Enantioselective bioreductive preparation of chiral halohydrins employing two newly identified stereocomplementary reductases

Article abstract of DOI:10.1039/c4ra16779a

Two robust stereocomplementary carbonyl reductases (DhCR and CgCR) were identified through rescreening the carbonyl reductase toolbox. Five reductases were returned through the activity and enantioselectivity assay for α-chloro-1-acetophenone and ethyl 4-chloro-3-oxo-butanate (COBE). Three reductases were stable at elevated substrate loading. Enzymatic characterization revealed that DhCR and CgCR were more thermostable. As much as 330 g COBE in 1 L biphasic reaction mixture was reduced to (S)- and (R)-3-hydroxy-4-chlorobutyrate by DhCR and CgCR (coexpressed with glucose dehydrogenase), with 92.5% and 93.0% yields, >99% ee, and total turnover numbers of 53800 and 108000, respectively. Six other α-halohydrins were asymmetrically reduced to optically pure forms at a substrate loading of 100 g L^-1. Our results indicate the potential of these two stereocomplementary reductases in the synthesis of valuable α-halohydrins for pharmaceuticals. This journal is

Full text of DOI:10.1039/c4ra16779a

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Enantioselective bioreductive preparation of chiral
halohydrins employing two newly identified
stereocomplementary reductases†
Cite this: RSC Adv., 2015, 5, 22703
a
a
a
Guo-Chao Xu,ab Hui-Lei Yu, Yue-Peng Shang and Jian-He Xu
*
*
Two robust stereocomplementary carbonyl reductases (DhCR and CgCR) were identified through
rescreening the carbonyl reductase toolbox. Five reductases were returned through the activity and
enantioselectivity assay for a-chloro-1-acetophenone and ethyl 4-chloro-3-oxo-butanate (COBE). Three
reductases were stable at elevated substrate loading. Enzymatic characterization revealed that DhCR and
CgCR were more thermostable. As much as 330 g COBE in 1 L biphasic reaction mixture was reduced to
(S)- and (R)-3-hydroxy-4-chlorobutyrate by DhCR and CgCR (coexpressed with glucose
dehydrogenase), with 92.5% and 93.0% yields, >99% ee, and total turnover numbers of 53 800 and
108 000, respectively. Six other a-halohydrins were asymmetrically reduced to optically pure forms at a
substrate loading of 100 g Lꢀ1. Our results indicate the potential of these two stereocomplementary
reductases in the synthesis of valuable a-halohydrins for pharmaceuticals.
Received 21st December 2014
Accepted 9th February 2015
DOI: 10.1039/c4ra16779a
www.rsc.org/advances
discovery and application of biocatalysts for chemical synthesis,
especially for value-added products, promise new synthetic
Introduction
methods due to their specialized and complicated spatial,
electronic and polar structures.7 The properties of enzymes
mean that they are highly diverse and enantioselective, easy to
use, environmentally benign and have a high atom economy.8
Dramatic improvement has been achieved employing enzymes,
accompanying the third wave of biocatalysis.9
Asymmetric reduction of prochiral ketones is one of the most
important fundamental and practical reactions for production
of chiral secondary alcohols, which can be transformed into
various functionalities of industrial relevance for pharmaceu-
ticals, agrochemicals and natural products.1 Optically active a-
halogenated alcohols constitute important building blocks in
the synthesis of pharmaceutical and liquid crystal products.2
Most importantly, the two opposite enantiomers may have
similar, different or even opposite effects. For example, ethyl (S)-
3-hydroxy-4-chlorobutyrate ((S)-CHBE) is a key chiral precursor
for HMG-CoA reductase inhibitors to lower cholesterol, whereas
(R)-CHBE is an important intermediate for L-carnitine, which
acts as an antioxidant.3,4 Both enantiomers of 2-chloro-1-(20,40-
dichlorophenyl)ethanol could be used in the synthesis of anti-
fungal agents, such as miconazole, econazole, and sertacona-
zole, with different antifungal proles and activities.5 Hence,
the enantioselective preparation of both a-halohydrins enan-
tiomers is of equal importance.
