Engineering of the Conformational Dynamics of Lipase to Increase Enantioselectivity

Article abstract of DOI:10.1021/acscatal.7b02404

In order to increase the R-enantioselectivity of Candida antarctica lipase B (CALB) toward (R)-3-t-butyl-dimethyl-silyloxy glutaric acid methyl monoester at 30 °C, we engineered CALB conformational dynamics. Based on structural analysis and molecular dynamics simulations, two key residues (D223 and A281) were identified, and three mutants (D223V, A281S, and D223V/A281S) were designed to decrease the conformational dynamics of the pocket and channel. Computational and experimental evaluations were performed for all mutants, with the D223V/A281S mutant exhibiting high R-enantioselectivity (>99.00%; increased from 8.00%) and high space-time yield (107.54 g L^-1 d^-1 a 5.70-fold increase).

Full text of DOI:10.1021/acscatal.7b02404

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Article
Engineering of the Conformational Dynamics
of Lipase to Increase Enantioselectivity
Bin Yang, Hongjiang Wang, Wei Song, Xiulai Chen, Jia Liu, Qiuling Luo, and Liming Liu
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b02404 • Publication Date (Web): 27 Sep 2017
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Engineering of the Conformational Dynamics of
Lipase to Increase Enantioselectivity
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Bin Yang,1,2 Hongjiang Wang,1,2 Wei Song,1,2 Xiulai Chen,1,2 Jia Liu,1,2 Qiuling Luo,1,2 Liming
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Liu1,2,
*
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1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China
2 Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi
214122, China
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* Eꢀmail: mingll@jiangnan.edu.cn.
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ABSTRACT: In order to increase the R-enantioselectivity of Candida antarctica lipase B (CALB)
toward (R)ꢀ3ꢀtꢀbutylꢀdimethylꢀsilyloxy glutaric acid methyl monoester at 30°C, we engineered
CALB conformational dynamics. Based on structural analysis and molecular dynamics simulations,
two key residues (D223 and A281) were identified, and three mutants (D223V, A281S, and
D223V/A281S) were designed to decrease the conformational dynamics of the pocket and channel.
Computational and experimental evaluations were performed for all mutants, with the D223V/A281S
mutant exhibiting high R-enantioselectivity (>99.00%; increased from 8.00%) and high spaceꢀtime
yield (107.54 g L−1 d−1; a 5.70ꢀfold increase).
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KEYWORDS: conformational dynamics, enantioselectivity, Candida antarctica lipase B, substrate
pocket, substrate channel
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INTRODUCTION
Recently, enzymes with high enantioselectivity have been widely used as biocatalysts to
produce optically pure, valuable compounds.1ꢀ6 However, in some cases, enzymes exhibit
satisfactory enantioselectivity only at low temperatures (even down to –80°C), with decreasing
enantioselectivity at higher temperatures.7,8 Therefore, lowꢀtemperature methods have been applied
to improve the enantioselectivity of some enzymes.9ꢀ12 From a practical standpoint, although
substantial efforts have been made to increase reaction rates at low temperatures, such as through the
use of immobilized enzymes on porous ceramics,9 the high costs and low yields associated with low
temperatures remain limiting factors for industrial applications.13 Some examples demonstrated the
plausibility of maintaining satisfactory enzyme enantioselectivity at higher temperature through
protein engineering14; however, simple and accurate strategies for protein engineering warrant
further discussion.
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Recently, there has been growing interest in the conformational dynamics of proteins, which
play an important role in enzyme catalysis,15ꢀ20 and engineering of enzyme conformational dynamics
has become an effective strategy for protein evolution, achieving significant results in terms of
relieving product inhibition21ꢀ23 and for the rational design of enzymes.24ꢀ26 Recent studies also
indicated that protein conformational dynamics, which are crucial for ligand recognition and binding,
might determine ligandꢀbinding orientations, thereby assuming responsibility for selectivity.19,27
Despite no direct reports on the relationship between enzyme conformational dynamics and
enantioselectivity, engineering of enzyme conformational dynamics as a strategy is expected to guide
improvements in enantioselectivity based on previous studies; however, the practical application of
such a strategy for enzyme design remains a major challenge.
