Benzoate Surfactants for Enhancing the Activity of Lipoprotein Lipase from Burkholderia Species in Organic Solvent

Article abstract of DOI:10.1002/bkcs.11877

Two benzoate surfactants were synthesized and examined as the additives for enhancing the activity of a lipoprotein lipase from Burkholderia species (BSLPL) in organic solvent. It was found that the benzoate surfactants enhanced the turnover number (k_cat) by four orders of magnitude and the catalytic efficiency (k_cat/K_m) by three orders of magnitude. These results strongly suggest that the favorable interaction between the aromatic rings of surfactant tails and the hydrophobic residues around the active site of enzyme may help BSLPL maintain highly active open conformation in organic solvent.

Full text of DOI:10.1002/bkcs.11877

Article
DOI: 10.1002/bkcs.11877
BULLETIN OF THE
Y. Oh
KOREAN CHEMICAL SOCIETY
Benzoate Surfactants for Enhancing the Activity of Lipoprotein Lipase
from Burkholderia Species in Organic Solvent
*
Yeonock Oh
Department of Chemistry, Pohang University of Science and Technology, Pohang 37673,
Republic of Korea. *E-mail: oyo0203@postech.ac.kr
Received June 17, 2019, Accepted August 21, 2019, Published online October 7, 2019
Two benzoate surfactants were synthesized and examined as the additives for enhancing the activity of a
lipoprotein lipase from Burkholderia species (BSLPL) in organic solvent. It was found that the benzoate
surfactants enhanced the turnover number (k ) by four orders of magnitude and the catalytic efficiency
cat
(
k /K ) by three orders of magnitude. These results strongly suggest that the favorable interaction
cat m
between the aromatic rings of surfactant tails and the hydrophobic residues around the active site of
enzyme may help BSLPL maintain highly active open conformation in organic solvent.
Keywords: Lipase, Activation, Surfactant, Organic solvent
Introduction
Experimental
1
,2
Enzymes are very useful catalysts in organic synthesis.
Material. All the commercially available starting materials
and reagents were purchased from Sigma-Aldrich Co. LLC.
(St. Louis, MO, USA), Acros Organics (Morris, NJ, USA),
and TCI Co., Ltd (Tokyo, Japan) and used without addi-
tional purification. Lipoprotein lipase from Burkholderia
species was purchased from Amano Enzyme Inc.
They are particularly useful as the catalysts for the synthe-
3–5
sis of optically active compounds.
Enzymes, however,
have a serious limitation that they display poor activities in
organic solvents which are common media for most of
organic reactions. The activities of enzymes in organic
media are several orders of magnitude lower than their
aqueous counterparts. Thus, it is of great importance to
develop practical procedures for enhancing the enzymatic
(Yokohama, Japan). All solvents were purified by purifying
system: Ultimate Solvent System, GlassContour from
Nikko Hansen & Co., Ltd. (Osaka, Japan). Gas chromatog-
raphy (GC) analyses for measurement of reaction conver-
sion were obtained using Agilent 6890 Series Gas
Chromatography System with Agilent HP-5 column
activity in organic solvent.
Several useful methods have been reported for enhancing
1
13
the enzymatic activity in organic solvent. They include the
(0.33 μm, 0.2 mm, 25 m). H NMR and C NMR spectra
6
–12
were recorded in CDCl solution at ambient temperature on
immobilization of enzymes on solid supporters
and the
3
1
3–17
a 300 MHz fourier-transform nuclear magnetic resonance
spectroscopy: AVANCE 300, Bruker (Billerica, MA,
USA). Chemical shifts are given in δ (ppm) relative to tet-
ramethylsilane as internal standard. Multiplicities were
recorded as s (singlet), d (doublet), q (quartet), m (multi-
plet), and brs (broad singlet). Molecular mass was mea-
sured by liquid chromatography-mass spectrometry: JMS-
T100 L (AccuTOF); Jeol (Tokyo, Japan). All enzyme prep-
arations were lyophilized by freeze dryer: FD-1000 or
FDU-1200, Eyela (Tokyo, Japan).
use of additives such as inorganic salts,
ionic
18–22
23–27
liquids,
or surfactants.
Recently, the Kim group
reported that the combination of dextrin and ionic surfac-
tants as the additives enhanced dramatically the activities of
synthetically useful lipase and subtilisin in organic sol-
28,29
vent.