Because of the demanding diversity in biomanufacturing,
there is a constant need for new biocatalysts with altered
performance, such as high catalytic efficiency, wide substrate
scope, high regio- and enantioselectivity, thermo- and pH-
stability and high tolerance to substrates, products, and
organic solvents.10 For specic reactions, classical enrichment
cultivation is an effective strategy for identifying microorgan-
isms harboring new active enzymes. Based on this, various new
and efficient tools have been established to shorten the period
for discovering enzymes with desired properties, including
metagenomics, shotgun insertion, mutation, genome database
mining, and isolation and purication approaches for the
original microorganisms.11 Recently, genome database mining,
which allows the screening of enzymes with similar sequences
through target-reaction-oriented screening and selection, has
produced various breakthroughs. Recombinant DNA tech-
nology has enabled the rapid increase in accessible genome
data (currently at a rate of about 200 genomes per month),
providing more and more sequences with unknown functions
and promoting the quick identication of naturally-evolved
enzyme libraries by genome mining.12 Various strategies have
been published for the prediction and rational selection of
The manufacturing industry is searching for efficient, green,
energy-saving and environmentally benign procedures.6 The
aState Key Laboratory of Bioreactor Engineering, East China University of Science and
Technology, and Shanghai Collaborative Innovation Center for Biomanufacturing
Technology, Shanghai 200237, China. E-mail: huileiyu@ecust.edu.cn; jianhexu@
ecust.edu.cn
bThe Key Laboratory of Industrial Biotechnology, Ministry of Education, School of
Biotechnology, Jiangnan University, Wuxi 214122, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra16779a
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genes from the genome database.13 However, the focus has not potential in the enantioselective preparation of halohydrins was
been on strategies to identify robust enantioselective enzymes. also investigated.
To work as an alternative to chemical synthesis on an
industrial scale, one biocatalyst should possess certain prop-
erties, such as tolerance to a minimum of 100 g Lꢀ1 substrate,
Results and discussion
Rescreening result with COBE substrate
>99% enantioselectivity, high operational stability, no byprod-
ucts and cofactor addition of <0.1 g Lꢀ1
. Our group has been
14
A carbonyl reductase toolbox was developed for robust hal-
oketone reductases. In our primary screening using 4 as the
target substrate, only three carbonyl reductases (KtCR, PgCR
and ClCR) were returned with higher activity (>0.5 U mgꢀ1) and
enantioselectivity (>99%).18 Among them, the highest activity
was determined with ClCR (1.5 U mgꢀ1). Further comparison at
increased substrate loading revealed the instability of PgCR and
ClCR, and only KtCR was selected (Table 2). Most of the
candidates were eliminated due to their low activity. However,
changing the screening substrate to ethyl 4-chloro-3-
oxobutanate (COBE, 22), an a-halogenated b-ketoester, resul-
ted in a different prole (Fig. 1). Twelve reductases were iden-
tied with specic activity of more than 0.5 U mgꢀ1, which was
higher than that with 4. These exciting results encouraged us to
analyze the enantioselectivity in the asymmetric reduction of 22
and 4. DhCR and CgCR, from Debaryomyces hansenii (Uniprot
accession no. Q6BQ25) and Candida glabrata (Q6FR42),
respectively, displayed stable performance, even at 200 mM of 4
(Table 2). Particularly for DhCR, the time and catalyst require-
ment was much less than for KtCR, although the apparent
specic activity of DhCR was only 38% that of KtCR, indicating
that low apparent activity has little connection with the
biotransformation efficiency. CgCR was the only reductase with
Prelog preference in the asymmetric reduction of prochiral
ketones. Reductases with relatively higher activity were more
liable to have unstable enantioselectivity. Harsh conditions,
such as high reagent concentration (substrate or product) and
high temperature, might affect the structural conformation of
the enzyme and decrease their activity and enantioselectivity.
Considering the activity and ee for two screening substrates,
DhCR and CgCR were regarded as interesting reductases, in
addition to KtCR. Hence, for the primary screening for activity
and operational stability in genome data mining, using at least
involved in the discovery and engineering of robust enzymes
with promising potential uses in organic synthesis of chiral
building blocks with pharmaceutical relevance, as shown in
Table 1. An NADH-dependent carbonyl reductase, ScCR, has
been identied from Streptomyces coelicolor with a substrate-
coupled cofactor regeneration. Its specic production rate was
36.8 gproduct/gdry-cell-weight in the asymmetric preparation of the
key chiral precursor for atorvastatin (Lipitor).15 For the synthesis
of chiral o-chloromandelic acids, key building blocks for clo-
pidogrel (Plavix), two biocatalysts, an NADPH-dependent
ketoreductase, CgKR1, and an novel nitrilase, LaN, have been
discovered in Candida glabrata and Labrenzia aggregata,
respectively.16,23 Their space-time yield reached >100 g Lꢀ1 dayꢀ1
for the asymmetric reduction of methyl o-chlorobenzoylformate
(CBFM) and enantioselective resolution of o-chloromandeloni-
trile (CMN), respectively. However, based on our previous
results in genome mining, as shown in Table 1, one biocatalyst
with desired characteristics was obtained from 8–30 candidates
(rejection rate of >87.5%). Nonetheless, most of the genes were
not investigated and little is known about their properties
because a single screen was run and the selection criteria were
generally limited to activity and enantioselectivity towards the
target substrate. In our previous work, only one substrate-
tolerant carbonyl reductase KtCR was screened out of 30
potential reductases for preparing chiral halohydrins from a-
chloro-1-acetophenone (CAPE, 4).18 We expected that the other
reductases might display similar or even higher catalytic
performance. To rescreen this library for robust reductases,
three further rounds of screening were proposed, covering
activity and enantioselectivity for substrates with altered
substituents as primary screening, operational stability against
substrate/product and thermostability. The application
Table 1 Enzymes identified through genome data mining
Characteristics: substrate, loading
Enzyme
ECa
Candidateb
[g Lꢀ1], ee [%], STY [g Lꢀ1 dayꢀ1 c
]
Ref.