Candida antarctica lipase B (CALB; EC 3.1.1.3) is a member of the α/βꢀhydrolase family,
possesses the catalytic triad S105ꢀH224ꢀD187, which lies between the two binding pockets (the
acylꢀbinding pocket and the alcoholꢀbinding pocket), and is widely used in both academic and
industrial production.28ꢀ35 The esterification of 3ꢀtꢀbutylꢀdimethylꢀsilyloxy (TBDMSO) glutaric
anhydride to (R)ꢀ3ꢀTBDMSO glutaric acid methyl monoester, which is a multifunctional chiral
building block of statins in the pharmaceutical industry, is catalyzed by CALB and was developed
and optimized in a previous study (Scheme 1).36 However, the CALB mutant, EF5, obtained by
manipulating the substrate pocket exhibited high enantioselectivity toward (R)ꢀ1 [98.50%
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enantiomeric excess (ee)] at 5°C and a low R-ee value of 8.00% at 30°C, which hindered industrial
application. Here, we engineered CALB conformational dynamics to increase its enantioselectivity at
30°C. The most successful mutant, D223V/A281S (>99.00 ee at 30°C), was identified based on
structural analysis and molecular dynamics (MD) simulations to decrease the conformational
dynamics of the pocket and channel, thereby increasing enantioselectivity.
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Scheme 1 The esterification scheme catalyzed by CALB.
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RESULTS AND DISCUSSION
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Effect of Temperature on the R-enantioselectivity of the EF5 Mutant
In our previous study, we obtained a CALB mutant, EF5.36,37 Catalysis by the mutant EF5
showed an ee value and yield for (R)ꢀ3ꢀTBDMSO glutaric acid methyl monoester of 98.50% and
13.20%, respectively, at 5°C. As shown in Figure 1A, increasing the temperature (from 5°C to 30°C)
reduced EF5 R-enantioselectivity from 98.50% to 8.00% and, conversely, increased the yield from
13.20% to 80%. Furthermore, we investigated and compared the effect of temperature on the initial
formation rates of the R- and S-enantiomers (Figure 1B), finding that the initial formation rates of the
Rꢀ and Sꢀenantiomers and the VR/VS value for EF5 were 93.90 ± 4.35 µmol h−1, 79.99 ± 3.18 µmol
h−1, and 1.17, respectively, at 30°C as compared with values of 17.05 ± 1.24 µmol h−1, 0.13 ± 0.01
µmol h−1, and 131.15, respectively, at 5°C. These results demonstrated that the lower values of VR/VS
at 30°C caused a lower R-enantioselectivity; therefore, it is critical to increase the
R-enantioselectivity of the protein at the highꢀyield temperature of 30°C.
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Figure 1. Effect of temperature on the enantioselectivity (A) and initial formation rate (B) of the EF5 mutant.
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Engineering CALB Conformational Dynamics
To understand how temperature affects the initial formation rates of the R- and S-enantiomers
and, therefore, the enantioselectivity, MD simulations were performed for the EF5 mutant at 5°C and
30°C on the basis of R- or S-enantiomerꢀbound structures. As shown in Figure 2, the
rootꢀmeanꢀsquare fluctuations (RMSFs) of αꢀcarbons were calculated to analyze changes in
dynamics, resulting in increases in the RMSFs around regions 1, 2, and 3, the components of the
CALB active pocket and channel, at 30°C as compared with those at 5°C. Combining the results of
Figure 2 and Figure 1B, we deduced that high temperature led to an increase in the conformational
dynamics of the active pocket and channel (Figure 2), which enhanced the catalytic rate of the
S-enantiomer (Figure 1B), thereby decreasing the R-ee value. Therefore, modulating conformational
dynamics around the CALB active pocket and channel provided a potential strategy for increasing
Rꢀenantioselectivity at 30°C.