These results encouraged me to explore the devel-
opment of new surfactants for inducing the higher
enzymatic activity in organic solvent.
Two anionic surfactants 1a-b as the benzoates with two
Synthesis of Benzoate Surfactants (1a-b). The synthesis
of 1a from 2a is described as the representative. Benzyl
alcohol (0.70 mL, 5.0 mmol), triethylamine (1.5 equiv),
and methanesulfonyl chloride (1.5 equiv) were dissolved in
anhydrous CH Cl (25 mL). The resulting mixture was
long chains at the two meta-positions (Figure 1) were syn-
thesized and tested as the additives for enhancing the activ-
ity of a lipoprotein lipase from Burkholderia species
(BSLPL) in organic solvent. It was found that the anionic
2
2
surfactants enhanced the catalytic efficiency of BSLPL in
organic solvent by three orders of magnitude. Furthermore,
the turnover number of anionic-surfactant-activated BSLPL
in organic solvent was higher than its aqueous counterpart.
ꢀ
stirred at 0 C for 30 min, warmed to room temperature
RT), and then stirred for 2 h to give mesylated product.
(
The reaction mixture was neutralized by 1 N HCl and con-
centrated under reduced pressure. The residue was extracted
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ppm): δ 23.3, 31.0, 33.2, 69.4, 69.6, 70.0, 70.4, 73.0,
1
26.0, 127.1, 134.7, 147.5.
To the solution of 3,5-dihydroxybenzoic acid methyl
ester (196 mg, 1.17 mmol), K CO3 (2.0 equiv), and
2
1
(
4
8-crown-6 (0.05 equiv) in acetone (10 mL) was added 5a
2.0 equiv), and the mixture was stirred under reflux for
8 h. After cooled down to RT, solid materials were
Figure 1. Structure of surfactants.
removed by filtration. The filtrate was evaporated under
reduced pressure, and the residue was purified by silica gel
chromatography to give 6a (530 mg, 74% yield): H NMR
1
with CH Cl (3 × 10 mL), and the combined organic layer
2
2
(
300 MHz, CDCl , ppm): δ 3.63–3.71 (m, 16H), 3.85 (t,
3
was washed with NaHCO solution. After dried over anhy-
3
J = 4.51 Hz, 4H), 3.88 (s, 3H), 4.12 (t, J = 4.36 Hz, 4H),
4.56 (s, 4H), 6.68 (s, 1H), 7.18 (s, 2H), 7.26–7.34 (m,
drous MgSO , the organic layer was evaporated to give 3a
4
1
3
1
10H). C NMR (75 MHz, CDCl , ppm): δ 52.2, 67.7,
3
(
770 mg, 91% yield): H NMR (300 MHz, CDCl , ppm): δ
3
6
1
9.4, 69.6, 70.7, 70.8, 73.2, 106.9, 108.0, 127.6, 127.7,
28.3, 131.9, 138.2, 159.7, 166.7.
6b (340 mg, 96% yield): H NMR (300 MHz, CDCl ,
1
3
2
.91 (s, 3H), 4.33 (s, 2H), 7.26–7.38 (m, 5H). C NMR
1
(
75 MHz, CDCl , ppm): δ 42.3, 66.6, 128.4, 129.8, 133.2.
3
3
1
3
b (740 mg, 89% yield): H NMR (300 MHz, CDCl ,
ppm): δ 1.24 (d, J = 7.02 Hz, 12H), 2.86–2.90 (m, 2H),
3.62–3.72 (m, 16H), 3.74 (t, J = 6.36 Hz, 4H), 3.87 (s,
3H), 4.11 (t, J = 6.36 Hz, 4H), 4.53 (s, 4H), 6.68–6.70
3
ppm): δ 1.25 (d, J = 6.87 Hz, 6H), 2.84–2.82 (m, 1H), 3.15
s, 3H), 4.58 (s, 2H), 7.16–7.38 (m, 4H). C NMR
1
3
(
(
1
1
3
75 MHz, CDCl , ppm): δ 23.5, 33.1, 46.2, 67.8, 126.3,
(m, 1H), 7.17–7.27 (m, 10H).
C NMR (75 MHz,
3
28.3, 135.0, 148.1.
CDCl , ppm): δ 24.0, 25.2, 33.9, 67.7, 69.3, 70.6, 70.7,
3
A mixture of triethylene glycol (2.0 equiv) and sodium
hydride (1.0 equiv) was stirred at RT in tetrahydrofuran
THF) (10 mL) for 2 h in Schlenk flask. Then, 3a (400 mg,
73.2, 106.9, 108.0, 126.4, 127.9, 131.9, 135.6,
148.3, 159.8.