1
2
3
4
5
6
7
8
9
10
ScCR
1
1
1
1
1
1
1
3
3
3
10
8
COBE, 600, >99, 304
CBFM, 300, >98.7, 261
OPBE, 206, >99, 700
CAPE, 154, >99, 283
QNCO, 242, >99, 916
TMP, 78.1, >99, 275
CNB, 14.8, >99 sel., 291
APA, 64.8, 99, 97.2
15
16
17
18
19
20
21
22
23
24
CgKR1
CgKR2
KtCR
13
30
17
15
24
17
13
22
ArQR
EsLeuDH
BaNTR1
rPPE01
LaN
CMN, 50.2, 96.5, 143
MDPEA, 40, 97, 38.6
BaE
a
EC classication, 1 as oxidoreductase, 3 as hydrolase. b Numbers of candidates. c STY: space-time yield. Substrates: COBE (ethyl 4-chloro-3-oxo-
butanate), CBFM (methyl o-chlorobenzoylformate), OPBE (ethyl 3-oxo-phenylbutanate), CAPE (a-chloro-1-acetophenone) QNCO (3-quinuclidinone),
CNB (4-cyanonitrobenzene), APA (a-acetoxyphenyl acetate), MDPEA (1-(30,40-methylenedioxyphenyl) ethyl ester), CMN (o-chloromandelonitrile).
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Table 2 Asymmetric reduction of a-chloroacetophenone with the five best carbonyl reductases
Enzyme
Substrate [mM]
Catalyst [kU Lꢀ1
]
Time [h]
Conv. [%]
ee [%], R/S
ClCR
10
100
200
10
100
200
10
100
200
10
100
200
10
1
10
40
1
10
20
1
10
40
1
10
10
1
12
12
24
12
12
12
12
12
24
6
6
6
12
12
8
>99
>99
80.8
>99
>99
>99
>99
>99
76.6
>99
>99
>99
>99
>99
>99
>99/S
95.6/S
80.0/S
>99/S
>99/S
>99/S
>99/S
92.4/S
60.5/S
>99/S
>99/S
>99/S
98.7/R
98.6/R
98.7/R
KtCR
PgCR
DhCR
CgCR
100
200
10
20
Table 3 Comparison of the enzymatic characteristics of KtCR, DhCR
and CgCR
two substrates with similar structures was better for under-
standing the enzyme performance comprehensively. However,
other characteristics, such as the substrate proles toward
different kinds of substrates and enzyme performance (ther-
mostability), were also considered.
Characteristic
KtCR
DhCR
CgCR
Molecular weight [kDa]
Numbers of subunit
Optimal pH
69.8
2
6.5
67.2
2
6.5
55
462
111
2.09
218 ꢂ 6
2.1 ꢂ 0.1
4.0 ꢂ 0.2
39.8
1
6.5
60
169
80.6
1.26
198 ꢂ 3
2.2 ꢂ 0.1
1.6 ꢂ 0.1
Optimal temperature [ꢁC]
Thermostability [h] 30 ꢁ
40 ꢁ
45
Enzymatic characteristics of three carbonyl reductases
C
C
C
18.0
11.8
0.163
190 ꢂ 5
Three recombinant reductases with an N-terminal His-tag were
puried to electrophoretic homogeneity by nickel affinity
chromatography. The specic activities toward 22 of puried
DhCR, KtCR and CgCR were 13, 11 and 8.0 U mgꢀ1, respectively.