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Figure 2. RMSFs calculated from MD simulations for the EF5 mutant at 5°C and 30°C.
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We selected mutation candidates based on the analysis of the CALB active pocket (Figure 3)29
and the following considerations: to avoid large alterations in mainꢀchain dynamics and secondary
structure distortion, candidate mutations were mainly inserted within the loop regions of the pocket,
excluding proline and glycine.22 We chose six residues (D134, A148, V149, I189, V190, and Q157)
in region 2 (the acylꢀbinding pocket) and five residues (T42, T43, W104, A281, and A282) in regions
1 and 3 (the alcoholꢀbinding pocket and channel). Furthermore, residues D223 and T186, which are
adjacent to the catalytically active H224 and D187, respectively, and might affect enantioselectivity,
were also selected.
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Alanine scanning is an important method for protein engineering. Here, candidate residues
(except for alanine) were computationally substituted with alanine, and MD simulations involving
these mutations were performed at 30°C, with A281 and A282 were computationally substituted with
serine, which exhibits a similar size and different properties than alanine. The results presented in
Figure S1 showed that among the mutations, the RMSFs for D223A and A281S showed clear
changes around regions 1 and 2 and regions 1 and 3, respectively. Therefore, residues D223 and
A281 were identified for further mutational analyses.
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Figure 3. The CALB active pocket. CALB is traditionally considered as having two binding domains capable of
harboring esters: one for the acid (acyl) moiety and the other for the alcohol moiety of the ester. Changes in pocket
conformation might result in different substrateꢀbinding modes, thereby showing different selectivity. The acylꢀ and
alcoholꢀbinding pockets are shown in purple and brown, respectively. D223 and T186 are shown in blue. The triad
D187ꢀH224ꢀS105 lies between the two pockets. Residues A281, A282, and I285 point toward the alcohol moiety of
the ligand and limit the size of the channel.
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With the key residues D223 and A281 as candidates, these amino acids were subject to further
mutation, resulting in 38 candidate mutants subsequently analyzed by MD simulation (Figure S6).
Among these, the RMSFs of D223V and A281S were reduced around regions 1 and 2 and regions 1
and 3, respectively (Figure S2). Moreover, an MD simulation was also conducted for the double
mutant D223V/A281S, with more obvious decreases in the RMSFs around regions 1, 2, and 3
observed as compared with those observed in the single mutants (Figure S3). In particular, the double
mutant D223V/A281S exhibited the largest decrease in RMSF of nearly 1.1 Å around region 2 as
compared with that in the EF5 mutant (ꢁRMSF; Figure 4). Therefore, we selected D223V, A281S,
and D223V/A281S as the optimal mutants expected to enhance enantioselectivity at 30°C by
decreasing the conformational dynamics of the pocket and channel.
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Figure 4. Differences in the RMSFs (ꢁRMSF) of the mutants EF5 and A281S, D223V, and D223V/A281S.
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Computational Evaluation of CALB Mutants
Changes in the pockets and channels of the selected CALB mutants (D223V, A281S, and
D223V/A281S) were investigated using MD simulations under a constant temperature of 30°C
(Figure 5). Compared with those of the EF5 mutant, the conformational dynamics of the pocket and
channel of the D223V/A281S mutant were reduced, resulting in decreases in the hydrogenꢀbond
distance between the mainꢀchain amide of Q157 and the carboxyl group of the Rꢀenantiomer from
5.70 Å to 3.00 Å and the distance between H224 (the HE2 atom) and S105 (the OG atom) from 2.60
Å to 2.30 Å (Figure 5A and B). These results clearly indicated that decreasing the conformational
dynamics resulting in strengthening of the structural constraints of the pocket and channel.
