Synthetically useful variants of industrial lipases from: Burkholderia cepacia and Pseudomonas fluorescens

Article abstract of DOI:10.1039/c7ob01823a

Industrial enzymes lipase PS (LPS) and lipase AK (LAK), which originate from Burkholderia cepacia and Pseudomonas fluorescens, respectively, are synthetically useful biocatalysts. To strengthen their catalytic performances, we introduced two mutations into hot spots of the active sites (residues 287 and 290). The LPS-L287F/I290A double mutant showed high catalytic activity and enantioselectivity for poor substrates for which the wild-type enzyme showed very low activity. The LAK-V287F/I290A double mutant was also an excellent biocatalyst with expanded substrate scope, which was comparable to the LPS-L287F/I290A double mutant. Thermodynamic parameters were determined to address the origin of the high enantioselectivity of the double mutant. The ΔΔH^? term, but not the ΔΔS^? term, was predominant, which suggests that the enantioselectivity is driven by a differential energy associated with intermolecular interactions around Phe287 and Ala290. A remarkable solvent effect was observed, giving a bell-shaped profile between the E values and the log&P or ? values of solvents with the highest E value in i-Pr₂O. This suggests that an organic solvent with appropriate hydrophobicity and polarity provides the double mutant with some flexibility that is essential for excellent catalytic performance.

Full text of DOI:10.1039/c7ob01823a

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: K. Yoshida, M.
Ono, T. Yamamoto, T. Utsumi, S. Koikeda and T. Ema, Org. Biomol. Chem., 2017, DOI:
10.1039/C7OB01823A.
This is an Accepted Manuscript, which has been through the
Volume 14 Number
1 7 January 2016 Pages 1–372
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-0520
COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
rsc.li/obc

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 7
View Article Online
DOI: 10.1039/C7OB01823A
Journal Name
ARTICLE
Synthetically useful variants of industrial lipases from
Burkholderia cepacia and Pseudomonas fluorescens
Kazunori Yoshida,a,b Masakazu Ono,a Takahiro Yamamoto,a Takashi Utsumi,a Satoshi Koikeda*b
Received 00th January 20xx,
Accepted 00th January 20xx
and Tadashi Ema*a
DOI: 10.1039/x0xx00000x
Industrial enzymes called lipase PS (LPS) and lipase AK (LAK), which originate from Burkholderia cepacia and Pseudomonas
fluorescens, respectively, are synthetically useful biocatalysts. To strengthen their catalytic performances, we introduced
two mutations into hot spots of the active sites (residues 287 and 290). The LPS_L287F/I290A double mutant showed high
catalytic activity and enantioselectivity for poor substrates for which the wild-type enzyme showed very low activity. The
LAK_V287F/I290A double mutant was also an excellent biocatalyst with expanded substrate scope, which was comparable
to the LPS_L287F/I290A double mutant. Thermodynamic parameters were determined to address the origin of the high
enantioselectivity of the double mutant. The ∆∆H‡ term, but not the ∆∆S‡ term, was predominant, which suggests that the
enantioselectivity is driven by a differential energy associated with intermolecular interactions around Phe287 and Ala290.
A remarkable solvent effect was observed, giving a bell-shaped profile between the E values and the log P or e values of
solvents with the highest E value in i-Pr2O. This suggests that organic solvent with appropriate hydrophobicity and polarity
provides the double mutant with some flexibility that is essential for the excellent catalytic performance.
www.rsc.org/
(a)
(b)
O
NH
BCL14595: Ile287
LPS: Leu287
LAK: Val287
–
O
Introduction
H
N
H
Phe287
CH3
O
Enzymes show high catalytic activity and stereoselectivity under
mild conditions. Industrial enzymatic processes are widely
accepted from the viewpoint of environmental harmony and
sustainability.1 Because of the limited diversity of natural
enzymes, new technologies have been developed to alter the
structure and property of enzymes.2 The directed evolution
method can evolve an enzyme stepwise using random
mutagenesis and a high-throughput screening system. Although
no information about the enzyme structure and the reaction
mechanism is necessary for directed evolution, a large number
of variants need to be screened. On the other hand, when the
enzyme structure and the reaction mechanism are known, a
rational design approach with site-directed mutagenesis is
effective and efficient.