Protein separation of the puried enzyme by SDS-PAGE resulted
in a single band for each enzyme, corresponding to a molecular
weight of 34, 35 and 40 kDa, for DhCR, KtCR and CgCR,
50 ꢁ
Ea [kJ molꢀ1
]
KM [mM] 2.3 ꢂ 0.2
kcat [sꢀ1
]
7.3 ꢂ 0.3
KM [mM] 3.2 ꢂ 0.1
1.3 ꢂ 0.1
3.7 ꢂ 0.2
kcat [sꢀ1
]
13.2 ꢂ 0.2 16.6 ꢂ 0.3 27.9 ꢂ 0.4
respectively (Fig. S1†), in agreement with their theoretical
values. Gel exclusion chromatography with a TSK G2000 SWx1
column showed a single peak for DhCR, KtCR and CgCR with an
elution volume corresponding to an apparent molecular mass
of 67.2 69.6 and 39.8 kDa, respectively, which indicated that
they were homodimeric and monomeric enzymes.
The effect of pH and temperature on the activity of three
carbonyl reductases was investigated (Fig. S2†). All displayed
their highest activity at around pH 6.5. The optimum temper-
atures of DhCR, CgCR and KtCR were 55, 60, and 45 ꢁC,
respectively, according to the temperature proles. The activity
ꢁ
of CgCR decreased rapidly above 60 C due to thermal inacti-
vation. For DhCR and CgCR, the relative activities at 30 ꢁC were
70.8% and 27.0% of the activity at the optimum temperature,
respectively. Thermal stabilities were investigated at different
temperatures. The half-lives of KtCR, DhCR and CgCR at 30, 40
and 50 ꢁC were 18, 462 and 169 h, 11.8, 111 and 80.6 h, and 0.16,
2.1 and 1.3 h, respectively, as shown in Table 3. DhCR and CgCR
were stable at 30 and 40 ꢁC, but unstable at higher
Fig. 1 Screening results for the 30 carbonyl reductases using two
haloketones (4 and 22) with different substituents. Blue columns:
activity towards CAPE, green columns: activity towards COBE.
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temperatures. The deactivation energies (Ea) of DhCR, CgCR and aromatic and aliphatic ketones and b-ketoesters, were selected
KtCR were 218 ꢂ 6, 198 ꢂ 8, and 190 ꢂ 8 kJ molꢀ1, respectively. to characterize the substrate spectra of DhCR and CgCR.
These results indicated that DhCR and CgCR were much more
stable at room temperature than at elevated temperatures.26
As shown in Fig. 2, different substrate proles were observed
with DhCR and CgCR. Among the tested substrates, no clear
The kinetic constants of the puried reductases for CAPE (4) preference was discovered for DhCR, whereas CgCR preferred b-
and COBE (22) were calculated from the Lineweaver–Burk ketoesters to aromatic and aliphatic ketones. In addition to the
double-reciprocal plot, as shown in Table 3. The kcat for 22 was increase of side-chain length of aromatic ketones, from aceto-
16.6 sꢀ1 for DhCR and 27.9 sꢀ1 for CgCR. Relative low KM values phenone to 1-butyrophenone (1–3), the specic activity of DhCR
for NADPH (<50 mM) for DhCR and CgCR guaranteed high effi- gradually decreased. CgCR displayed higher catalytic activity for
ciency, even when no external cofactors were added (Table S3†). 1-butyrophenone than for 1-propiophenone. Due to the
Although the kcat values for 4 of DhCR and CgCR were lower than electron-imbalance, a-substituted acetophenone derivatives (4–
that of KtCR, they were much more efficient in the asymmetric 6, 9–12) were more easily reduced by reductases. Although CN
reduction of COBE and stable to high temperatures. Their was a strong electron-withdrawing group, benzoylacetonitrile
substrate specicity and catalytic performance in the prepara- (6) was difficult to reduce because of the steric hindrance of the
tion of chiral halohydrins were investigated further.
CN group. 20-Chloroacetophenone (7) was generally a poor
substrate for carbonyl reductases. However, CgCR displayed
higher activity toward 7. The specic activity ratio of 40-chlor-
oacetophenone (8) to 7 for CgCR was 3.2, much lower than 88.3
for DhCR, indicating that CgCR could accept much larger
aromatic ketones with ortho substituents on the phenyl ring.
