_083723433.1 Pseudomonas pachastrellae
36% with HheC
244 aa
-
45% with HheB
64.5 3.2/(S)
226 aa
7
8
41% with HheB
NDc
550 4.9
–
OUR79898.1
Alphaproteobacteria incertae sedis isolate 46_93_T64 41% with HheC
>99/(R)
243 aa
Highest observed ee of residual epoxide (ees)
aThe specific activity was expressed in U/g cell wet weight (cww)
b–: no enantioselectivity
cND no detectable activity
1 3

F. Xue et al.
20 min at 4 °C. The supernatant was fractionated by the
addition of ammonium sulfate between 30 and 70% satu-
ration. The active precipitate was collected by centrifuga-
tion at 12,000×g for 20 min and dissolved in the buffer.
The as-prepared solution was loaded onto a HiTrap Phe-
nyl Sepharose column, which had been equilibrated with
1.0 M (NH4)2SO4/20 mM Tris–SO4 buffer (pH 8.0). After
washing the column with the same buffer, the adsorbed
proteins were eluted with Tris–SO4 buffer (20 mM, pH
8.0) supplemented with a linear gradient of ammonium
sulfate (from 1.0 to 0 M). The active fractions were com-
bined and dialyzed against the Tris–SO4 buffer (20 mM,
pH 8.0). Finally, the fractions with HHDH activity were
loaded onto a Superdex 75 column, pre-equilibrated with
Tris–SO4 buffer (20 mM, pH 8.0), and the enzyme was
eluted with the same buffer at a flow rate of 0.5 mL/min.
Aliquots containing pure HHDH were collected and used
for subsequent studies. SDS-PAGE was applied to check
the purity of the protein [24], and the protein concentra-
tion was determined using a Bradford protein assay kit
with bovine serum albumin as the standard.
2.6 Analytical Methods
The ee and yields of halohydrins, epoxides and azido alco-
hol were determined by chiral GC analysis on a capillary
BGB-175 column, or HPLC analysis on Chiralcel AS-H
and Chiralcel OD-H columns (250×4.6 mm, particle size
5 µm, Daicel Chemical Industries, Tokyo, Japan) under the
conditions described in the Supplementary information. The
enantiomers of the epoxides was assigned by comparison
of their elution order on the GC or HPLC columns with
published data [21, 25–29]. The enantiomers of the azido
alcohols were identified by analogy according to the reduced
epoxides. The ees and eep were derived from the remaining
epoxide and formed azido alcohols of the two enantiomers
as [ee(%)=(S−R)/(S+R)×100]. R and S are the concen-
trations of (R) and (S)-enantiomer, respectively. E values
were calculated by formula E=ln[(1−c)(1−ees)]/ln[(1−c)
(1+ees)] (c, conversion) [30].
2.7 Sequential Kinetic Resolution
of (R,S)‑1‑Chloro‑3‑Phenoxy‑2‑Propanol
Freshly prepared E. coli cells expressing AbHHDH were
resuspended to a density of 5 g/L cell dry weight (cdw) in
0.2 M Tris–SO4 buffer (pH 7.0, 7.5 or 8.0) containing (R,S)-
1-chloro-3-phenoxy-2-propanol (20–150 mM) and 1 equiv
of NaN3 to a 10 mL system in a shake-flask. Reactions were
carried out at 20 °C and 150 rpm. Reaction progress was
periodically monitored by taking samples (0.5 mL) from
the reaction mixture. The samples were extracted with ethyl
acetate (1 mL), dried over Na2SO4 and analyzed by HPLC.