N
δ+
O
H
H
NH
H
N
X
Ala290
N
H
H
H
O
Ser87
X
Ile290
R1
O
O
O
N
O
H
Ph
Ser87
HN
O
R2
H
H
O
O
δ–
HN
H
O
N
N
O
HN
bulkiness
R1 > R2 : faster-reacting enantiomer
R1 < R2 : slower-reacting enantiomer
Fig. 1 (a) The transition-state model to explain the enantioselectivity of lipase toward
secondary alcohols (residues 287 and 290 are added to the original version). (i) The C–O
bond of the substrate takes the gauche conformation with respect to the breaking C–O
bond, which is due to the stereoelectronic effect. (ii) The H atom attached to the
stereocenter of the substrate is syn-oriented toward the carbonyl O atom to minimize
the torsional strain. Enantioselectivity is explained by the conformational requirements
and repulsive interactions and/or strains. Typically, the (R)-enantiomer reacts faster
because, in this favorable conformation shown in blue, the larger substituent (R1) can be
directed toward external solvent without severe strain and/or steric hindrance. (b) The
catalytic activity of the BCL14595_I287F/I290A double mutant for (R)-1-phenyl-1-
hexanol is enhanced by introducing attractive CH/p interactions and removing steric
hindrance.
OH
OCOR
OH
lipase
+
Ar
Ar
Ar
acylating agent
organic solvent
rac
(R)
(S)
OCOR
Ar
OH
OCOR
lipase
H2O
+
Ar
Ar
rac
(R)
(S)
Scheme 1. Typical good substrates for lipases, where R and Ar designate the alkyl group
and the aromatic/large substituent, respectively.
Lipases are synthetically useful biocatalysts that show high
catalytic activity and enantioselectivity for a broad range of
unnatural substrates in both water and organic solvent.1a,3 In
particular, they exert high enantioselectivity for various
secondary alcohols (Scheme 1). We performed mechanistic
studies based on kinetic and thermodynamic analysis, X-ray
crystal structures, and MO calculations and proposed a
transition-state model (Fig. 1a) to explain the origin of
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 7
ARTICLE
Journal Name
enantioselectivity of lipases for secondary alcohols.4 The and NMR, and the reaction was stopped by filtratiVoienw. AArtciceletaOtnelin2e
transition-state model essentially represents a mechanism by and alcohol
were separated byDOsI:il1i0c.a1039g/eCl7OBco01lu82m3An
1
which the slower-reacting (S)-enantiomer is disfavored. We also chromatography. The enantiomeric purity was determined by
introduced point mutation(s) into the active site of a lipase to GC, HPLC, or NMR, and the E value was calculated according to
rationally control the enantioselectivity.5
the literature.8 The results are shown in Tables 1 and 2, where
We redesigned a Burkholderia cepacia NBRC14595 lipase, the reaction rates can be compared because the same amounts
BCL14595, to create a more useful double mutant, which is of enzyme and substrate were used in all cases.
herein called BCL14595_I287F/I290A.5b,c The wild-type
BCL14595 showed very low activity for 1-phenyl-1-hexanol,
while the BCL14595_I287F/I290A double mutant showed high
activity and enantioselectivity for this secondary alcohol. We
OH
OAc
OH
lipase
AcOCH=CH2
+
R
R
'
R
R
'
R
R'
i-Pr2O, 30 °C
rac-1
2
1
OH
OH
OH
OH
have proposed that the reaction is accelerated by the CH/
p
interactions between Phe287 and the alkyl chain of the (R)-
enantiomer (Fig. 1b).5b,c,6 On the other hand, steric repulsion
takes place between Phe287 and the benzene ring of the (S)-
enantiomer (not shown). Phe287 is thus considered to have
dual mode interactions with the two enantiomers, improving
both catalytic activity and enantioselectivity. In addition, the
I290A mutation removes steric hindrance to accelerate the
reaction of the (R)-enantiomer (Fig. 1b). The wild-type
BCL14595 showed very low activity with an E value of 5 for 1-
phenyl-1-hexanol, while the BCL14595_I287F/I290A double
mutant showed much higher activity with a high E value of >200.
We employed E. coli for the heterologous expression of the
BCL14595 gene and successfully converted a denatured protein
(inclusion body) into an active enzyme by in vitro refolding with
a separately overproduced activator (chaperon), which is
however unsuitable for large-scale preparation.5 In contrast, an
industrial enzyme called lipase PS (LPS, Amano Enzyme Inc.),
which is a homologous protein of BCL14595 with 13 different
amino acid residues (96% homology), is produced on a large
scale with a B. cepacia expression system. Active LPS can be
secreted into a culture broth.7 Here we prepared both the
LPS_wild-type enzyme and the LPS_L287F/I290A double mutant
using the B. cepacia expression system and compared their
catalytic properties. We also investigated another industrial
enzyme called lipase AK (LAK, Amano Enzyme Inc.), which
originates from Pseudomonas fluorescens. LAK has 35 different
amino acid residues (89% homology) as compared with
BCL14595 or LPS. We compared the enzymatic characteristics
of the LAK_wild-type enzyme and the LAK_V287F/I290A double
mutant. Substrate mapping revealed excellent catalytic
performances (expanded substrate scope) of the
LPS_L287F/I290A and LAK_V287F/I290A double mutants. The
temperature effect and solvent effect were investigated to
address the origin of the high enantioselectivity of the double
mutant.