Compared with aryl ketones, heteroaryl ketones were more
Substrate proles of stereocomplementary DhCR and CgCR
No activity with NADH but full activity with NADPH was detec-
ted using puried DhCR and CgCR (data not shown), indicating
both were NADPH-dependent carbonyl reductases. Twenty-ve
prochiral ketones (1 to 25) with various substituents, covering
Fig. 2 Substrate profiles of DhCR (B) and CgCR (D). Specific activities are shown in logarithmic form in the spider web diagram. The activities
equal to or lower than 0.01 U mgꢀ1 purified protein are shown as 0.01 U mgꢀ1. Values are listed in Table S4 in ESI.†
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difficult to reduce. Aliphatic ketones were better substrates for for statin side chains. In contrast, (R)-CHBE was produced with
DhCR than for CgCR, suggesting that DhCR could be used to CgCR as an intermediate for L-carnitine 22, and was not stable
prepare chiral aliphatic secondary alcohols. CgCR showed in the aqueous phase, especially under alkaline conditions, and
higher activity to b-ketoesters than DhCR, especially to ethyl may be toxic to biocatalysts.31 Biphasic systems are usually used
4,4,4-triuro-3-oxo-butonate (23). The highest activity of DhCR to minimize the loss of the substrate and product. In the
was found towards 2,30,40-trichloroacetophenone (11, 33 U toluene/aqueous system, the partition coefficients of 22 and
mgꢀ1). Ethyl 2-chloro-3-oxo-butonate (25, 19 U mgꢀ1) was the CHBE were 21.3 and 2.8, respectively, and DhCR, CgCR and
best of the tested substrates for CgCR. Interestingly, DhCR BmGDH retained their high activity; therefore, toluene was
showed the opposite enantioselectivity to CgCR, although low selected as the organic phase.25 The intracellular amount of
enantioselectivity was observed with CgCR for several substrates NADP+ was also quantied to calculate total turnover number
(20–98% ee, Table S4†). In the asymmetric reduction of pro- (TTN) of these two complementary reductases. There was 1.86 ꢂ
chiral ketones, DhCR obeyed the anti-Prelog rule whereas CgCR 0.13 mmol gꢀ1 NADP+ in DCW of E. coli BL21 (Table S5†), which
complied with Prelog priority. The preparation of both enan- was slightly higher than the reported amount of 0.39–0.79 mmol
tiomers of chiral a-halohydrins, which are usually of equal gꢀ1 NADP+ in DCW.32 Less than 0.1 mmol gꢀ1 of NADPH was
importance, could be achieved with these two stereo- detected, whereas 0.44 ꢂ 0.02 mmol gꢀ1 NAD+ in DCW was
complementary reductases.
In the regions conserved between DhCR and CgCR proteins, between E. coli BL21 harbouring DhCR and CgCR coding genes.
which are members of the SDR and AKR families, respectively, High substrate loading is desirable for practical applica-
calculated in vivo for E. coli BL21. There was less of a difference
typical SDR and AKR sequence motifs, such as cofactor binding, tions, usually at more than 100 g Lꢀ1. Hence, optimization was
catalytic, substrate binding and structure stabilizing residues, carried out to increase the substrate concentration and TTN of
were found, as illustrated in Table S1 and Fig. S4 and S5.†27 This reductases in the toluene/aqueous biphasic system. Within 6 h,
also indicated that DhCR and CgCR belonged to the SDR and 0.2 M of 22 was asymmetrically reduced with >99% conversion
AKR families, respectively. Further consensus analysis with by using 10 g Lꢀ1 DhCR and 5 g Lꢀ1 CgCR. In addition to the
homologous proteins showed that they were members of the increase of the substrate/catalyst ratio, as much as 330 g Lꢀ1
SDR51C and AKR1B10 subfamilies, based on online database (660 g Lꢀ1 in the toluene phase) of 22 could be fully reduced
nomenclature searches (http://www.sdr-enzymes.org/, http:// with no addition of an external cofactor (Table S6†). The prep-
www.med.upenn.edu/akr/).28
aration of (S)- and (R)-CHBE was scaled up to 1 L, and 330 g of 22
A characteristic glycine-rich Rossmann-fold scaffold was were added to the 0.5 L toluene phase and fully reduced within
found in the N-terminal of DhCR for binding the NADP+ dinu- 24 h (Fig. 3). Aer purication, the molar isolation yield of (S)-
cleotide.29 The Rossmann-fold sequence in DhCR was and (R)-CHBE was calculated to be 92.5% and 93.0% respec-
TGSSGGIGWA, sharing the motif of the classical or extended tively, eep was >99%. The substrate/catalyst (S/C) ratio was 33 for
subfamily. The length of the extended subfamily is 30 residues CgCR and 16.5 for DhCR.
longer than the 250 amino acid residues of the classical SDR,
As shown in Fig. 4 and Table S7,† much research has focused
and DhCR also had the conserved adenine ring binding and on the preparation of (S)-CHBE, because of its widespread use
active site motifs of the extended subfamily (Table S2†), DhCR in the preparation of statin side-chains in the pharmaceutical
was a member of the extended group.
industry. All the reductases that produced (S)-isomers belonged
No Rossmann-fold scaffold motif was found in CgCR. to the SDR family. Except for CmMR from Candida magnolia,
However, as shown in Table S1,† typical motifs for AKR were most reductases displayed excellent enantioselectivity. The
found in CgCR, which indicated that CgCR was a potential
member of AKR superfamily.30 Among the consensus
sequences, the catalytic tetrad, Asp-Tyr-Lys-His (50-55-80-111),
appeared in the N-terminal of CgCR. Thr26, Asp50, Asn167,
Gln190, Ser263 and Arg268 played important roles in cofactor
binding. Other residues, such as Gly23, Gly25, Gly45, Asp106,
Pro113, Gly165 and Pro187, might play a structural role in
forming the barrel core, because they were found in the b-
strand, a-helix and short loop regions of the barrel.
