Enhancing enantioselectivity of Candida antarctica lipase B towards chiral sec-alcohols bearing small substituents through hijacking sequence of A homolog

Article abstract of DOI:10.1016/j.tetlet.2021.153186

Candida antarctica lipase B (CAL-B) exhibits extraordinary enantioselectivity towards most chiral sec-alcohols but not towards sec-alcohols bearing substituents smaller than a propyl group (i.e., (±)-but-3-yn-2-ol (E = 4) and (±)-butan-2-ol (E = 7)). Previously, we reported a homologous enzyme (lipase from Pseudozyma brasiliensis GHG001, PBL) of CAL-B, which exhibited high enantioselectivity of CAL-B towards (±)-but-3-yn-2-ol (E > 200). Based on the result, we hypothesized that the comparison of their local sequence or structure would provide a clue for enhancing the enantioselectivity of CAL-B. In this paper, we report enhancing enantioselectivity of CAL-B towards (±)-but-3-yn-2-ol and (±)-butan-2-ol through the substitution of the local sequence of CAL-B with that of PBL. The sequence-substituted mutant of CAL-B exhibited much higher enantioselectivity towards (±)-but-3-yn-2-ol (E > 200) and (±)-butan-2-ol (E > 25).

Full text of DOI:10.1016/j.tetlet.2021.153186

Journal Pre-proofs
Enhancing Enantioselectivity of Candida antarctica Lipase B towards Chiral
sec-Alcohols Bearing Small Substituents Through Hijacking Sequence of A
Homolog
Seonghyeon Yi, Seongsoon Park
PII:
S0040-4039(21)00403-2
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2021.153186
TETL 153186
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Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
25 March 2021
15 May 2021
17 May 2021
Please cite this article as: Yi, S., Park, S., Enhancing Enantioselectivity of Candida antarctica Lipase B towards
Chiral sec-Alcohols Bearing Small Substituents Through Hijacking Sequence of A Homolog, Tetrahedron
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Enhancing Enantioselectivity of Candida
antarctica Lipase B Towards Chiral sec-
Alcohols Bearing Small Substituents
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Through Hijacking Sequence of A Homolog
Seonghyeon Yi and Seongsoon Park*