1a
1b
1c
1d
OH
OH
OH
Cl
1e
1g
1f
OH
OH
OH
R
CF3
EtO2C
1j: R = H
1k: R = Ph
1h
1i
OH
OH
N
TMS
S
1l
1m
Scheme 2 Lipase-catalyzed kinetic resolution of 1.
Table 1 Substrate scope of the LPS_L287F/I290A double mutant and the LPS_wild-type
enzymea
Time
(h)
1
L287F/I290A
Wild-type
Entry
1
2
3
4
5
6
7
8
1
1a
1b
1c
1d
1e
1f
1g
1h
1i
c (%)b
Ec
>200
>200
90
>200
>200
>200
>200
105
c (%)b
Ec
>200
119
>200
>130
>200
>200
>200
43
117
–
–
–
–
40
50
37
50
51
40
45
39
40
16
50
49
49
39
4
2
1
42
35
53
10
15
2.5
96
24
2
46
36
24
19
49
9d
9
31
10
11
12
13
1j
1k
1l
>200
>200
>200
113
5d
3
2
5d
1m
1.5
10d
a Reaction conditions: immobilized lipase (200 mg, 0.5% (w/w) enzyme/Toyonite-
200M), 1 (0.50 mmol), vinyl acetate (1.0 mmol), molecular sieves 3A (three pieces),
b
c
dry i-Pr2O (5 mL), 30 °C. Conversion calculated from c = ee(1)/(ee(1) + ee(2)).
Calculated from E = ln[1 – c(1 + ee(2))]/ln[1 – c(1 – ee(2))]. d Conversion calculated
from 1H NMR.
The results of kinetic resolution of
enzyme and the LPS_L287F/I290A double mutant are shown in
Table 1. Alcohols 1a with a small substituent such as the
methyl group were resolved well by the double mutant as well
as the wild-type enzyme in most cases (entries 1–5). The double
mutant exhibited superior activity for 1a and a comparable E
value as compared with the wild-type enzyme (entry 1).
1 with the LPS_wild-type
Results and discussion
The recombinant enzymes prepared and purified as described
in the ESI were immobilized on Toyonite-200M according to the
–
e
literature.5 A mixture of secondary alcohol
1, the immobilized
enzyme, and molecular sieves 3A in i-Pr2O was stirred at 30 °C
for 30 min, and vinyl acetate was added to start the reaction
(Scheme 2). The progress of the reaction was monitored by TLC
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 7
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
ARTICLE
Table 2 Substrate scope of the LAK_V287F/I290A double mutant and the LAK_wild-type
Although the double mutant showed slightly lower activity for
1b than the wild-type enzyme, the enantioselectivity of the
former was improved (entry 2). In the case of 1c, the catalytic
activity of the double mutant was improved but with a drop of
the E value (entry 3). We consider that the pocket comprising
Phe287 and Ala290 (Fig. 1b) attracts the methylene chain of (S)-
View Article Online
enzymea
DOI: 10.1039/C7OB01823A
Time
(h)
1
V287F/I290A
Wild-type
Entry
1
2
3
4
5
6
7
8
1
1a
1b
1c
1d
1e
1f
1g
1h
1i
c (%)b
Ec
c (%)b
Ec
>200
78
50
42
49
43
25
43
43
48
41
50
46
50
48
>200
>200
55
>200
>200
>200
>200
90
>200
>200
>200
>200
134
47
50
47
50
49
35
26
45
43
4d
4
2.5
0.25
10
24
3
60
12
1.5
4
>200
>200
>200
>200
>200
4
30
–
–
–
1c to enhance the reactivity of (S)-1c
enantioselectivity. Alcohols 1f with a substituent that is
slightly larger than the methyl group were also examined. The
double mutant achieved higher conversions for 1f and higher
, lowering the
–
i
–
h
enantioselectivity for 1h than the wild-type enzyme (entries 6–
8). This outcome for 1h was unexpected because a fluorine-
containing substrate has previously exhibited a dropped
9
10
11
12
13
1j
1k
1l
enantioselectivity because of the lack of CH/p
interactions.5b,c
6d
6d
The double mutant showed lower activity and
enantioselectivity for 1i than the wild-type enzyme (entry 9). It
is likely that the cleft comprising Phe287 and Ala290 (Fig. 1b)
cannot accommodate well the ethyl ester group of (R)-1i. To our
delight, the double mutant showed much higher activity and
3
1.5
1m
27
8
a Reaction conditions: immobilized lipase (200 mg, 0.5% (w/w) enzyme/Toyonite-
200M), 1 (0.50 mmol), vinyl acetate (1.0 mmol), molecular sieves 3A (three pieces),
b
c
dry i-Pr2O (5 mL), 30 °C. Conversion calculated from c = ee(1)/(ee(1) + ee(2)).