2.5 Substrate Specificity and Enantioselectivity
of the AbHHDH
The substrate specificity of recombinant AbHHDH was
determined by measuring the dehalogenation rates of
seven different halohydrins as well as monitoring the
ring-opening reaction of nine different epoxides in the
presence of azide. Analytical-scale reactions were carried
out in 2 mL Eppendorf tubes with a total reaction volume
of 400 µL. Stock solutions comprising halohydrins 1–7 in
ethanol were added to a final concentration of 20.0 mM
into Tris–SO4 buffer (100 mM, pH 8.0). The epoxide
ring-opening reactions were carried out in 100 mM
Tris–SO4 buffer (pH 7.5) containing 20 mM epoxides
9–16 and 40 mM NaN3. The reactions were started by
the addition of enzyme in Tris–SO4 buffer (pH 8.0 or 7.5)
and incubated at 30 °C and 150 rpm. Samples were col-
lected at regular time intervals, extracted twice with 1 mL
ethyl acetate and dried over with anhydrous Na2SO4. The
conversion, activity and enantiomeric excess (ee) values
were determined using HPLC or GC analysis. One unit
of enzyme activity was defined as the amount of enzyme
that catalyzed the formation of 1 µmol of epoxide per
min under the described assay conditions. The specific
activities were expressed in U/mg, and all experiments
were conducted in triplicate. Activity assay of the control
contained cell-free extracts obtained using the pET28a
vector without the HHDH gene in E. coli BL21(DE3),
cultured in the same way.
2.8 Nucleotide Sequence Accession Number
The nucleotide sequence of AbHHDH has been depos-
ited in the GenBank database under the accession number
MH782148.
3 Results and discussion
3.1 Identification and screening of HHDHs
A genome mining approach was adopted to search for robust
HHDHs that might be able to catalyze the azide-mediated
ring opening reaction of epoxides to corresponding azido
alcohols with high enantioselectivity. In total, 8 HHDHs
with 36–46% identity with HheC or HheB were selected
from the NCBI database and overexpressed in E. coli BL21
(DE3) to form a HHDH toolbox. After testing their activities
and enantioselectivities toward (R,S)-phenyl glycidyl ether
as a representative substrate (Table 1), HHDH from an alp-
haproteobacterium 46_93_T64 exhibited the high specific
1 3

Exploring the Biocatalytic Scope of a Novel Enantioselective Halohydrin Dehalogenase from…
activity [550 U/g cell wet weight (cww)] and enantioselec-
tivity (99% ee). Therefore, the AbHHDH was chosen for
further studies.
without the HHDH gene in E. coli BL21(DE3), did not
show any activity toward any of the tested substrates. The
activity and enantioselectivity of AbHHDH in the dehalo-
genation of different halohydrins (1–7) are summarized in
Table 2. Almost all the tested substrates were accepted by
AbHHDH. The highest activity was measure for the dehal-
ogenation of 1,3-dibromo-2-propanol (17.2 U/mg), while
1,3-dichloro-2-propanol, (R,S)-2-bromo-1-phenylethanol
and (R,S)-2,3-dibromo-2-propanol were converted with good
specific activities, reaching 48.8%, 12.2% and 6.9% relative
activity, respectively. With the substrate (R,S)-2-chloro-
1-phenylethanol, only a low activity could be detected, and
there was no detectable activity were observed with (R,S)-
2,3-dichloro-propanol. (R,S)-3 is efficiently dehalogenated
by HheC but was a poor substrate for all other currently
characterized HHDH. These results indicate that a halogen
in the α position relative to the hydroxyl group seems to be
beneficial with respect to the specific activity of the HHDHs.
For instance, the activity of AbHDH toward (R,S)-compound
4 was less than 6.9% of that toward the prochiral halohy-
drin 2 (Table 2). Furthermore, the activity with bromine-
substituted compounds were higher than with equivalent
chlorine-substituted compounds. The reason why AbHHDH
prefers brominated alcohols as substrates is that chlorine has
a higher electronegativity than bromine, so that the strength
of the carbon-bromine bond is much weaker than that of the
carbon-chlorine bond [2, 18]. The specific activities toward
the aromatic substrates (R,S)-2-chloro-1-phenylethanol and
(R,S)-1-chloro-3-phenoxy-2-propanol were generally low.
One of the reasons could be steric hindrance, which has been
observed to affect the reactivity substantially.