Furthermore, we found that one important hydrogen bond between H224 and the oxygen of the
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Sꢀenantiomer was lost (Figure S7). Therefore, we compared the distances between H224 (the HE2
atom) and the oxygen (the O2 atom) of the Rꢀenantiomer (distance d1) and the Sꢀenantiomer (distance
d2), as well as the corresponding binding energies. As shown in Table S1, the CALB mutants (D223V,
A281S, and D223V/A281S) exhibited decreases in d1, increases in d2, and larger differences in the
binding energies of the Rꢀ and Sꢀenantiomers as compared with those of the EF5 mutant. When
compared with the corresponding values for the EF5 mutant (Figure 5), the d1 value in the
D223V/A281S mutant decreased from 3.50 Å to 2.60 Å, whereas the d2 value increased from 3.80 Å
to 4.30 Å. The distances separating the catalytic serine and the new ester O2 of the Rꢀenantiomer
(distance d3) and the Sꢀenantiomer (distance d4) showed similar changing trends as those of d1 and d2,
respectively (Table S4). The binding energy to the Rꢀenantiomer was –410.27 ± 9.30 kJ/mol, and that
of the Sꢀenantiomer was –392.78 ± 12.65 kJ/mol; therefore, the difference in the binding energies of
the two enantiomers increased to 17.49 kJ/mol from 3.86 kJ/mol in the EF5 mutant. The oxyanion
hole (Q106 and T40) plays an important role in stabilizing the transition state of the reaction. As
shown in Figure S7, the Rꢀenantiomer is favorably stabilized by formation of two hydrogen bonds
with Q106 and T40, respectively, whereas this occurs in the Sꢀenantiomer with only with T40. These
results clearly indicated that decreasing conformational dynamics led to a strengthening of the
structural constraints of the pocket and channel, which might represent the improvement in
R-enantioselectivity.
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Figure 5. Comparison and analysis of differences between R/Sꢀenantiomerꢀbound EF5 and D223V/A281S. (A) The
RꢀenantiomerꢀEF5 complex, (B) the RꢀenantiomerꢀD223V/A281S complex, (C) the SꢀenantiomerꢀEF5 complex,
and (D) the SꢀenantiomerꢀD223V/A281S complex. The hydrogenꢀbond distance between Q157 and the carboxyl
group of the Rꢀenantiomer is shown in red (A, B). The distance between H224 (the HE2 atom) and S105 (the OG
atom) is shown in blue (A, B). The distances (d1/d2) between H224 (the HE2 atom) and the bound R/Sꢀenantiomer
oxygen (the O2 atom) are shown in yellow (A–D).
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Experimental Evaluation of CALB Mutants
The mutants D223V, A281S, and D223V/A281S were constructed through siteꢀdirected
mutagenesis (Figure S4), and the initial formation rates of the R- and S-enantiomers of each mutant
were detected at 30°C (Table 1). As illustrated in Table 1, the VS values of the A281S, D223V, and
D223V/A281S mutants significantly decreased from 79.99 ± 3.18 in the EF5 mutant to 3.14 ± 0.04,
2.00 ± 0.01, and 0.46 ± 0.01 ꢂmol h−1, respectively. However, compared with that of the EF5 mutant,
the VR values decreased only slightly (Table 1). Additionally, the VR/VS values increased from 1.17
(the EF5 mutant) to 29.78 (A281S), 46.71(D223V), and 200.11(D223V/A281S). Overall, we
hypothesized that decreases in the dynamics of the pocket and channel resulting from mutations at
sites 223 and 281 might lead to a sharp decline in the initial formation rate of the Sꢀenantiomer,
thereby increasing Rꢀenantioselectivity at 30°C.
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Table 1 The initial formation rates of R- and Sꢀenantiomers catalyzed by CALB mutants at 30°C.