1
Tetrahedron Letters
Enhancing Enantioselectivity of Candida antarctica Lipase B towards Chiral sec-
Alcohols Bearing Small Substituents Through Hijacking Sequence of A Homolog
Seonghyeon Yi and Seongsoon Park
Department of Chemistry, Center for NanoBio Applied Technology, Sungshin Women’s University, Seoul 01133, Republic of Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Candida antarctica lipase B (CAL-B) exhibits extraordinary enantioselectivity towards most
chiral sec-alcohols but not towards sec-alcohols bearing substituents smaller than a propyl group
(i.e., ()-but-3-yn-2-ol (E = 4) and ()-butan-2-ol (E = 7)). Previously, we reported a
homologous enzyme (lipase from Pseudozyma brasiliensis GHG001, PBL) of CAL-B, which
exhibited high enantioselectivity of CAL-B towards ()-but-3-yn-2-ol (E > 200). Based on the
result, we hypothesized that the comparison of their local sequence or structure would provide a
clue for enhancing the enantioselectivity of CAL-B. In this paper, we report enhancing
enantioselectivity of CAL-B towards ()-but-3-yn-2-ol and ()-butan-2-ol through the
substitution of the local sequence of CAL-B with that of PBL. The sequence-substituted mutant
of CAL-B exhibited much higher enantioselectivity towards ()-but-3-yn-2-ol (E > 200) and ()-
butan-2-ol (E > 25).
Available online
Keywords:
Candida antarctica lipase B
homologous enzyme
kinetic resolution
lipase
protein engineering
2021 Elsevier Ltd. All rights reserved.
Candida antarctica lipase B (CAL-B; also referred to as
Pseudozyma antarctica lipase B) is one of the most extensively
used biocatalysts in the preparation of enantiopure sec-alcohols
because of its extraordinary enantioselectivity, high chemo-
to resolve (±)-but-3-yn-2-ol has been continued. However, most
hydrolases exhibited low to moderate enantioselectivity (E = 1.0-
1
3, 14
26) as CAL-B does.
To improve enantioselectivity of a
hydrolase toward (±)-but-3-yn-2-ol, Bornscheuer and coworkers
have employed a combined approach of directed evolution and
site-direct mutagenesis on an esterase from Pseudomonas
1
tolerance, and thermo-stability. It has been known that CAL-B is
able to efficiently distinguish the difference in the size between a
methyl group and a substituent larger than an ethyl group, and
thus exhibits high enantioselectivity towards a broad range of
sec-alcohols (mostly E > 200; E, enantiomeric ratio defined by
1
5
fluorescens (PFE). They have improved the enantioselectivity
of PFE from E = 3 to 96 through screening over 7,000 clones, but
the enantiomeric ratio has not reached to 200. In this paper, we
report a successful result for enhancing enantioselectivity of
CAL-B towards (±)-but-3-yn-2-ol.
2
3
Shi et al. ). However, CAL-B does not obviously exhibit high
enantioselectivity towards all sec-alcohols. Especially, CAL-B
exhibits poor enantioselectivity towards sec-alcohol bearing
substituents smaller than a propyl group. For instance, the
enantiomeric ratios of CAL-B are 7 and 4 towards (±)-butan-2-ol
and (±)-but-3-yn-2-ol, respectively, while exhibiting E > 200
toward (±)-pentan-2-ol. To date, it has not been reported to
improve enantioselectivity of CAL-B towards sec-alcohols
bearing small substituents, and thus improving its
enantioselectivity towards such sec-alcohols has still remained a
challenging task.
Recently, we have found a homolog (lipase from Pseudozyma
brasiliensis GHG001, PBL) of CAL-B possessing higher
enantioselectivity towards (±)-butan-2-ol (E > 200) and (±)-but-
3
-yn-2-ol (E = 17), although its catalytic activity is much lower
1
6, 17
than that of CAL-B.
This result was rather surprising and
unexpected because PBL possesses a 72% sequence identity with
CAL-B, and thus it was expected that they share a common
structure and function or selectivity. Hence, we hypothesized that
the comparison of local sequences between PBL and CAL-B can
provide a clue to improve the enantioselectivity of CAL-B
towards (±)-butan-2-ol and (±)-but-3-yn-2-ol.
In addition, enantiopure but-3-yn-2-ol has been used as a
4
useful building block to synthesize various compounds including
bioactive compounds, such as cytotoxic butenolide (-)-
The enantioselectivity of lipases generally follows the
5
6
7
8
akolactone, (-)-Enigmazole A, Fenleuton, Frondosin B, HIV
1
8
9
10
Kazlauskas rule,
which provides an estimation of
protease inhibitor,
Meayamycin,
iodoalynes (+)-pancratistatin,
enantioselectivity of a lipase towards chiral sec-alcohols (see
below Figure 1). The rule is based on the structural features of
lipase related to the substrate-binding pockets. CAL-B also
follows the rule and exhibit (R)-selectivity. CAL-B possesses two
1
1
12
and tetrahydronaphtyridine.
Hence, the
preparation of enantiopure but-3-yn-2-ol has garnered much
attention, and the effort for identifying or developing a hydrolase
—
——