A protein BLAST search was conducted with AbHHDH
as the search sequence, which showed that it has moderate
sequence similarity to known HHDH, reaching 41% identity
with Hhe C from Agrobacterium radiobacter AD1 [31], 36%
identity with HheA from Corynebacterium sp. N-1074 [32],
42% identity with TmHHDH from Tistrella mobilis ZJB1405
[21], 56% identity with PlHHDH from Parvibaculum lava-
mentivorans DS-1 [19]. Sequence analysis indicated that the
cloned HHDH gene contained an open reading frame with
732 bp encoding a 243 amino-acid protein. Multiple align-
ments of AbHHDH and other known HHDHs showed that
it shares the N-terminal motif TX4[FY]XG for nucleophile
binding and the catalytic triad of Ser135-Tyr148-Arg152
(Fig. S1) [1]. The expression of HHDH was clearly visible
in the SDS-PAGE of the soluble fraction of the E. coli cells
taken at 10 h. When a cleavable His-tag was fused to the
N- or C-terminus of AbHHDH, it was almost completely
inactivated. Therefore, it was purified to apparent homoge-
neity by salting out with ammonium sulfate, hydrophobic
chromatography and gel chromatography which yielded
52 mg/L purified AbHHDH. The purified enzyme had a
molecular weight of 27 kDa (Fig. S2), which was in good
agreement with the theoretical value (26.2 kDa) calculated
using ExPASy. The specific activity of AbHHDH for the
azidolysis of (R,S)-phenyl glycidyl ether was determined to
be 27.5 U/mg protein.
3.2 Substrate specificity and enantioselectivity
Our interest was focused on the enantioselectivity of AbH-
HDH in the conversion of various halohydrins. AbHHDH
catalyzed the transformation of prochiral 1,3-dichloro-2-pro-
panol and 1,3-dibromo-2-propanol into (S)-epichlorohydrin
(ee=78.3%) and (S)-epibromohydrin (ee=62.9%). Asym-
metric synthesis using prochiral halohydrins is particularly
attractive because the theoretical yield of one enantiomer in
Substrate specificity and enantioselectivity are among the
most important enzyme characteristics for biocatalysis. To
map the substrate spectrum of this newly mined HHDH,
various halohydrins and epoxides of diverse structure were
employed. The activity assay of the control, using a cell-
free extracts obtained by expression of the pET28a vector
Table 2 Activity and
Substrate Activity (U/mg)
Time (min) Conversion(%) Halohydrins
Epoxide product
enantioselectivity of AbHHDH
toward various halohydrins
substrates
ees/configuration eep(%)/configuration
1
2
3
4
5
6
7
8.4 1.6
17.2 1.9
ND
2
1
–
5
21.2 0.5
30.4 0.7
–
5.7 0.2
–
65.5 1.2
67.0 1.3
–
–
–
–
78.3 1.5/(S)
62.9 1.2/(S)
–
67.6 0.9/(S)
59.4 0.7/(R)
52.8 1.1/(R)
2.8 0.3/(S)
1.2 0.1
(2.0 0.1)×10−2 660
51.1 1.7/(S)
>99/(S)
5.7 0.3/(S)
2.1 0.2
20
(3.7 0.2)×10−1 120
The reactions were performed with halohydrins 1–7 (20 mM) and purified AbHHDH (0.2 mg/mL) in Tris–
SO4 buffer (200 mM, pH 8.0) at 30 °C. The specific activity was expressed in U/mg. The ee value was
determined by chiral HPLC and GC analysis, and the identity of the enantiomers was determined by com-
parison to the literature. ND: Not detectable due to very low activity
1 3

F. Xue et al.
Table 3 Results for the enzymatic resolution of epoxides 8–17 on an
this case is 100% [33]. Compared with other HHDHs, the
enantioselectivity of AbHHDH toward prochiral halohydrins
was similar to that of HHDH from T. mobilis ZJB1405 and
different from that of the HheB and HheB8 from Corynebac-
terium sp. N-1074 and Bradyrhizobium erythrophlei [21,
22]. The epoxides produced by HheC and HheA were almost
racemic, suggesting that enantioselective dehalogenation of
prochiral halohydrins is not a general property of all HHDHs
[34–36]. Furthermore, the enantioselectivity of AbHHDH
in the conversion of racemic haloalcohols 3–7 was studied
by chiral GC and HPLC. AbHHDH was able to convert
(R,S)-2,3-dibromo-2-propanol into (S)-epibromohydrin.