enantioselectivity for 1j
–
m
, for which the wild-type enzyme Calculated from E = ln[1 – c(1 + ee(2))]/ln[1 – c(1 – ee(2))]. d Conversion calculated
from 1H NMR.
showed very low activity (entries 10–13). The trimethylsilyl
group or the thiazole ring had a good influence on the outcome
(entries 12,13).
(a)
(b)
(c)
Leu287
Phe287
Leu287
Ile290
Phe287
Ala290
Asp264
His286
Ile290
Ala290
Ser87
Val287
Phe287
(d)
(e)
(f)
Val287
Ile290
Phe287
Ala290
Asp264
His286
Ile290
Ala290
Ser87
Fig. 2 Electrostatic potential maps of the active sites of (a) the LPS_wild-type enzyme, (b) the LPS_L287F/I290A double mutant, (d) the LAK_wild-type enzyme, and (e) the
LAK_V287F/I290A double mutant. Superimposed views of the active sites of (c) the LPS_wild-type enzyme (blue) and the LPS_L287F/I290A double mutant (red) and (f) the LAK_wild-
type enzyme (blue) and the LAK_V287F/I290A double mutant (red). Each of (a)–(c) and (d)–(f) is seen from the same direction.
The results of kinetic resolution of
1
with the LAK_wild-type
7). The E values of the double mutant for 1h–i were much
enzyme and the LAK_V287F/I290A double mutant are shown
in Table 2. The double mutant and the wild-type enzyme
showed comparable enantioselectivities for 1a (entry 1). The
double mutant showed higher enantioselectivity for 1b than
the wild-type enzyme (entry 2), whereas the former gave a
lower E value for 1c than the latter (entry 3). The double
improved (entries 8–9). Interestingly, the V287F/I290A double
mutations in LAK enhanced enantioselectivity for 1i (Table 2,
entry 9) although the L287F/I290A double mutations in LPS
decreased enantioselectivity (Table 1, entry 9). The
LAK_V287F/I290A double mutant is a useful biocatalyst
because 1h
type enzyme.9,10 Furthermore, the double mutant exhibited
high activity and enantioselectivity for 1j , for which the
–i are reported to be poor substrates for a wild-
mutant showed excellent enantioselectivity for 1d
wild-type enzyme did (entries 4,5). The double mutant showed
higher activity for 1f than the wild-type enzyme (entries 6–
–e as the
–
m
–
g
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 7
ARTICLE
Journal Name
Table 3 Temperature effect in the kinetic resolution of 1m with the LPS_L287F/I290A
wild-type enzyme showed poor activity and enantioselectivity
(entries 10–13).
View Article Online
double mutant and the LPS_wild-type enzymea
DOI: 10.1039/C7OB01823A
We performed molecular modeling (MOE, MOLSYS Inc.) to
understand the catalytic behaviors of the wild-type enzymes
and the double mutants of LPS and LAK. The structure of LPS
was obtained by refining the X-ray crystal structure (PDB: 1OIL),
and that of LAK was constructed by homology modeling using
LPS as a template. The double mutants of LPS and LAK were
then generated from the corresponding wild-type enzymes.
The active sites of these lipases are shown in Fig. 2. The
LPS_wild-type enzyme with Leu287 has a narrow pocket as
compared with the LAK_wild-type enzyme with Val287, which
can account for a tendency that LPS is more enantioselective
than LAK (Tables 1 and 2). The active-site pockets of the double
mutants of LPS and LAK are deeper around residue 290 than
those of the corresponding wild-type enzymes, the former of
which can accommodate the substituents that are larger than
the methyl group. Although it is reasonable that the double
mutants of LPS and LAK with similar pockets in size and shape
showed similar catalytic properties, the irregular behaviors of
LPS and LAK toward 1i (entry 9 in Tables 1 and 2) may result
from the different electrostatic potentials of their active sites
(Fig. 2).