Optimization of the asymmetric reduction of COBE to chiral
CHBEs
DhCR and CgCR were coexpressed with glucose dehydrogenases
separately to provide cofactor regeneration systems. The cata-
lytic performance in the asymmetric reduction of COBE (22)
into optically active CHBE with DhCR and CgCR was systemat-
ically studied as shown in Table S6.† DhCR could asymmetri-
Fig. 3 Asymmetric reduction of 330 g COBE to both enantiomers of
chiral CHBE with DhCR and CgCR in a 1 L reaction mixture (toluene/
cally reduce substrate 22 into (S)-CHBE, an important synthon aqueous biphasic system).
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With no exogenous NADP+, as much as 330 g Lꢀ1 of the
substrate could be asymmetrically reduced within 24 h using
recombinant DhCR, which provided a much more efficient and
environmentally friendly reductase for preparing chiral (S)-
CHBE. The TTN and space-time yield (STY) of DhCR was 53 800
and 305 g Lꢀ1 dayꢀ1, respectively, calculated based on intra-
cellular NADP+/NADPH. Compared with (S)-isomers, fewer
reductases have been reported for producing (R)-CHBE. A
carbonyl reductase from Sporobolomyces salmonicolor, SsCR,
could catalyze the full reduction of 300 g Lꢀ1 of 22, with only
91.7% (R) ee.34 Of the reductases that produce (R)-isomers, the
gene YueD from Bacillus subtilis was reported to show the
highest ee value at a substrate loading of 214 g Lꢀ1 (fed-batch).
However, it required 1 mM NADP+,35 which would increase its
cost and hinder its application. With no external cofactors,
330 g Lꢀ1 of 22 was asymmetrically reduced to optically pure (R)-
CHBE, and the S/C, TTN and STY of CgCR were 33 108 000 and
614 g Lꢀ1 dayꢀ1, respectively.
Fig. 4 Comparison of the catalytic performance of selected ketor-
eductases in the asymmetric reduction of COBE to chiral CHBE. Black
bar (-): total turnover number, red bar (-): space-time yield, blank
circle (B): ee.
highest substrate loading and TTN of the cofactor were achieved
with carbonyl reductase ScCR and CmS1 from Streptomyces
coelicolor and C. magnolia.15,31,33 However, the requirement for
an external cofactor is a disadvantage for its wide use in organic
synthesis.
Enzymatic preparation of both halohydrin enantiomers
High substrate specicity is an important characteristic of
biocatalysts. Different substituents at different positions may
imbue substrates with distinct electronic, hydrophobic, polar,
and spatial properties. Unlike classical chemical catalysts, there
Table 4 Efficient bioreductive preparation of various chiral halohydrins at high substrate loading
DhCR
CgCR
Chiral blocks in
Concn.
Catal.a Time Yield ee [%], Concn.
Catal.b Time Yield ee [%], pharmaceutically
Substrate
[g Lꢀ1
]
[g Lꢀ1
]
[h]
24
24
[%]
R/S
[g Lꢀ1
]
[g Lꢀ1
]
[h]
24
—
[%]
R/S
98/R
—
relevant products
100
30
88
>99/S
>99/S
30.8
20
85
c
174
30
90
—
—
—
189
30
30
24
12
89
89
>99/S
>99/S
94.5
100
20
20
24
12
90
88
>99/R
>99/R
44.4
100
30
20
12
24
90
92
>99/S
>99/S
100
20
10
24
12
87
93
>99/R
>99/R
330
330
(660)d
(660)d
184
30
24
89
>99/S
184
10
24
92
>99/R
a
Catalyst, dry cells of recombinant E. coli BL21/pET28-bmgdh-dhcr. b Catalyst, dry cells of recombinant E. coli BL21/pET28-bmgdh-cgcr. c Reaction
was not performed due to the low ee value (54%). d 330 g Lꢀ1 in the reaction mixture and 660 g Lꢀ1 in organic phase.