This indicated that the (R)-2,3-dibromo-2-propanol was
converted preferentially, followed by a slower conversion
of the (S)-enantiomer. Similar to the (R)-selective HheC,
AbHHDH also preferentially converted the (R)-enantiomer
of (R,S)-2-chloro-1-phenylethanol [37]. So far, HheC still
seems to be the only enzyme exhibiting a comparably high
R-enantioselectivity in the conversion of (R,S)-2-chloro-
1-phenylethanol (E=65), while other reported HHDHs are
either non-selective or display only low enantioselectivity
toward haloalcohol 5 (E≤4.7) [18, 37]. In the dehalogena-
tion reaction of (R,S)-2-chloro-1-phenylethanol, AbHHDH
showed a moderate enantioselectivity with an E value of
6.43. We also investigated the enantioselectivity of AbH-
HDH in the dehalogenation of (R,S)-2-bromo-1-phenyleth-
anol. The (R)-enantiomer was converted preferentially, fol-
lowed by a much slower conversion of the (S)-enantiomer.
(S)-2-bromo-1-phenylethanol was obtained with > 99% ee
and 34.5% yield (E=27.7). Thus, AbHHDH exhibited a rela-
tively high enantioselectivity among the reported HHDHs,
with the 2nd highest R-enantioselectivity of all known
HHDHs in literature [1, 2]. From this point of view, AbH-
HDH may be of considerable interest in biocatalysis.
analytical scale
Substrate Activity (U/mg) ees/configuration Analytical yield (%)
8
9
23.7 0.6
5.7 0.3/(R)
–
–
(2.7 0.1)×10−1 17.6 0.6/(S)
10
11
12
13
14
15
16
17
27.2 0.3
27.6 0.6
25.1 0.7
26.0 0.7
20.1 0.5
5.5 0.2
2.6 0.1
1.7 0.1
>99/(R)
16.6 0.4
–
–
–
23.4 0.3
–
28.5 0.7
–
56.1 1.2/(R)
81.1 1.7/(R)
35.7 0.5/(R)
>99/(R)
32.4 0.8/(R)
>99/(R)
None
The reactions were performed with epoxides 8–17 (20 mM), 50 mM
NaN3 and purified AbHHDH (0.2 mg/mL) in Tris–SO4 buffer
(200 mM, pH 7.5) at 30 °C. The specific activity was expressed in U/
mg. The ee value was determined by chiral HPLC and GC analysis,
and the identity of the enantiomers was determined by comparison to
literature
not perform well with (R,S)-naphthyl glycidyl ether (2.6
U/mg) and benzyl glycidyl ether (1.7 U/mg), which might
be due to steric hindrance. Moreover, AbHHDH displayed
only poor activity on styrene oxide.
To further confirm the enantiopreference of the enzyme,
its selectivity in the ring-opening reaction of epoxides was
also investigated, as shown in Table 3. AbHHDH showed
moderate enantioselectivity for substrates 10, 15, and 16,
while poor enantioselectivity was observed for substrates
8, 9 and 17. In contrast to (R)-selective HheA, AbHHDH
preferentially converted the (S)-enantiomer of epoxide 10
[10]. The substituent and its position at the phenyl ring
also affected the enantioselectivity of HHDH in the bio-
catalytic azidolysis of epoxides. Compared to the epoxides
with a substitution at the para- or ortho position (11, 13),
the epoxide with a substitution at the meta position (12)
was more selectively converted (higher ee). It is worth not-
ing that a nitro group at the 2′-position greatly increased
the enantioselectivity to 99% ee, while the ee values were
32.4% with 2′-CH2CH3 substituents and 56.1% ee with
2′-CH3 substituents. It was also found that a larger side-
chain (17) could decrease the enantioselectivity to some
extent, which is in agreement with earlier reports [25].
Thus AbHHDH was able to catalyze the enantioselec-
tive conversion of various prochiral and racemic halo-
hydrins, as well as epoxides, yielding enantiomerically
enriched (S)-epoxides, (S)-halohydrins and (R)-epoxides.
This preference is significantly different from other well-
known HHDHs such as HheC and HheA, demonstrating
the unique substrate specificity and special synthetic appli-
cations of AbHHDH.