LPS
T (°C)
30
35
40
45
50
30
35
40
Time (h)
c (%)b
50
33
41
44
50
40
41
Ec
57
46
39
36
26
4.9
4.1
3.9
3.6
3.3
L287F/I290A
L287F/I290A
L287F/I290A
L287F/I290A
L287F/I290A
wild-type
wild-type
wild-type
wild-type
wild-type
2
1
1
1
1
7
5
4
4
4
39
44
53
45
50
a Reaction conditions: immobilized lipase (200 mg, 0.5% (w/w) enzyme/Toyonite-
200M), 1m (0.50 mmol), vinyl acetate (1.0 mmol), dry i-Pr2O (5 mL), molecular
b
sieves 3A (three pieces). Conversion calculated from c = ee(1m)/(ee(1m) +
ee(2m)). c Calculated from E = ln[1 – c(1 + ee(2m))]/ln[1 – c(1 – ee(2m))].
4.5
4.0
3.5
y = 3531.6x - 7.6143
R² = 0.96812ꢀ
3.0
ꢀ
2.5
The mechanism of enantioselectivity can be inspected by
thermodynamic analysis.4c Plot of ln E against 1/T according to
equation 1 gives the ∆∆H‡ and ∆∆S‡ values.11
2.0
1.5
1.0
0.5
y = 1811.1x - 4.4212
R² = 0.95392ꢀ
ln E = –∆∆H‡/(RT) + ∆∆S‡/R
(eq 1)
The ∆∆H‡ and ∆∆S‡ values represent the differences in
activation enthalpy (∆H‡) and entropy (∆S‡), respectively,
between the faster-reacting and slower-reacting enantiomers
(equations 2,3).
0.00308 0.00313 0.00318 0.00323 0.00328
1/Tꢀ
Fig. 3 Temperature effect on the enantioselectivity in the kinetic resolution of 1m with
the LPS_L287F/I290A double mutant (circle) and the LPS_wild-type enzyme (square) in
i-Pr2O.
∆∆H‡ = ∆H‡fast – ∆H‡
∆∆S‡ = ∆S‡fast – ∆S‡
(eq 2)
(eq 3)
slow
slow
The ∆H‡ value involves a change of the energy associated with
covalent bonds, strain, and intermolecular interactions, while
the ∆S‡ value is associated with a change of the disorder of the
system. If the enantioselectivity of the double mutant is
enhanced by the additional attractive interaction and steric
repulsion (Fig. 1), the ∆∆H‡ value for the double mutant should
be negatively larger than that for the wild-type enzyme. We
selected a combination of LPS (wild-type enzyme and the
L287F/I290A double mutant) and 1m because of the moderate
to good E values and determined the thermodynamic values
(∆∆H‡ and ∆∆S‡) from the E values at 30–50 °C according to
equation 1. The results are summarized in Tables 3 and 4 and
Fig. 3.
Table 4 Thermodynamic parameters for the kinetic resolution of 1m with the
LPS_L287F/I290A double mutant and the LPS_wild-type enzyme in i-Pr2O
∆∆H‡
(kcal·mol–1)
–7.02
∆∆S‡
(cal·K–1·mol–1)
–15.1
∆∆G‡
(kcal·mol–1)a
–2.43
L287F/I290A
wild-type
–3.60
–8.8
–0.94
a Calculated from ∆∆G‡ = ∆∆H‡ – 303∆∆S‡.
In both cases, the ∆∆H‡ value is a dominant factor in the
∆∆G‡ value, which indicates that enantioselectivity is driven by
a differential energy associated with covalent bonds, strain,
and intermolecular interactions (Table 4). The ∆∆H‡ value of
the double mutant is two times negatively larger than that of
the wild-type enzyme. The attractive interaction between
Phe287 and the alkyl chain of (R)-1m would decrease the
∆H‡ value, and steric repulsion between Phe287 and the
fast
thiazole ring of (S)-1m would increase the ∆H‡slow value, both
of which give a negatively larger ∆∆H‡ value (equation 2). Table
4 also indicates a partial compensation effect; the ∆∆H‡ value,
which becomes negatively larger, is counterbalanced by the
∆∆S‡ value, which also becomes negatively larger.4c Steric
repulsion between Phe287 and (S)-1m favors the ∆∆H‡ term
because the ∆H‡slow value becomes larger, whereas it disfavors
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 7
Organic & Biomolecular Chemistry
Please do not adjust margins
Journal Name
the ∆∆S‡ term because the ∆S‡
ARTICLE
value increases with an
(Fig. 4a). A bell-shaped profile with a peak at i-Pr2O is also seen
slow
View Article Online
DOI: 10.1039/C7OB01823A
increase in the disorder of the system. The CH/
p
interaction
when the E values are plotted against the e values (Fig. 4b). It
between Phe287 and (R)-1m as well as the removal of steric
hindrance between Ala290 and (R)-1m also favor the ∆∆H‡
term because the ∆H‡fast value becomes smaller, whereas they
disfavor the ∆∆S‡ term because the ∆S‡fast value decreases with
a decrease in the disorder of the system. Therefore, the trends
observed for the ∆∆H‡ and ∆∆S‡ values are consistent with the
transition-state model (Fig. 1).