(
2
To the solution of 6a (0.5 mmol) in ethanol (1.0 mL)
was added aqueous KOH (2.0 equiv). After stirring for 3 h
at RT, the reaction mixture was neutralized by 1 N HCl
and concentrated under reduced pressure. The residue was
extracted with methylene chloride, and the solvent was
.4 mmol) was added dropwise, and the mixture was stirred
ꢀ
at 60 C for 12 h. After cooling at RT, distilled water was
added and the mixture was stirred in air for 5 min. After
extracting the mixture with CH Cl , the organic layer was
2
2
dried with anhydrous MgSO . The crude product was puri-
evaporated. The product 7a was obtained (290 mg, 98%
4
1
fied by column chromatography, leading to the desired
yield): H NMR (300 MHz, CDCl , ppm): δ 3.64–3.72 (m,
3
1
product 4a (480 mg, 83% yield): H NMR (300 MHz,
16H), 3.86 (m, 4H), 4.13 (t, J = 4.36 Hz, 4H), 4.56 (s, 4H),
13
CDCl , ppm): δ 3.48–3.82 (m, 12H), 4.57 (s, 2H),
6.71 (s, 1H), 7.23 (s, 2H), 7.26–7.34 (m, 10H). C NMR
3
1
3
7
6
.23–7.36 (m, 4H). C NMR (75 MHz, CDCl , ppm): δ
1.5, 70.0, 70.3, 70.4, 73.1, 127.6, 127.7, 128.4, 138.1.
b (560 mg, 83% yield): H NMR (300 MHz, CDCl ,
(75 MHz, CDCl , ppm): δ 67.7, 69.4, 69.6, 70.7, 70.8,
3
3
73.2, 107.5, 108.5, 127.6, 127.7, 128.3, 131.3, 138.2,
1
4
159.8,
170.6.
TOF-MS
(ESI+)
calculated
for
3
+
ppm): δ 1.24 (d, J = 6.87 Hz, 6H), 2.78–3.03 (m, 1H),
[C H O + Na] 621.26757, found 621.26873.
33 42 10
13
1
3.55–3.82 (m, 12H), 4.53 (s, 2H), 7.13–7.36 (m, 4H).
C
7b (330 mg, 99% yield): H NMR (300 MHz, CDCl ,
3
NMR (75 MHz, CDCl , ppm): δ 23.3, 33.2, 61.3, 70.0,
ppm): δ 1.23 (d, J = 7.02 Hz, 12H), 2.84–2.91 (m, 2H),
3.63–3.73 (m, 16H), 3.86 (t, J = 6.36 Hz, 4H), 4.11
(t, J = 6.36 Hz, 4H), 4.53 (s, 4H), 6.72–6.71 (m, 1H),
3
7
0.3, 70.4, 73.0, 126.0, 127.1, 134.7, 147.5.
To the solution of 4a (720 mg, 3 mmol) and triphenyl
13
phosphine (1.25 equiv) in anhydrous THF (10 mL) was
7.17–7.28 (m, 10H). C NMR (75 MHz, CDCl , ppm): δ
3
ꢀ
added carbon tetrabromide (1.25 equiv) at 0 C, and the
24.0, 33.9, 67.7, 69.3, 70.7, 73.2, 107.6, 108.5, 126.4,
mixture was stirred at RT for 3 h. The mixture was filtered
through celite-pad to remove insoluble materials and then
concentrated under reduced pressure. The crude product
127.9, 131.1, 135.5, 148.4, 159.8, 170.6. TOF-MS (ESI+)
+
calculated for [C H O + Na]
705.36147, found
3
9 54 10
705.36329.
was purified by silica gel chromatography to afford 5a
To the solution of 7a (0.3 mmol) in THF (0.5 mL) was
added KOH (0.1 M, 2.3 mL). After stirring for 1 h at RT,
the reaction mixture was concentrated under reduced pres-
sure and further dried in vacuum for 24 h to afford 1a
1
(
3
(
710 mg, 78% yield): H NMR (300 MHz, CDCl , ppm): δ
3
.46 (t, J = 6.39 Hz, 2H), 3.64 (m, 2H), 3.68 (s, 6H), 3.81
13
t, J = 6.35 Hz, 2H), 4.57 (s, 2H), 7.26–7.35 (m, 5H).