Initial formation rate [ꢂmol h−1]
Mutants
VR/VS
VR
VS
EF5
A281S
93.90 ± 4.35
93.52 ± 8.02
93.42 ± 2.35
92.05 ± 4.82
79.99 ± 3.18
3.14 ± 0.04
2.00 ± 0.01
0.46 ± 0.01
1.17
29.78
46.71
200.11
D223V
D223V/A281S
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The kinetic parameters of the CALB mutants were determined using optically pure R- and
S-enantiomers as substrates and compared, with the results are listed in Table 2. For the
D223V/A281S mutant, the kcat, KM, and kcat/KM values toward the Rꢀenantiomer were 5.60% higher,
34.00% lower, and 58.00% higher, respectively, than the corresponding values in the EF5 mutant.
The kcat, KM, and kcat/KM values toward the Sꢀenantiomer exhibited a 13.94% decrease, 30.86ꢀfold
increase, and 35.00ꢀfold decrease, respectively, as compared with the corresponding values in the
EF5 mutant. Furthermore, the esterification reactions (Scheme 1) catalyzed by the EF5, D223V,
A281S, and D223V/A281S mutants were investigated at a 3ꢀL scale at 30°C, with the results listed in
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Table 3. The conversion yields of (R)ꢀ3ꢀTBDMSO glutaric acid methyl monoester were >80.00%
using the CALB mutants at 30°C, and for the D223V/A281S mutant, the R-ee value and the
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spaceꢀtime yield were >99.00% and 107.54 g L−1 d−1
, respectively, at 30°C, whereas the R-ee value
and the spaceꢀtime yield of the EF5 mutant were 8.00% and 110.11 g L−1 d−1 at 30°C. As expected,
the experimental results demonstrated that increased R-enantioselectivity was achieved with the
D223V/A281S mutant at 30°C, consistent with results from MD simulations.
Table 2 Kinetic parameters of CALB mutants
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Rꢀenantiomer
Sꢀenantiomer
kcat/KM
[mM−1
s−1]
kcat/KM
[mM−1
s−1]
Mutant
Fold
Fold
kcat [s−1]
KM [mM]
kcat [s−1]
KM [mM]
change
change
EF5
4.96 ± 0.24
5.02 ± 0.33
5.13 ± 0.36
0.60 ± 0.01
0.43 ± 0.03
0.42 ± 0.02
0.40 ± 0.04
8.29
1.00
1.41
1.47
1.58
4.59 ± 0.15
4.52 ± 0.39
4.01 ± 0.12
3.95 ± 0.46
0.64 ± 0.02
11.59 ± 1.07
15.42 ± 0.98
19.75 ± 3.81
7.12
1.00
0.06
0.04
0.03
A281S
D223V
11.67
12.21
13.10
0.39
0.26
D223V/ A281S 5.24 ± 0.47
0.20
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Table 3 Production of Rꢀenantiomers by CALB mutants
Mutants
Temperature
Time [h]
eeR [%]
Conv. [%]
Spaceꢀtime yield
[g·L−1·d−1]
[
oC
5
]
EF5
EF5
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12
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12
98.50
8.00
70.49
82.17
81.23
79.75
80.25
18.89
30
30
30
30
110.11
108.85
106.87
107.54
A281S
93.50
95.80
>99.00
D223V
D223V/A281S
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CONCLUSIONS
In conclusion, the conformational dynamics of the CALB pocket and channel were engineered
to successfully overcome the low enantioselectivity of the EF5 mutant at high temperatures toward
(R)ꢀ3ꢀTBDMSO glutaric acid methyl monoester and increase the ee value at 30°C. The most
successful mutant, D223V/A281S, obtained following structural analysis and MD simulations
showed a high R-ee value (>99.00%), explained by computational evaluation, and a high spaceꢀtime
yield (107.54 g L−1 d−1). Mutations caused decreases in the conformational dynamics of the pocket,
thereby enhancing enzyme enantioselectivity toward the Rꢀform isomer, even at higher temperature.