is likely that the polarity of the solvent also affects the protein
flexibility and that the highest E value is achieved in the solvent
giving the lipase appropriate flexibility.
Conclusions
Industrial enzymes called lipase PS (LPS) and lipase AK (LAK),
which originate from Burkholderia cepacia and Pseudomonas
fluorescens, respectively, are synthetically useful biocatalysts.
To strengthen their catalytic performances, we introduced two
mutations into the hot spots of the active sites (residues 287
and 290). The LPS_L287F/I290A double mutant showed high
catalytic activity and enantioselectivity for poor substrates for
which the wild-type enzyme showed very low activity. This
double mutant also exhibited high catalytic activity and
enantioselectivity for good substrates of the wild-type enzyme.
Clearly, the substrate scope of the double mutant has been
broadened. It should be emphasized again that sterically
demanding substrates possessing two bulky substituents on
both sides are usually poor substrates.15 The
LAK_V287F/I290A double mutant is also an excellent
biocatalyst with expanded substrate scope, which was
comparable to the LPS_L287F/I290A double mutant. Although
the two double mutants were equally excellent on the whole,
some differences were also observed between them. It is
therefore recommended that the better one be selected on a
case-by-case basis. The enantioselectivity of the
LPS_L287F/I290A double mutant was driven by the differential
activation enthalpy (∆∆H‡), and this ∆∆H‡ value for the double
mutant was negatively larger than that for the wild-type
enzyme, both of which suggest that attractive interactions
and/or steric repulsion are used for chiral discrimination in the
transition state. Bell-shaped profiles with a peak at i-Pr2O were
obtained when the E values for the double mutant were
Table 5 Solvent effect in the kinetic resolution of 1m with the LPS_L287F/I290A double
mutanta
Solvent
1,4-dioxane
acetone
THF
Et2O
i-Pr2O
toluene
cyclohexane
hexane
log P
–1.1
–0.23
0.49
0.85
1.9
2.5
3.2
3.5
e
2.2
21
7.5
4.3
3.4
2.4
2.0
1.9
Time (h)
c (%)b
38
11
19
37
50
50
42
Ec
9
48
48
48
11
2
5
1
0.5
7
11
27
57
42
50
24
38
a
Reaction conditions: LPS_L287F/I290A double mutant (200 mg, 0.5% (w/w)
enzyme/Toyonite-200M), 1m (0.50 mmol), vinyl acetate (1.0 mmol), dry organic
solvent (5 mL), molecular sieves 3A (three pieces), 30 °C. b Conversion calculated
c
from c = ee(1m)/(ee(1m) + ee(2m)). Calculated from E = ln[1 – c(1 +
ee(2m))]/ln[1 – c(1 – ee(2m))].
(a) 60
(b)
60
50
40
30
20
10
0
50
40
30
20
10
0
-2
-1
0
1
2
3
4
0
2
4
6
8
10 12 14 16 18 20 22
log P
permittivity (e)
Fig. 4 The solvent effect in the LPS_L287F/I290A-catalyzed kinetic resolution of 1m. (a)
The correlation between the E value and the log P value of the solvent. (b) The
correlation between the E value and the permittivity (e) of the solvent.
plotted against the log P or e values of organic solvents, which
suggests that appropriate protein flexibility is essential for the
excellent catalytic performances. The LPS_L287F/I290A and
LAK_V287F/I290A double mutants will find many applications
in the kinetic resolution and dynamic kinetic resolution of
various chiral alcohols.5c,16
The solvent effect is often remarkable and even provides a
valuable insight into the mechanism of biocatalysis.12 We
therefore investigated the solvent effect on the kinetic
resolution of 1m with the LPS_L287F/I290A double mutant
(Table 5). The best solvent was found to be i-Pr2O. The log P
value, which is the logarithm of a partition coefficient P of a
solvent between 1-octanol and water, is a measure of
hydrophobicity of the solvent.13 Table 5 indicates that the E
value and the reaction rate sharply decreased with a decrease
of the log P value. The relationships between the E value and
Conflicts of interest
There are no conflicts of interest to declare.