In the reverse reaction, which is epoxide-ring opening,
different terminal epoxides can be accepted. However,
there were significant differences in the activity and enan-
tioselectivity among the accepted substrates. The substitu-
ents at the phenyl ring had an effect on the activity, but no
general trend was observed. The position of the substituent
at the phenyl ring also affected the enzyme activity, which
increased from 3′<4′<2′ for the CH3 group. AbHHDH
showed high activity in the kinetic resolution of (R,S)-phe-
nyl glycidyl ether (27.2 U/mg) but only low activity toward
(R,S)-1-chloro-3-phenoxy-2-propanol (0.37 U/mg). This
could imply that AbHHDH preferentially catalyzes epox-
ide ring opening of (R,S)-10 using azide as a nucleophile,
compared to the elimination of a chloride ion from the
corresponding (R,S)-7. Although, the methyl- substituted
phenyl glycidyl ether derivatives were well accepted by
the AbHHDH, their nitro- and methoxy- derivatives were
transformed at a slower rate. Moreover, the enzyme did
1 3

Exploring the Biocatalytic Scope of a Novel Enantioselective Halohydrin Dehalogenase from…
OH
OH
Cl-
O
Cl
N3
O
b:fast
O
(S)-10
O
a:fast
O
Tris-buffer
AbHHDH NaN3
Tris-buffer
AbHHDH
(R)-7
(S)-18
OH
OH
Cl-
O
Cl
N3
b:slow
O
a:slow
O
Tris-buffer
AbHHDH NaN3
Tris-buffer
AbHHDH
(R)-18
(R)-10
(S)-7
Fig. 2 Sequential kinetic resolution of (R,S)-1-chloro-3-phenoxy-2-propanol
reaction is reversible, the released chlorine opens the epox-
ides, resulting in an equilibrium within an hour, producing
racemic epoxides. This issue can be solved by removing the
produced epoxide or the chloride. We tested the ring-closure
reaction of AbHHDH in the presence of NaN3, allowing the
AbHHDH itself to eliminate the (R,S)-compound 10 by cata-
lyzing a successive step. The advantage of the sequential
reaction is that the enantioselectivity of the first step can
be improved. The accumulation of (R,S)-epoxide 10 can be
avoided, and (S)-7 is obtained in the presence of azide in a
one-pot reaction starting from (R,S)-7. When this reaction
was carried out, the conversion rate of (R,S)-7 increased
markedly, resulting in the formation of 1-azido-3-phenoxy-
2-propanol. Only a small amount of (R,S)-10 transiently
accumulated. Since the intermediate epoxide (S)-10 was
efficiently and irreversibly converted by the AbHHDH to
(R)-1-azido-3-phenoxy-2-propanol, the conversion of (R)-
7 was drawn to completion. The reaction rate of the (S)-
enantiomer was slower, and this resulted in a high optical
purity of the remaining halohydrin (>99% ee). As shown in
Fig. 3, the sequential kinetic resolution of 20 mM (R,S)-7
by using resting E. coli cells reached an ee of >99% within
13 min, while the final yield of enantiopure (S)-7 and the
enantiomeric ratio (E) were 30.7% (theoretical yield=50%)
and 21.3, respectively.
3.3 Sequential kinetic resolution catalyzed
by halohydrin dehalogenase
Compound 7, (R,S)-1-chloro-3-phenoxy-2-propanol was
chosen as the substrate to investigate the sequential kinetic
resolution process. The reactions that can occur starting
from a halohydrin and N3− are depicted in Fig. 2. The ring
closure of (R,S)-7 yields (R,S)-phenyl glycidyl ether (10),
which can again act as a substrate for the enzyme. Ring
opening of (R,S)-10 by N3− results in the formation of the
corresponding azido alcohol.
Firstly, kinetic resolution of (R,S)-10 catalyzed by AbH-
HDH was tested in the presence of NaN3. When the kinetic
resolution was conducted at 20 mM (R,S)-10, a moderate
enantioselectivity (E = 9.98) was obtained, leading to the
formation of 16.7% (R)-10 with an ee>99%, and the enan-
tiopurity of the product was very low (data not shown). The
azidolysis of (R,S)-10 leads to 1-azido-3-phenoxy-2-pro-
panol, which was established through a comparison with the
1H NMR spectrum reported in the literature [38]. The enan-
tiomers of 1-azido-3-phenoxy-2-propanol were assigned by
analogy according to the reduced epoxides configuration
and comparing the retention times with reference materials
[28]. To date, limited numbers of HHDHs were reported to
catalyze azidolysis of (R,S)-10. HHDH from Arthrobacter
sp. AD2 was reported to catalyze the ring-opening of (R,S)-
10 with low (S)-enantioselectivity (E=2) [10]. In contrast
to the (S)-selective AbHHDH, the recombinant HHDH from
Bradyrhizobium erythrophlei catalyzed the azidolysis of
(R,S)-10, yielding (S)-10 with 99% ee [22].