By contrast, a recent study showed that mutations increased the flexibility around the binding pocket
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and induced higher activity in the thermophilic enzyme alcohol dehydrogenase at ambient
temperature.38 Although the design methods for mutants differed, these two studies both achieved
changes in the flexibility of the active pocket by mutation, thereby adjusting the effect of temperature
on enzyme properties. Our results lay the foundation for the industrialization of (R)ꢀ3ꢀTBDMSO
glutaric acid methyl monoester and provided insights for enzyme design guided by the engineering
of conformational dynamics to solve issues related to enzymes exhibiting satisfactory
enantioselectivity only at low temperatures and decreasing enantioselectivity at higher temperatures.
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EXPERIMENTAL SECTION
Materials
The expression plasmid pGAPZαA was purchased from Novagen (Madison, WI, USA). The
host strain Pichia pastoris GS115 was purchased from Invitrogen (Carlsbad, CA, USA). The
restriction enzymes (XbaI, XhoI, DpnI, and AVrII), T4 DNA ligase, primerSTAR polymerase,
plasmid miniprep kit, and agarose gel DNA purification kit were supplied by TaKaRa (Dalian,
China). The 3ꢀTBDMSO glutaric anhydride was purchased from Henan Yuchen Fine Co., Ltd.
(Henan, China). Hexane and isopropanol [highꢀperformance liquid chromatography (HPLC)ꢀgrade]
were purchased from Fisher Chemical (Fairlawn, NJ, USA). The (R)ꢀ3ꢀTBDMSO glutaric acid
methyl monoester standard and racemic 3ꢀTBDMSO glutaric acid methyl monoester were gifted
from Chanyoo Pharmatech Co., Ltd. (Nantong, China). A Daicel chiralpak ADꢀH column (4.6
× 250
mm) was purchased from Daicel Chiral Technologies Co., Ltd. (Shanghai, China). Other chemicals
and solvents (analytical grade) were obtained from local suppliers.
Molecular Modeling and MD Simulations
The theoretical structures of CALB were downloaded from the Protein Data Bank (PDB:
1TCA), and structures of the mutants were obtained by homology modeling.39 The threeꢀdimensional
structures of product enantiomers [(R)ꢀ1 and (S)ꢀ1] were obtained using the ChemOffice Ultra 11.0
program (CambridgeSoft; Perkin Elmer, Billerica, MA, USA). The tetrahedral intermediate, used as
the transition state, was manually bound in the active site. Partial charges and forceꢀfield parameters
for the substrates were automatically generated using the antechamber program using the general
AMBER force field (http://ambermd.org/). Nonpolar hydrogen atoms were added to the enzyme
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using the GROMACS 4.5.5 simulation package (http://www.gromacs.org/). The protonation state of
residues was set according to pH 7.0. Na+ counterions were added to neutralize the system, and the
whole system was immersed in a cubic box of TIP3P water molecules, which was extended 10 Å
from the dissolved atoms in all three dimensions. MD simulations were performed with GROMACS
4.5.5 and an AMBER03 force field following the three main steps of energy minimization, system
equilibration, and production protocols.40,41 Following steepestꢀdescent energy minimization, 10ꢀns
simulations were run at different temperatures in 2ꢀfs steps. All simulations were performed
individually for both (R)ꢀ or (S)-1ꢀbound structures, and the binding energies and distances for
hydrogen bonds were calculated. The enzyme and product were assigned to separate energy groups,
and the binding energies between them were calculated as total interꢀgroup potential energy.