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are marked differences between biocatalysts for diverse (Madison, WI, USA). Competent cells of E. coli strains, Dh5a and
substrates. The catalytic activity and enantioselectivity may vary BL21(DE3), were purchased from Tiangen (Shanghai, China).
according to changes in substrates. An ideal reductase should
display high activity, enantioselectivity, and substrate tolerance,
and, most importantly, a broad substrate scope. Although it is
impossible to nd one enzyme for all substrates, as an alter-
native tool in the organic synthesis of chiral compounds, a
series of substrates containing similar functional groups should
be catalyzed. Several a-substituted prochiral ketones with high
activity and enantioselectivity in the substrate spectra analysis
of DhCR and CgCR were chosen to test their applicability.
All the tested substrates were reduced at 100 g Lꢀ1 substrate
loading to optically pure a-halohydrins as illustrated in Table 4.
All the a-halohydrins are important chiral building blocks with
pharmaceutical relevance. For example, (S)-a-chloro-1-
acetophenol could be used for sotalol,36a (S)-2,2,2-tri-
uroacetophenol for liquid crystals and anticonvulsant phar-
maceuticals,36b,c (S)-2,40-dichloroacetophenol for adrenergic
receptor agonists,36d (S)-2,30,40-trichloroacetophenol for ser-
traline,36e (R)-2,20,40-trichloroacetophenol for econazole,5 and
ethyl (R)-4,4,4-triuro-3-hydroxybutyrate for beoxatone.36f The
efficient preparation of optically pure a-halohydrins shows that
DhCR and CgCR are promising catalytic enzymes.
General remarks for gene cloning, expression and purication
of proteins
Gene cloning and construction of recombinant plasmids
(pET28a-CRs) was reported in our previous work.18 The E. coli
BL21(DE3) cells harbouring recombinant pET28a-CRs weꢀre1
cultivated at 37 ꢁC in LB medium containing 50 mg mL
kanamycin. When the OD600 of the culture reached 0.5–0.6,
IPTG was added to a nal cꢁoncentration of 0.3 mM, and culti-
vation was continued at 25 C for a further 15 h. The recombi-
nant E. coli BL21(DE3) cells were collected and puried as
previously described.16,18
Co-expression of reductase and GDH
The genes coding for KtCR, DhCR and CgCR with independent
promoters were separately cut from pET28-ktcr, pET28-dhcr,
pET28-cgcr with BglII and XhoI. The pET28-bmgdh vector was
digested with BamHI and XhoI. The fragments of ktcr, dhcr and
cgcr with independent promoters were ligated with the linear
pET28-bmgdh vector to form pET28-bmgdh-ktcr, pET28-bmgdh-
dhcr and pET28-bmgdh-cgcr plasmids.25 The resulting plas-
mids were transformed into E. coli BL21 and over-expressed as
mentioned above.
Conclusions
In summary, two robust haloketone reductases (DhCR and
CgCR) were identied from the recently developed carbonyl
reductase toolbox through rescreening and characterization.
Activity and enantioselectivity are usually the key criteria for
chiral biocatalysis. In previous work, the carbonyl reductase
KtCR was discovered with high activity and enantioselectivity.
However, 29 other candidates were identied by reassessing the
enzymatic properties. Hence, a three-round screening strategy
was proposed to recheck the missed reductases. Ethyl 4-chloro-
Enzyme activity assay
Reductase activity was detected spectrophotometrically at 30 ꢁC
through monitoring the change of NAD(P)H absorbance at 340
nm. The reaction mixture consisted of 2 mmol substrate (4 or 22,
unless otherwise stated), 0.1 mmol NADPH or NADH, 50 mmol
sodium phosphate buffers (pH 6.5), and an appropriate amount
of enzyme in a total volume of 1 mL. One unit of enzyme activity
was dened as the amount of enzyme that catalyzed the
oxidation of 1 mmol of NADPH per minute under these
conditions.
3-oxobutanate,
a
different haloketone from a-chloro-1-
acetophenone, was used to retest all 30 reductases. Twelve
were returned with activity of more than 0.5 U mgꢀ1. Five with
high enantioselectivity were selected to go through the
substrate tolerance assay. Only KtCR, DhCR and CgCR were
stable enough, and their enzymatic properties were compared.
Aer rescreening their thermostability, DhCR and CgCR, which
had opposite enantioselectivity for the asymmetric reduction of
prochiral ketones, were identied. At a 1 L scale, both reduced
330 g of 22 into chiral CHBEs, with 92.5% and 93.0% yields,
respectively, and >99% ee. The S/C ratios were 33 for CgCR and
16.5 for DhCR. Seven chiral halohydrins with pharmaceutical
relevance were asymmetrically prepared. Our results provide
key evidence for the stereocomplementary enzymes DhCR and
CgCR as potential robust reductases in organic synthesis.