The reaction steps shown in Fig. 2 can be influenced
by several factors such as pH and the concentration of the
organic substrates. The pH of the reaction medium influ-
ences the rate of enzyme-catalyzed ring-closure and ring-
opening reactions and the position of the equilibrium
between these reactions. To identify suitable reaction con-
ditions, the influence of pH on all aspects of the sequential
kinetic resolution process was investigated. Table 4 shows
that the conversion rate of the (R,S)-7 increased as the pH
Bioconversions of (R,S)-7 in the absence of azide were
subsequently carried out. At the initial stage of the reaction,
AbHHDH converted (R)-7 to the corresponding epoxide in
preference to the other enantiomer. However, as the enzyme
1 3

F. Xue et al.
to 150 mM. Enantiopure (S)-7 with a high optical purity
(ee>99%) was readily obtained from 40 to 150 mM (R,S)-
7 within 120 min. As shown in Table 5, the final yield of
(S)-7 decreased as more (R,S)-7 was used initially, while
the reaction time required for > 99% ee was prolonged. A
yield of 27.8% enantiopure (S)-7 could be obtained from
40 mM (R,S)-7 after 22 min incubation, while the optical
yield from the conversion of 60 mM (R,S)-7 was 26.4%
after 40 min incubation. When the initial concentration
was further increased up to 150 mM, (S)-7 with 99% ee
was prepared with 22.1% yield. These results suggest that
HHDH-catalyzed sequential kinetic resolution can be used
to synthesize useful chiral building blocks.
10
8
(S)-7
6
(R)-7
(R)-10
(S)-10
(R)-18
(S)-18
4
2
0
0
2
4
6
8
10
12
14
Time (min)
4 Conclusions
Fig. 3 Biotransformation of (R,S)-3-chloro-1-phenoxy-2-propanol
with recombinant E. coli expressing AbHHDH in the presence of
NaN3. The reactions were performed with (R,S)-3-chloro-1-phenoxy-
2-propanol (20 mM), 50 mM NaN3 and resting cells (5 g cdw/L) in
Tris–SO4 buffer (200 mM, pH 7.5) at 30 °C
In summary, a toolbox comprising several novel HHDHs
was developed by data mining approaches. A novel HHDH,
which exhibited a high activity and good enantioselectivity
toward (R,S)-phenyl glycidyl ether, was successfully dis-
covered from an alphaproteobacterium. AbHHDH exhibits
varied activities (0.02–27.6 U/mg) toward the tested halo-
hydrins and epoxides. AbHHDH catalyzed the conversion of
prochiral halohydrins 1 and 2 to (S)-epichlorohydrin and (S)-
epibromohydrin with 78.3% ee and 62.9% ee, demonstrat-
ing its potential for the direct synthesis of chiral epoxides.
Among all the test racemic halohydrins, HHDH exhibited
the highest enantioselectivity toward (R,S)-2-bromo-1-phe-
nylethanol, producing (S)-2-bromo-1-phenylethanol with
99% ee and 34.5% yield. To the best of our knowledge, AbH-
HDH has the second-highest R-enantioselectivity toward
halohydrins of all the HHDHs reported to date, and thus
may be of considerable interest in biocatalysis. In addition,
AbHHDH catalyzed the ring opening of epoxides with low
to moderate enantioselectivity. The best result was obtained
in the preparation of (S)-benzyl glycidyl ether, reaching 99%
ee with 28.5% yield. Furthermore, this is also the first study
to our knowledge to describe a sequential kinetic resolu-
tion process using AbHHDH, which provides a green and
Table 4 Effects of pH on the enantioselectivity and conversion rates
of AbHHDH
pH Conversion
rate (U/g cdw)
Time (min) ees
Analytical yield
E
7.0 169 2.9
7.5 412 3.4
8.0 645 4.6
27
13
9
>99
>99
>99
30.9 0.6
30.8 0.5
26.4 0.7
21.6
21.4
16.5
The specific activity was expressed in U/g cell dry weight (cdw)
E values were calculated as E=ln[(1−c)(1−ees)]/ln[(1−c)(1+ees)]
of the reaction mixture was increased from 7.0 to 8.0. The
enantiomeric excess was not affected by the pH over the
range 7.0–8.0, but the yield of (S)-7 decreased.