Corresponding author. Tel.: +82-2-920-7646; e-mail: spark@sungshin.ac.kr

2
Tetrahedron
Table 1. Enantioselective transesterification of (±)-butan-2-ol and (±)-but-3-yn-2-ola
5
D-CAL-B
CAL-B-42-47
Time Conv.
CAL-B-T42V/S47N
CAL-B-T42V
Time Conv.
CAL-B-S47N
Time Conv.
Substrate
Acyl donor Time Conv.
Time Conv.
b
E
E
E
E
E
(h)
(%)
(h)
45
(%)
(h)
53
(%)
(h)
44
(%)
(h)
26
(%)
2
3
2
3
8
41.9
5.7
7.2
5.4
4.0
38.7
12.9
25.7
30.5
13.2
24.9
41.8
6.7
8.0
4.4
3.2
33.9
14.0
4
44.2
52.2
39.0
35
48
25
31.5
37.6
40.6
48
40.1
32.1
39.4
20
46.3
37.0
40.0
20
23
17
43.9
40.4
45.1
30.7
rac-1a
rac-1b
16
4
> 200 60
> 200 43
> 200 48
> 200 20
> 200
> 200
aReaction condition: enzyme, 20 mg (1%, immobilized on Celite); rac-1a or 1b, 0.1 mmol; acyl donor (vinyl acetate 2 or vinyl
butanoate 3), 0.3 mmol, MTBE, 1 mL; 25ºC.
bEnantioselectivity was represented by the enantiomeric ratio defined by Sih et al.2
binding pockets to accept two different substituents (large and
CAL-B 39 GTGTTGPQSF 48
a)
small substituents) at the chiral carbon atom of sec-alcohol. One
is referred to as the large binding pocket, which is the entrance of
the substrate, and the other is referred to as the medium binding
pocket. The medium binding pocket is located in the deep inside
of CAL-B. The rule suggests that the medium binding pocket
accepts the smaller substituent rather than the larger one. The
previous experimental results demonstrated that CAL-B can
PBL
GTGVDGRQNF
b)
3
accept a substituent smaller than a propyl group. It means that
CAL-B cannot distinguish the difference in the size between a
methyl group and an ethyl or acetylene group. Hence, we
hypothesized that the difference of the sequences corresponding
to the medium binding pockets between PBL and CAL-B would
account for their distinct enantioselectivity. The medium binding
pocket of CAL-B is composed of Thr40, Thr42, Ser47, and
Trp104, while the corresponding residues of PBL are Thr40,
Val42, Asn47, and Trp104. Because the residue 104 is identical
in both enzymes, we compared the sequences of the region
between the residues 39-48. In the region between the residues
4
2T/V
47S/N
Figure 1. a) The sequence alignment of the stereospecific pocket between
CAL-B and PBL. b) A section image of an overlapped structure of CAL-B
and PBL. The catalytic triad (Asp187-His224-Ser105) and the residues (39-
3
9 and 48, the residues 42, 43, 45, and 47 of PBL are not
identical to those of CAL-B (Figures 1a and S1). Among these
residues, the side chains of the two residues 42 and 47 are
oriented to the active site (Figure 1b). As an initial approach, we
decided to replace the sequence of the residues 42-47 of CAL-B
with the corresponding sequence of PBL.
4
8) of the stereospecific pocket are represented by a stick model. The
stereospecific pocket residues of CAL-B are colored by yellow, while the
corresponding residues are colored by atomic elements. The arrow indicates
the stereospecificity pocket.
The corresponding gene for the residues 42-47 of PBL was
synthesized and introduced to replace the corresponding gene of
the template CAL-B (5D-CAL-B), which we previously prepared
to improve expression levels with identical enantioselectivity and
activity of the wild type CAL-B. The mutant gene was
transformed and functionally expressed in E. coli (Top10)
4
4
2
-5) (Table 1 and Figure S3). The enantioselectivity of CAL-B-
2-47 is similar to that of PBL towards (±)-butan-2-ol (E = 13 or
1
7
7) and (±)-but-3-yn-2-ol (E >200). The result indicates that
stereospecificity pocket influences the
altering the
1
9
enantioselectivity of CAL-B. It is worth noting that this high
enantioselectivity (E > 200) toward (±)-but-3-yn-2-ol has not
been achieved to date.
(
Figure S2). After immobilizing the mutant enzyme (CAL-B-42-
4
2
7) on celite, we have conducted transesterification of (±)-butan-
-ol and (±)-but-3-yn-2-ol with vinyl acetate or vinyl butyrate to
Besides, we decided to identify which residue is essential for
the improved enantioselectivity. We selected two residues (T42
and S47) of CAL-B as a candidate because the residues are
different from those of PBL (V and N, respectively). Moreover,
the side chains of the residues are oriented to the active site and
contributed to forming the stereospecificity pocket. Hence, we
hypothesized that the substitution of these two residues
presumably influences the binding of the substrate. We have
prepared a double mutant (CAL-B-T42V/S47N) and measured
determine the enantioselectivity. Interestingly, the mutant
enzyme exhibited higher enantioselectivity towards both
substrates compared to 5D-CAL-B. The enantioselectivity of the
mutant CAL-B-42-47 towards (±)-butan-2-ol and (±)-but-3-yn-2-
ol was noticeably improved about 3-4 times (E = 13 or 26) and
4
0-50 times (E > 200; > 99% ee for (R)-4b and (R)-5b,
respectively, compared to those of the 5D-CAL-B (E = 6-7 and