C
1
NMR (75 MHz, CDCl , ppm): δ 30.3, 69.4, 70.5, 70.6,
quantitatively (200 mg, 99% yield): H NMR (300 MHz,
3
7
0.7, 71.2, 73.2, 127.6, 127.7, 128.3, 138.2.
CDCl , ppm): δ 3.24–3.65 (m, 16H), 3.76 (m, 4H), 4.03
3
1
5b (630 mg, 90% yield): H NMR (300 MHz, CDCl ,
(t, J = 4.36 Hz, 4H), 4.56 (s, 4H), 6.58 (s, 1H), 7.12
3
13
ppm): δ 1.24 (d, J = 7.02 Hz, 6H), 2.81–2.98 (m, 1H),
.40–3.74 (m, 10H), 3.82 (t, J = 6.36 Hz, 2H), 4.53
(s, 2H), 7.14–7.24 (m, 10H). C NMR (75 MHz, CDCl3,
ppm): δ 67.5, 69.3, 69.6, 70.5, 70.8, 73.1, 107.2, 108.1,
127.4, 127.7, 128.3, 131.2, 138.1, 159.8, 171.6.
3
1
3
(s, 2H), 7.13–7.34 (m, 4H). C NMR (75 MHz, CDCl3,
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Benzoate Surfactants for Lipase Activation in Organic Solvent
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1
1
b (210 mg, 99% yield): H NMR (300 MHz, CDCl ,
3
ppm): δ 1.20 (d, J = 7.02 Hz, 12H), 2.81–2.90 (m, 2H),
.53–3.62 (m, 16H), 3.75 (t, J = 6.36 Hz, 4H), 4.02
t, J = 6.36 Hz, 4H), 4.43 (s, 4H), 6.72–6.61 (m, 1H),
3
(
1
3
7
2
1
.07–7.18 (m, 10H). C NMR (75 MHz, CDCl , ppm): δ
3
4.0, 33.9, 67.7, 69.8, 70.8, 73.2, 107.7, 108.5, 126.5,
27.9, 131.2, 135.5, 148.4, 158.8, 174.6.
Kinetics of BSLPL for the Reaction of para-Nitrophenyl
Acetate with 1-Phenylethanol. The kinetic parameters
(
Vmax and K ) of BSLPL preparation for the alcoholysis of
m
p-nitrophenyl acetate with 1-phenylethanol were obtained
through two sets of experiments to determine Vmax and Km
separately. The Vmax value of BSLPL preparation was
determined with solutions containing 1-phenylethanol
(
0
(
0.1–0.7 M), BSLPL preparation (commercial BSLPL
.25 mg), and p-nitrophenyl acetate (0.5 equiv) in toluene
1 mL). The Vmax/K value was determined with solutions
Scheme 1. Synthesis of surfactants.
m
containing 1-phenylethanol (0.2 M), BSLPL preparation
alcohol 4a. The treatment of 4a with CBr and Ph P followed
4
3
(
(
commercial BSLPL 0.25 mg), and p-nitrophenyl acetate
0.025–0.3 M) in toluene (1 mL). The rate measurement at
by the coupling with methyl 3,5-dihydroxybenzoate afforded
a, which was hydrolyzed and then converted finally to the
target surfactant 1a as the potassium salt. The synthesis of
b as the isopropyl derivative of 1a was accomplished simi-
6
each substrate concentration was performed three times as
ꢀ
follows. The solution was shaken at 250 rpm at 25 C. An
1
aliquot was sampled periodically, filtered through a short
silica pad, and analyzed by GC to determine the conversion
percent against time. The Vmax and Vmax/Km values were
then estimated from the nonlinear curve fitting of average
initial rates. The Km value was calculated from the Vmax
larly according to the procedure described for the synthesis
of 1a except that p-isopropylbenzyl alcohol was employed
as the starting material.