To further test the feasibility of HHDH catalyzing
enantioselective resolution of (R,S)-7, resting cells of E.
coli expressing AbHHDH were incubated at pH 7.0 with
(R,S)-7 at various substrate concentrations ranging from 40
Table 5 Enantioselective
Substrate (mM)
Time (min)
Conv. (%)
ees(%)/con- eep(%)/configuration
Yield of (S)-7 (%)
conversion of (R,S)-3-chloro-
1-phenoxy-2-propanol using
resting cell of E. coli expressing
AbHHDH
figuration
40
60
80
100
150
22
40
62
95
120
72.0 0.5 >99/S
72.2 0.7 >99/S
76.0 1.0 >99/S
77.2 1.1 >99/S
77.8 0.9 >99/S
38.8 0.7/S
35.7 0.6/S
31.8 0.9/S
36.4 1.0/S
37.6 0.8/S
27.8 0.4
26.4 0.3
23.5 0.6
22.7 1.0
22.1 1.2
Conditions: reactions (1 mL) were performed aerobically in Tris–SO4 buffer (200 mM, pH 7.5) containing
(R,S)-3-chloro-1-phenoxy-2-propanol (40–150 mM) and 1 equiv of NaN3. The reactions were agitated at
150 rpm and 30 °C. Conversion and yield were determined by HPLC analysis. The identities of the enanti-
omers were determined by comparing to the literature
1 3

Exploring the Biocatalytic Scope of a Novel Enantioselective Halohydrin Dehalogenase from…
14. Chen SY, Yang CX, Wu JP et al (2013) Adv Synth Catal 355:3179
efficient synthesis route for (S)-1-chloro-3-phenoxy-2-pro-
panol, an intermediate for the preparation of β-adrenergic
blockers, in highly enantioenriched form (>99% ee). These
features make this a very attractive enzyme for potential
applications in the biocatalytic preparation of enantiopure
halohydrins, epoxides or azido alcohols.
15. Ma SK, Gruber J, Davis C et al (2010) Green Chem 12:81
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25:338
17. SchallmeyM KoopmeinersJ, Wells E et al (2014) Appl Environ
Microbiol 80:7303
18. Koopmeiners J, Halmschlag B, Schallmey M et al (2016) Appl
Microbiol Biotechnol 100:7517
19. Wan NW, Liu ZQ, Huang K et al (2014) RSC Adv 4:64027
20. Xue F, Liu ZQ, Wan NW et al (2014) Appl Biochem Biotechnol
174:352
Acknowledgements This project was sponsored by the National Natu-
ral Science Foundation of China (No. 21606192), the Natural Science
Foundation of the Jiangsu Higher Education Institutions of China (No.
16KJB180029) and the China Postdoctoral Science Foundation (No.
2016M601795).
21. Xue F, Liu ZQ, Wang YJ et al (2015) J Mol Catal B-Enzym
115:105
22. Xue F, Gao J, Zhang L et al (2018) Catal Lett 148:1181
23. Shapenvoa DS, Belyatskii MK, Panicheva LP (2010) Russ J Org
Chem 46:1017
Compliance with Ethical Standards
24. Liu ZQ, Wu L, Zhang XJ et al (2017) J Agr Food Chem 65:3721
25. Zhao J, Chu YY, Li AT et al (2011) Adv Synth Catal 353:1510
26. Wu K, Wang HL, Sun HH et al (2015) Appl Microbiol Biotechnol
99:9511
Conflict of interest The authors declared that they have no conflicts of
interest to this work.
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29. Xu Y, Xu JH, Pan J et al (2004) J Mol Catal B 27:155
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Affiliations
Feng Xue1,2 · Xiangju Ya1,3 · Yuansong Xiu1 · Qi Tong1 · Yuqi Wang1 · Xinhai Zhu1 · He Huang2
1
2
3
School of Marine and Bioengineering, Yancheng
Institute of Technology, Yancheng 224051,
People’s Republic of China
School of Environmental and Chemical Engineering, Jiangsu
University of Science and Technology, Zhenjiang 212003,
People’s Republic of China
College of Pharmaceutical Science, Nanjing Tech University,
Nanjing 210009, People’s Republic of China
1 3