Enzyme characterization
The optimum pH of KtCR, DhCR and CgCR was determined in
the following buffers (nal concentration, 50 mM): sodium
citrate (pH 4.0–6.0), sodium phosphate (pH 6.0–8.5), and
glycine-NaOH (pH 8.5–10.0) using above mentioned enzyme
activity assay protocol. The optimum temperature was deter-
mined under the standard conditions at various temperatures
(25–80 ꢁC). Thermal stability was determined by incubating the
puried enzyme (0.1 mg mLꢀ1) at the desired temperature (30,
40 or 50 ꢁC) followed by periodically measuring the residual
activity. The kinetic constant analysis of the puried enzyme
was performed as previously described.15
Experimental
Conversion and enantioselectivity analysis
Materials
The enantioselectivity was determined by examining the
Prochiral ketones were all from commercial sources (TCI and reduction of prochiral ketones using an NADPH regeneration
Aladdin Inc.). Strains used as genome donors were purchased system consisting of puried reductase and externally added
from CGMCC. The pET28a vector was obtained from Novagen glucose dehydrogenase (GDH). The reactions were carried out
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in a reaction mixture (0.4 mL) comprising 50 mM sodium The organic phase was combined, dried over anhydrous Na2SO4
phosphate buffer (pH 6.5), 10 mM carbonyl substrates, 0.2 U of and evaporated under vacuum.
the puried reductase, 0.4 U of BmGDH, 20 mM glucose and 0.5
Asymmetric preparation of six a-halohydrins was conducted
+
ꢁ
mM NADP with shaking for 12 h at 30 C and 900 rpm. Each using the same protocol as COBE (22). The reaction mixture
reaction mixture was extracted twice with ethyl acetate. The consisted of 1.0 mmol sodium phosphate buffer (5–10 mL, pH
conversion and ee value were determined by GC analysis 6.5), 2.0–10.0 mmol of substrates in toluene (5 mL), glucose (1.5
equipped with a CP-Chirasil-DEX CB (Varian, USA; 25 m ꢃ 0.25 equiv.) and appropriate amounts of dry cells as listed in Table 4.
ꢁ
mm ꢃ 0.39 mm) column or HPLC analysis using a Chiralcel OD- The reaction was performed with magnetic agitation at 30 C,
H column (Daicel Co., Japan; 4.6 ꢃ 250 mm) as described in our 200 rpm and titrated with 1.0 M Na2CO3 to keep the pH at 6.5
previous work.16,18
until completion. Reactions were stopped and extracted as
above.
Quantication of intracellular NADP+/NADPH
The intracellular amount of NADP+/NADPH of E. coli BL21 was
quantied by using HPLC (Shimadzu 2010, Shimadzu Scientic
Instruments, Japan) equipped with Shim-pack VP-ODS C18
column (Shimadzu Scientic Instruments, Japan; 4.6 ꢃ 250
mm). Dry cells (0.10 g) or wet cells (0.50 g) of E. coli BL21 har-
bouring pET28a-bmgdh-dhcr or pET28a-bmgdh-cgcr plasmids
was weighed and fully dispersed in 10 mL KPB (pH 7.0, 10 mM).
The mixture was disrupted with sonication (400 W, 3 s sonica-
tion, pause of 7 s) in an ice/water bath and centrifuged at 12 000
rpm for 30 min. Aer that, the upper aqueous phase was
ltered, diluted, and injected into the HPLC, which was per-
formed with 3% aqueous acetonitrile containing 0.025 mM
N,N,N-triethylamine as the mobile phase at a ow rate of 1.0 mL
minꢀ1, monitored at 254 nm, and at 30 ꢁC. The retention times
of NAD(H) and NADP(H) were analysed with standards, which
were 4.785, 4.555, 7.371 and 8.639 min for NADP+, NAD+, NADH
and NADPH, respectively, as shown in Fig. S3.†
Acknowledgements
This work was nancially supported by the National Natural
Science Foundation of China (no. 21276082), Ministry of
Science and Technology, P. R. China (nos 2011CB710800 and
2011AA02A210), China National Special Fund for State Key
Laboratory of Bioreactor Engineering (no. 2060204) and Huo
Yingdong Education Foundation.
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General protocol for the asymmetric reduction of 4 and 22 into
optically active a-chloro-1-acetophenol (CAPL) and ethyl 4-
chloro-3-hydroxybutanate (CHBE) was carried out with the
whole cell reaction of E. coli harbouring pET28-bmgdh-ktcr,
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