the log P value or permittivity (e
) are plotted in Fig. 4.14 We
Acknowledgements
We are grateful to Toyo Denka Kogyo Co. for the kind gift of
Toyonite-200M.
speculate that hydrophilic solvent such as 1,4-dioxane
deprives the lipase of the essential water, which lowers the
protein flexibility that is essential for the catalytic activity.12a In
contrast, hydrophobic solvent such as hexane enables the
lipase to retain the essential water, which keeps the flexibility
of the protein. The E value was the highest in i-Pr2O, where the
lipase is considered to have the most appropriate flexibility
Notes and references
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 6 of 7
ARTICLE
Journal Name
1
(a) U. T. Bornscheuer and R. J. Kazlauskas, Hydrolases in
combination of mutation and chemical modificVaietwioAnrt,icslee eO:nl(inde)
DOI: 10.1039/C7OB01823A
(a) M. Nishio, M. Hirota and Y. Umezawa, The CH/p
Organic Synthesis, Wiley-VCH, Weinheim, 2nd edn, 2006; (b)
Biocatalysis in the Pharmaceutical and Biotechnology
Industries, ed. R. N. Patel, CRC, Florida, 2007; (c) K. Faber,
Biotransformations in Organic Chemistry, Springer-Verlag,
Berlin, 6th edn, 2011; (d) Practical Methods for Biocatalysis
and Biotransformations 3, ed. J. Whittall, P. W. Sutton and
W. Kroutil, John Wiley & Sons, West Sussex, 2016.
T. Ema and H. Inoue, Chem. Lett., 2015, 44, 1374–1376.
6
Interaction: Evidence, Nature, and Consequences; Wiley-
VCH: New York, 1998; (b) M. Nishio, Y. Umezawa, J. Fantini,
M. S. Weiss and P. Chakrabarti, Phys. Chem. Chem. Phys.,
2014, 16, 12648–12683.
Y. Hirose, K. Kariya, Y. Nakanishi, Y. Kurono and K. Achiwa,
Tetrahedron Lett., 1995, 36, 1063–1066.
C.-S. Chen, Y. Fujimoto, G. Girdaukas and C. J. Sih, J. Am.
Chem. Soc., 1982, 104, 7294–7299.
A. Ghanem and V. Schurig, Tetrahedron: Asymmetry, 2003,
14, 2547–2555.
7
8
9
2
(a) M. Bocola, N. Otte, K.-E. Jaeger, M. T. Reetz and W. Thiel,
ChemBioChem, 2004,
5
, 214–223; (b) Z. Qian and S. Lutz, J.
Am. Chem. Soc., 2005, 127, 13466–13467; (c) J. D. Carballeira,
P. Krumlinde, M. Bocola, A. Vogel, M. T. Reetz and J.-E.
Bäckvall, Chem. Commun., 2007, 1913–1915; (d) M. T. Reetz,
S. Prasad, J. D. Carballeira, Y. Gumulya and M. Bocola, J. Am.
Chem. Soc., 2010, 132, 9144–9152; (e) H. B. Brundiek, A. S.
Evitt, R. Kourist and U. T. Bornscheuer, Angew. Chem. Int. Ed.,
2012, 51, 412–414; (f) Q. Wu, P. Soni and M. T. Reetz, J. Am.
Chem. Soc., 2013, 135, 1872–1881; (g) Y. Wikmark, M. S.
10 P. Chen and W. Yang, Tetrahedron Lett., 2014, 55, 2290–
2294.
11 T. Sakai, I. Kawabata, T. Kishimoto, T. Ema and M. Utaka, J.
Org. Chem., 1997, 62, 4906–4907.
12 (a) A. M. Klibanov, Nature, 2001, 409, 241–246; (b) T. Itoh, E.
Akasaki, K. Kudo and S. Shirakami, Chem. Lett., 2001, 262–
263; (c) T. Itoh, S. Han, Y. Matsushita and S. Hayase, Green
Humble and J.-E. Bäckvall, Angew. Chem. Int. Ed., 2015, 54
4284–4288.
,
3
Recent examples: (a) W. Greschner, B. Neumann, H.-G.
Stammler, H. Gröger and D. Kuck, Angew. Chem. Int. Ed.,
2015, 54, 13764–13768; (b) B. Yang, C. Zhu, Y. Qiu and J.-E.