All the steps for the syntheses of 1a-b were straightfor-
ward and provided good yields. This procedure thus can
serve as the protocol for the synthesis of other benzoate
surfactants and related analogues.
and Vmax/K values.
m
Kinetics of BSLPL for the Reaction of para-Nitrophenyl
Acetate with Benzyl Alcohol. The kinetic parameters
(
Vmax and K ) of BSLPL preparation for the alcoholysis of
Activity of Surfactant-coated BSLPL. Surfactant-coated
BSLPL preparations (BSLPL-1a and -1b) were obtained by
freeze-drying aqueous solutions containing BSLPL, dextrin,
and benzoate surfactant at an 1:2:1 weight ratio in the 1:1
(v/v) water–1,4-dioxane mixture. It is noted that dextrin
was added to improve the powdery nature of BSLPL prepa-
rations in organic solvent. The activities and kinetic param-
eters of BSLPL-1a and -1b were then determined by
measuring the initial rates for the BSLPL-catalyzed reac-
tions of p-nitrophenyl acetate with alcohol (1-phenylethanol
or benzyl alcohol) in toluene. The results are described in
Tables 1 and 2.
First, all the data from Tables 1 and 2 indicate that the
activity of BSLPL in organic solvent is dramatically
enhanced by benozoate surfactants. The kcat values of
surfactant-coated BSLPL-1a and -1b for the reactions of p-
nitrophenyl acetate with 1-phenylethanol in toluene
are four orders of magnitude greater than that of their
surfactant-free counterpart (compare the entries 1–3 in
Table 1). As a result, the kcat/Km values of BSLPL-1a
and -1b are ca 4000~5000 times higher than that of
surfactant-free BSLPL.
m
p-nitrophenyl acetate with benzyl alcohol were obtained
through two sets of experiments to determine Vmax and Km
separately. The Vmax value of BSLPL preparation was
determined with solutions containing benzyl alcohol
(
0
(
0.125–0.625 M), BSLPL preparation (commercial BSLPL
.25 mg), and p-nitrophenyl acetate (0.5 equiv) in toluene
1 mL). The Vmax/K value was determined with solutions
m
containing benzyl alcohol (0.2 M), BSLPL preparation
(
(
commercial BSLPL 0.25 mg), and p-nitrophenyl acetate
0.05–0.25 M) in toluene (1 mL). The rate measurement at
each substrate concentration was performed three times as
follows. The solution was shaken at 250 rpm at 25 C. An
ꢀ
aliquot was sampled periodically, filtered through a short
silica pad, and analyzed by GC to determine the conversion
percent against time. The Vmax and Vmax/Km values were
then estimated from the nonlinear curve fitting of average
initial rates. The Km value was calculated from the Vmax
and Vmax/K values.
m
Results and Discussion
Synthesis of Benzoate Surfactants. As described in
Scheme 1, the synthesis of benzoate surfactant 1a started from
benzyl alcohol, which was mesylated and then reacted with
triethylene glycol in the presence of NaH to give monobenzyl
Hydrolysis and alcoholysis of p-nitrophenyl acetate were
performed in buffer and organic solvents, respectively, to
investigate the effect of benzoate surfactants on the enzyme
activity (Table 2). The activity of lyophilized BSLPL is
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Table 1. Kinetic parameters of BSLPL preparations for the reaction of p-nitrophenyl acetate with 1-phenylethanol in organic solvent.
kcat (s−1
a
)
Km (M)
a
kcat/K (s M−1
−1
)
(kcat/K )
m rel
Entry
BSLPL preparation
m
b
−2
−2
−1
1
BSLPL
BSLPL-1a
BSLPL-1b
1.7 × 10
1.8 × 10
2.1 × 10
8.7 × 10
2.2 × 10
2.2 × 10
2.0 × 10
1
2
−1
−1
2
3
3
2
3
a
8.2 × 10
4.1 × 10
4.8 × 10
2
2
9.5 × 10
BSLPL preparations containing BSLPL (25%, w/w), dextrin (50%, w/w), and surfactant (25%, w/w) were lyophilized in 1:1 (v/v)
water–1,4-dioxane. The reactions were performed with solutions containing substrate (0.1–0.7 M), BSLPL preparation (1 mg), and p-nitrophenyl
ꢀ
acetate (0.5 equiv) at 25 C. Rate measurements were done three times for each enzyme preparation. The kinetic parameters were then obtained
using the nonlinear curve fitting of average rates. The error range of the kinetic parameter values is Æ1% for entries 2 and 3.
b
30
From Lee et al.
