aggregation inhibitor. To date, several synthesis routes
have been developed for R-CMM, including
hindered the binding of the substrate with the esterase.
The completion of racemization as indicated by an
S-CM conversion of 50% was achieved when the
racemase concentration was 8.4 µM (esterase/racemase
concentration ratio of 1: 21). Considering the
requirement for complete racemization in the recycling
process, the esterase/racemase concentration ratio was
fixed at 1: 21 in the following experiments.
Subsequently, the concentrations of esterase and
racemase were fixed at 0.4 and 8.4 µM respectively to
further explore the effect of racemase addition on the
apparent enantioselectivity. As shown in Table 1, under
similar conversion of substrates, the apparent
1
2, 13
hydrocyanation of o-chlorobenzaldehyde,
hydrolysis
of o-chloromandelonitrile, asymmetric reduction of
1
4
1
5, 16
methyl o-chlorobenzpylformate,
of methyl o-chloromandelate and diastereomeric salt
resolution
kinetic resolution
1
7
1
8, 19
. However, those methods generally suffer
from drawbacks of process risk (toxic HCN), high cost
expensive NADP or chemical resolving agent) and/or
low yield.
To overcome the above drawbacks, herein an
+
(
alternative route for R-CMM production as depicted in
Fig. 1B was proposed. Using the racemic (R,S)-CMM as
the substrate, under the catalysis of a S-selective
enantiomeric excess value (ee
coexistence of esterase and racemase was higher than
that in the presence of solely esterase, leading to the
significant enhancement in the apparent
s
) of substrates in the
hydrolase,
(
S-CMM
S)-o-chloromandelic acid (S-CM), while the target
is
hydrolyzed
into
compound R-CMM remains almost untouched.
Subsequently, a racemase catalyzes the transformation
of S-CM into the racemized (R,S)-CM, which is then
enantioselectivity (Table 1). Hence, sequential reaction
of hydrolysis and racemization was proven to be an
efficient strategy for further improvement of enzymatic
enantioselectivity.
2
0
chemically esterified into the starting material (Fig. 2).
In our previous work, an esterase BioH mutant
L123V/L181A/L207F) with a moderate enantiomeric
ratio (E value) of 73 was obtained by rational design.
And a mandelate racemase mutant (V22L) with a
(
Finally, the whole recycling process was conducted at
the optimal esterase/racemase concentration ratio of
1:21, and an initial (R,S)-CMM concentration of 25 mM.
After 2 h reaction, the remaining esters in the reaction
solution were extracted with ethyl acetate, and the target
compound R-CMM was recovered with an ee value of >
97% (Fig. S5). The extraction raffinate was then
adjusted to pH 2 using 4 M HCl, and extracted with
ethyl acetate again to retrieve the fully racemized
o-chloromandelic acid (Fig. S6), which was further
3
-1 -1
catalytic efficiency of 2 × 10 M s towards
o-chloromandelic acid was successfully designed based
on the strategy described in another publication of our
2
1
group.
In the present study, we aimed to explore whether the
enantioselectivity of this esterase could be further
improved by sequential hydrolysis and racemization,
and the whole recycling process would be validated for
R-CMM production.
Both the recombinant esterase and mandelate
racemase were overproduced and purified from E. coli
stain BL21 by metal ion affinity chromatography
following the Novagen protocols. The effects of reaction
2
2
2
esterified applying SOCl as the catalyst and reused as
the starting material. After 5 cycles, R-CMM was
obtained with a yield of 88%. In comparison with the
existing methods, the present synthesis route was more
cost-effective since no toxic or expensive agent was
used. Moreover, the raw material was utilized more
efficiently compared to traditional chemical resolution.
In the present study, the apparent enantioselectivity of
esterase BioH was successfully improved from 73 to
162 using sequential hydrolysis and racemization
reactions, validating the effectiveness of this strategy in
enhancing the enantiomeric purity of the target
compound. Moreover, sequential hydrolysis and
racemization might also be a powerful tool for
improving the resolution efficiency of other enzymes
with moderate enantioselectivity.
conditions
(pH,
temperature
and
substrate
concentration) on the activities of both enzymes were
explored, as well as the influence of pH on the
self-hydrolysis of methyl-o-chloromandelate (Fig.
S1-S4). The optimal reaction condition was figured out,
and the subsequent experiments were all carried out at
2
o
5 C in HEPES buffer (0.1mM, pH 7.7), containing
2
MgCl (3.3 mM), and (R,S)-CMM (10 mM).
As the next step, the effect of the mandelate racemase
dosage on the catalytic efficiency of the esterase BioH
was investigated. In this experiment, the finial
concentration of esterase was fixed at 0.4 µM, while the
dosage of racemase varied from 0 to 12 µM. As shown
in Fig. 3, with the increase of the racemase dosage, the
conversion of S-CMM gradually increased. At a
racemase concentration of 7.2 µM (esterase/racemase
concentration ratio of 1: 18), the conversion reached the
maximum (58%), which was 2.5-fold higher than the
corresponding conversion in the absence of racemase
Acknowledgments
This work was financially supported by the Natural Science
Foundation of China (Grant No. 21176215), the Program for
Zhejiang Leading Team of S&T Innovation (Grant no. 2011R50007),
the Fundamental Research Funds for the Central Universities (Grant
No. 2014QNA4025) and Zhejiang Provincial Natural Science
Foundation of China (Grant No. LQ14B060005).
(
24%). Therefore, sequential racemization of S-CM
Supplementary Material
facilitated by adding a proper amount of racemase could
notably accelerate the hydrolysis rate of S-CMM.
However, with further increase of racemase dosage, the
conversion of S-CMM began to drop. A possible
explanation might be the blocking of the esterase active
site by the excessive racemase molecules, which
The experimental section and optimization of the reaction
conditions for esterase BioH and mandelate racemase can be
found in the online version.
Gu, Jiali