Bäckvall, Angew. Chem. Int. Ed., 2016, 55, 5568–5572; (c) Á.
Mourelle-Insua, M. López-Iglesias, V. Gotor and V. Gotor-
Fernández, J. Org. Chem., 2016, 81, 9765–9774; (d) A.
Sevenich, G.-Q. Liu and A. J. Arduengo, III, B. F. Gupton and
T. Opatz, J. Org. Chem., 2017, 82, 1218–1223; (e) K. G.
Hugentobler, H. Sharif, M. Rasparini, R. S. Heath and N. J.
Turner, Org. Biomol. Chem., 2016, 14, 8064–8067; (f) T.
Görbe, K. P. J. Gustafson, O. Verho, G. Kervefors, H. Zheng, X.
Chem., 2004,
Nakamura, Green Chem., 2004,
Matsushita, Y. Abe, S. Han, S. Wada, S. Hayase, M. Kawatsura,
S. Takai, M. Morimoto and Y. Hirose, Chem. Eur. J., 2006, 12
6
, 437–439; (d) T. Matsuda, T. Harada and K.
6
, 440–444; (e) T. Itoh, Y.
,
9228–9237; (f) T. Matsuda, J. Biosci. Bioeng., 2013, 115, 233–
241; (g) H. N. Hoang and T. Matsuda, Tetrahedron Lett., 2015,
56, 639–641; (h) Y. Kitamoto, Y. Kuruma, K. Suzuki and T.
Hattori, J. Org. Chem., 2015, 80, 521–527; (i) H. N. Hoang, Y.
Nagashima, S. Mori, H. Kagechika and T. Matsuda,
Tetrahedron, 2017, 73, 2984–2989.
13 The log P values were taken from the literature: C. Laane, S.
Zou, E. V. Johnston and J.-E. Bäckvall, ACS Catal., 2017,
7
,
Boeren, K. Vos and C. Veeger, Biotechnol. Bioeng., 1987, 30,
1601–1505; (g) S. Wedde, P. Rommelmann, C. Scherkus, S.
Schmidt, U. T. Bornscheuer, A. Liese and H. Gröger, Green
Chem., 2017, 19, 1286–1290.
81–87.
14 The
e values were taken from the literature: CRC Handbook
of Chemistry and Physics, ed. D. R. Lide, CRC, Boca Raton,
Florida, 75th edn, 1994.
4
5
(a) T. Ema, J. Kobayashi, S. Maeno, T. Sakai and M. Utaka,
Bull. Chem. Soc. Jpn., 1998, 71, 443–453; (b) T. Ema, M.
Jittani, K. Furuie, M. Utaka and T. Sakai, J. Org. Chem., 2002,
67, 2144–2151; (c) T. Ema, K. Yamaguchi, Y. Wakasa, A. Yabe,
R. Okada, M. Fukumoto, F. Yano, T. Korenaga, M. Utaka and
T. Sakai, J. Mol. Catal. B: Enz., 2003, 22, 181–192.
15 (a) T. Yıldız and A. Yusufog˘lu, Tetrahedron: Asymmetry,
2011, 22, 1347–1352. (b) J. Cho, J. Lee, J. Park and M.-J. Kim,
Tetrahedron: Asymmetry, 2015, 26, 840–845.
16 For reviews on dynamic kinetic resolution with lipases, see:
(a) O. Pàmies and J.-E. Bäckvall, Trends Biotechnol., 2004, 22
,
(a) T. Ema, T. Fujii, M. Ozaki, T. Korenaga and T. Sakai, Chem.
Commun., 2005, 4650–4651; (b) T. Ema, S. Kamata, M.
130–135; (b) B. Martín-Matute and J.-E. Bäckvall, Curr. Opin.
Chem. Biol., 2007, 11, 226–232; (c) Y. Ahn, S.-B. Ko, M.-J. Kim
and J. Park, Coord. Chem. Rev., 2008, 252, 647–658; (d) J. H.
Lee, K. Han, M.-J. Kim and J. Park, Eur. J. Org. Chem., 2010,
999–1015.
Takeda, Y. Nakano and T. Sakai, Chem. Commun., 2010, 46
,
5440–5442; (c) T. Ema, Y. Nakano, D. Yoshida, S. Kamata and
T. Sakai, Org. Biomol. Chem., 2012, 10, 6299–6308. For the
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 7
Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C7OB01823A
Table of Contents Entry
The double mutants of industrial lipases, LPS_L287F/I290A and LAK_V287F/I290A,
exhibited high catalytic activity and enantioselectivity for originally poor substrates.