3
Table 2. Specific activity of CAL-B variants
a)
b)
Gln106
Ser105
His224
-1
-1
a
Thr40
specific activity (μmol·min ·mg )
Enzyme
D-CAL-B
CAL-B-S47N
(R)-but-3-yn-2-ol
(S)-but-3-yn-2-ol
5
.7 Å
4.8 Å
b
d
5
0.51 ± 0.12
0.25 ± 0.08
Ser47
Asn47
c
0.21 ± 0.02
0.0018 ± 0.0005
aThe specific activity was calculated based on the amount of
protein.
Figure 2. A proposed tetrahedral structure with (S)-but-3-yn-2-ol. a) 5D-
CAL-B. b) The CAL-B-S47N mutant. The substrate ((S)-but-3-yn-2-ol), the
catalytic residues (His224 and Ser105), and the oxyanion hole (Thr40 and
Gln106) are represented by a stick model. The solid lines represent the
distances between the Oγ atom of serine or the Oδ1 atom of asparagine and
the terminal carbon atom of the acetylene group.
bReaction condition for 5D-CAL-B: 5D-CAL-B, 10 mg (1%,
immobilized on Celite); (R)- or (S)-but-3-yn-2-ol, 0.1 mmol;
vinyl butanoate, 0.3 mmol, MTBE, 4 mL.
cReaction condition for CAL-B-S47N: CAL-B-S47N, 40 mg
(
1%, immobilized on Celite); (R)- or (S)-but-3-yn-2-ol, 0.1
The template 5D-CAL-B enzyme exhibited about 50% lower
specific activity for the slow enantiomer ((S)-but-3-yn-2-ol)
compared to the fast enantiomer ((R)-but-3-yn-2-ol), whereas the
CAL-B-S47N enzyme exhibited about 120 times lower specific
activity toward the slow enantiomer compared to the fast
enantiomer. These results clearly demonstrate that the
introduction of asparagine at the residue 47 makes the reaction
for the slower enantiomer much slower. Hence, it can be
concluded that the binding of the slower enantiomer in the CAL-
B-S47N mutant at the transition state is less favorable than that in
the template CAL-B.
mmol; vinyl butanoate, 0.3 mmol, MTBE, 4 mL.
dAll reactions were conducted at least three times and
averaged. The errors were represented by the standard deviation.
the enantioselectivity towards (±)-butan-2-ol as well as (±)-but-3-
yn-2-ol. As expected, the double mutant (CAL-B-T42V/S47N)
exhibited as high enantioselectivity as CAL-B-42-47 (Table 1).
This indicates that either both residues or one of the residues
serves the key role for the improved enantioselectivity. Hence,
we have prepared two single mutants (CAL-B-T42V and CAL-
B-S47N). The two mutant enzymes were successfully expressed,
and their enantioselectivity was measured. The two mutants
exhibited distinct enantioselectivity. The enantioselectivity of
CAL-B-T42V towards (±)-butan-2-ol and (±)-but-3-yn-2-ol was
E = 7-8 and 3-4, while the enantioselectivity of CAL-B-S47N
was E = 14-30 and >200, respectively. The result clearly
indicates which residue significantly influences the
enantioselectivity of CAL-B. Introducing asparagine at the
residue 47 noticeably improved the enantioselectivity of CAL-B,
especially toward (±)-but-3-yn-2-ol. The significantly improved
enantioselectivity toward (±)-but-3-yn-2-ol by introducing
asparagine at the residue 47 may be elucidated by one of the
following two rationales. One of the clarifications is that the
reaction rate for the fast enantiomer by the mutant enzyme
increased more than that by the template enzyme. On the other
hand, the reaction with the slow enantiomer by the mutant
enzyme becomes much slower than that by the template enzyme.
This can be clarified by measuring kinetic parameters for both
enantiomers. However, obtaining the kinetic parameters for the
slow enantiomer is difficult because the reaction rate of the slow
enantiomer is too low to be detected at the low concentration of
the substrate. Hence, we have compared the specific activity
towards the fast and slow enantiomers (Table 2). The specific
activities of the template 5D-CAL-B and CAL-B-S47N mutant
enzymes have been measured by the reactions towards (R)-but-3-
yn-2-ol and (S)-but-3-yn-2-ol with vinyl butanoate. Since the
CAL-B-S47N enzyme is less active than the 5D-CAL-B enzyme
Molecular modeling was conducted to understand the
influence of introducing asparagine at the residue 47 on the
improved enantioselectivity of the CAL-B-S47N mutant. After
the generation of the tetrahedral intermediates of the wild type
CAL-B and CAL-B-S47N mutant enzymes for the slow
enantiomer, the structures were energy minimized. However,
molecular modeling did not provide a rationale for explaining the
molecular basis of the improved enantioselectivity. Energy-
minimized structures for both enzymes are not much distinct
from each other except the distances between the terminal carbon
atom of the acetylene group and the Oγ atom of serine or the Oδ1
atom of asparagine (Table S1 in supporting information).
Although the distance between the Oδ1 atom of asparagine and
the terminal carbon of the acetylene group (4.8 Å) is shorter than
the distance between the Oγ atom of serine and the terminal
carbon atom (5.7 Å) (Figure 2), both distances are considered as
beyond optimum distance for van der Waals interaction. This
implies that asparagine itself may not significantly influence the
binding of the slow enantiomer because the side chain of
asparagine is still far from the slow enantiomer. One reasonable
hypothesis for the improved enantioselectivity is that water
molecules may hydrogen bond to the side chain of asparagine
and thus interrupt binding of the slow enantiomer at the transition
2
0
state.
In the current study, we demonstrated that the
enantioselectivity of CAL-B towards sec-alcohols bearing small
substituents can be improved by utilizing information from
homologous enzymes of CAL-B. Nature has divergently evolved
enzymes and so has produced various homologous enzymes.
Each evolved homologous enzyme may possess specific
characteristics for certain purposes. This implies that the detailed
comparison of the local sequence between homologous enzymes
may provide a clue to alter or improve enzyme function. We
adapted the sequence information of the medium binding pocket
from one of the homologous enzymes of CAL-B and replaced the
sequence of CAL-B with that. The created mutant enzyme
(
see Table 1), a larger amount of the CAL-B-S47N enzyme was
used to obtain reliable reaction rates. Reaction was carried out at
5°C in methyl tert-butylether (MTBE). The reaction mixtures
2
were retrieved within a particular interval of time, and the
reaction progress was analyzed by gas chromatography (GC).
The specific activities were calculated based on the amount of
protein used instead of the total amount of immobilized enzymes.