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Construction, Purification, and Immobilization of the CALB Mutants
Polymerase chain reactions (PCR) were performed using the EF5 plasmid (pGAPZαAꢀEF5) as
a template and primers (Table S2). DpnI (1 ꢂL of 10 U ꢂL−1) was added to 25 ꢂL of the PCR
reaction mixture and incubated for 3 h at 37°C to eliminate the template plasmid. The digested
product was transformed into Escherichia coli JM109 cells for amplification. Plasmid pGAPZαA
mutants were obtained from E. coli JM109 cells, linearized by AvrII, purified, and transformed into
P. pastoris GS115. P. pastoris GS115 was inoculated into yeast extract peptone dextrose medium
(10 g L−1 yeast extract, 20 g L−1 peptone, and 20 g L−1 glucose) and grown at 30°C on a rotary shaker
(200 rpm) for 2 days. The supernatant was harvested by aspiration and purified on a nickelꢀaffinity
column (Ni Sepharose 6 Fast Flow; Amersham Biosciences, Freiburg, Germany) to analyze the
kinetic parameters.42 Enzyme purity was tested by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDSꢀPAGE) (Figure S4). The supernatants of the mutants were mixed with D101
macroporous resin (Shanghai Hualing Resin Factory, Shanghai, China) for immobilization at 37°C
for 5 h. The resin was filtered and dried under a vacuum, and the immobilized enzyme was used for
esterification in the organic phase.
Measurement of the Initial Formation Rate of Each Enantiomer for Esterification
For the esterification (ring opening), 1.23 mM 3ꢀTBDMSO glutaric anhydride and 1.23 mM
methanol were dissolved in acetonitrile (5 mL), followed by ultrasonic dispersion and the addition of
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enzyme (400 mg). The mixed system was shaken (200 rpm/min) at 5°C or 30°C, and at appropriate
times, samples were analyzed by HPLC. Several data points were collected to determine the initial
formation rate of each enantiomer. The activities of the immobilized mutant enzymes were
maintained at the same level.
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Measurement of the Kinetic Parameters of CALB Mutants
The kinetic parameters, including KM and kcat, of the R/Sꢀenantiomers were calculated by
measuring the initial rates of product formation at different concentrations of R/Sꢀenantiomers (1–20
mM) at 30°C (ring forming). Samples were withdrawn, extracted, and analyzed by HPLC, and all
assays were performed at least three times. The data were plotted, and KM and kcat values were
obtained by the double reciprocal method.
Determination of the ee Values and Conversion Rates of CALB Mutants
The reaction (ring opening) was performed with immobilized EF5 and new mutants (80 g L−1)
in acetonitrile containing 60 g L−1 3ꢀTBDMSO glutaric anhydride with methanol for 12 h at different
temperatures (5–30°C). The activity of each immobilized mutant enzyme was maintained at the same
level. The concentrations of (R)ꢀ3ꢀTBDMSO glutaric acid methyl monoester and the
enantioselectivity were determined by HPLC using a mobile phase consisting of 96% hexane and 4%
isopropanol with 0.02% (v/v) trifluoroacetic acid and filtered through a 0.22ꢀµm membrane before
use. Analysis (210 nm) was performed by injecting a 20ꢀµL sample into the chromatograph, with a
detection temperature of 25°C, a 1ꢀmL/min flow rate, and a sample detection time of 15 min. The ee
value was defined as follows: eeR = (R – S) / (R + S) × 100%, where R and S represent the
concentrations of the R- and Sꢀenantiomers, respectively.
CONFLICTS OF INTEREST
The authors declare no competing financial interest.
FUNDING SOURCES
Financial support from the National Natural Science Foundation of China (21422602), the National
Natural Science Foundation of China (21676118), the Special Foundation for State Key Research
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and Development Program of China (2016YFD0400801), and Jiangsu Province “333 Highꢀlevel
Talents Cultivating Project” (BRA2016365) is gratefully acknowledged.
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SUPPORTING INFORMATION
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Supporting results, including details of RMSFs of the candidates (Figures S1–S3), SDSꢀPAGE of
pure CALB mutants (Figure S4), chiral HPLC data of the esterification (Figure S5), details of
RMSDs of the candidates (Figure S6), hydrogenꢀbond interaction data in D223V/A281S (Figure S7),
hydrogenꢀbond distances and the binding energy of Rꢀ or Sꢀtetrahedral intermediates in CALB
mutants (Tables S1 and S4), primers used in this work (Table S2), information about mutant
immobilization (Table S3).
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