4
Tetrahedron
9
.
Yanada, R.; Koh, Y.; Nishimori, N.; Matsumura, A.; Obika, S.;
Mitsuya, H.; Fujii, N.; Takemoto, Y. J. Org. Chem, 2004, 69,
2
exhibits more than 50 times higher enantioselectivity toward
±)-but-3-yn-2-ol. Besides, we identified that the substitution of
(
417-2422.
0. Ko, H.; Kim, E. Park, J. E.; Kim, D.; Kim, S. J. Org. Chem. 2004,
9, 112-121.
serine 47 with asparagine is responsible for the improved
enantioselectivity through the preparation of a series of mutants.
However, improving the enantioselectivity by altering the residue
1
6
11. Gartshore, C.; Tadano, S.; Chanda, P. B.; Sarkar, A.; Chowdari,
N. S.; Gangwar, S.; Zhang, Q.; Vite, G. D.; Momirov, J.; Boger,
D. L. Org. Lett. 2020, 22, 8714-8719.
4
7 is unlikely to be rationally expected because the residue 47 is
located rather far away from the reaction center, although the
residue is deep inside of CAL-B. Hence, the current result is not
readily achievable by a conventional rational design approach for
protein engineering. And also, the directed evolution may not be
able to obtain the current results by screening a small number of
clones because many results show that most residues identified
by directed evolution are found on the surface of an enzyme.
Therefore, the current approach would be valuable in improving
or altering enzyme functions.
1
2. Hartner, F. W.; Hsiao, Y.; Eng, K. K.; Rivera, N. R.; Palucki, M.;
Tan, L.; Yasuda, N.; Hughes, D. L.; Weissman, S.; Zewge, D.;
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8723-8730.
13. Nakamura, K.; Takenaka, K.; Ohno, A. Lipase-catalyzed kinetic
resolution of 3-butyn-2-ol. Tetrahedron: Asymmetry 1998, 9,
4429-4439.
2
1
1
4. Baumann, M.; Hauerb, B. H.; Bornscheuer, U. T. Tetrahedron:
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15. Schmidt, M.; Hasenpusch, D.; Khler, M.; Kirchner, U.;
Wiggenhorn, K.; Langel, W.; Bornscheuer, U. T. ChemBioChem
2
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Acknowledgments
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This work was supported by the Sungshin Women's
University Research Grant of 2018-1-11-045/1.
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5
Highlights

Candida antarctica lipase B (CAL-B)
exhibits high enantioselectivity towards
sec-alcohols.




However, CAL-B shows low
enantioselectivity towards sec-alcohols
bearing small substituents.
A recently found homologous enzyme
of CAL-B exhibited high
enantioselectivity.
Substitution with the local sequence of
the homolog may improve the
enantioselectivity of CAL-B.
Enantioselectivity of the created mutant
CAL-B was higher than 50 times toward
but-3-yn-2-ol.