Table 2. Kinetic parameters of BSLPL preparations for the hydrolysis and alcoholysis of p-nitrophenyl acetate.
cat (s−
1 a
)
K
a
(M)
k
cat/K
(s−1 M−1
)
Entry
BSLPL preparation
BSLPL
ROH
k
m
m
b
2
−4
−4
−4
−1
−1
−1
5
1
H O
2.8 × 10
2.5 × 10
2.9 × 10
6.4 × 10
7.3 × 10
1.8 × 10
4.8 × 10
6.8 × 10
4.5 × 10
2.5 × 10
5.2 × 10
1.4 × 10
5.7 × 10
3.7 × 10
6.4 × 10
2.6 × 10
1.4 × 10
2
c
2
5
2
3
4
BSLPL
BSLPL-1b
BSLPL
2
H O
2
5
H O
2
b
−2
2
−1
3
PhCH OH
2
5
6
a
BSLPL-1b
2
PhCH OH
d
−1
LPS
PhCH OH
1.3
2
BSLPL-1b containing BSLPL (25%, w/w), dextrin (50%, w/w), and 1b (25%, w/w) was lyophilized in 1:1 (v/v) water–1,4-dioxane. The reactions
were performed with solutions containing substrate (0.125–0.625 M), BSLPL preparation (1 mg), and p-nitrophenyl acetate (0.5 equiv) in toluene at
ꢀ
2
5 C. Rate measurements were done three times for each enzyme preparation. The kinetic parameters were then obtained using the nonlinear curve
fitting of average rates. The error range of the kinetic parameter values is Æ1% for entries 2, 3, 5, and 6.
b
c
d
30
From Lee et al.
BSLPL was lyophilized in 1:1 (v/v) water–1,4-dioxane.
Amano Lipase PS from Burkholderia cepacia.
lower than that of crude BSLPL (compare the entries 1 and
). The decrease in enzymatic activity is caused by denatur-
(compare the entries 1 and 5). This observation implies that
the benzoate surfactants can enhance the turnover number
of BSLPL in organic solvent over the aqueous level.
2
ation of the active site of lipase during lyophilization.
Surfactant-coated BSLPL, however, does not decrease in
activity even after lyophilization compared with BSLPL in
aqueous solution (entries 1 and 3). These results mean that
the surfactants may act as a lyophilization stabilizer and
thus help the lipase to keep the active form during lyophili-
zation. The large increases of kcat and k /K values
It has been known that some lipases have a lid carrying
hydrophobic residues around the active site. The molecular
31
dynamics simulation studies suggest that the lipase with a
lid exists mainly in closed conformation in the aqueous
solution to minimize the solvent-accessible surface area of
hydrophobic residues around the active site but maintain
poorly active open conformation in organic solvent.
Although the open conformation population of BSLPL can
be enhanced by the closed-open conformation changes in
the hydrophobic organic solvents, the substrate binding site
of BSLPL may be greatly distorted in hydrophobic sol-
vents, resulting in poor activity. The results described
above, however, strongly suggest that the surfactant-coated
BSLPL should exist in the highly active open conformation
in organic solvent. It is believed that the hydrophobic aro-
matic rings at the tails of surfactant 1a or 1b can stabilize
cat
m
by benzoate surfactants are also observed for the reaction
of p-nitrophenyl acetate with benzyl alcohol in toluene
(compare the entries 4 and 5). Compared with LPS (Amano
Lipase PS from Burkholderia cepacia), a commercially
available immobilized BSLPL, LPS shows about five times
higher activity than surfactant-free BSLPL. The activity of
LPS, however, was negligibly small relative to that of
BSLPL-1b (compare entries 4–6). Furthermore, the kcat
value of the surfactant-coated enzyme (BSLPL-1b) in this
reaction was even superior to its aqueous counterpart
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Benzoate Surfactants for Lipase Activation in Organic Solvent
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the active open conformation by providing the favorable
interaction with the hydrophobic residues around the active
site. The benzoate surfactants can increase the flexibility of
the substrate binding site of BSLPL by coating on surface
of lipase. And also the hydrophilic parts of benzoate surfac-
tants can stabilize the lipase in organic solvent by providing
the aqueous environment around the hydrophilic surface of
lipase, thus inducing the enzyme to display such a high
turnover number in organic solvent.
9. D. N. Tran, K. J. Balkus, ACS Catal. 2011, 1, 956.
1
0. C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan,
R. Fernandez-Lafuente, Enzyme Microb. Technol. 2007,
4
0, 1451.
1
1
1
1
1
1
1
1. D. M.-R. D. Temino, W. Hartmeier, M. B. Ansorge-
Schumacher, Enzyme Microb. Technol. 2005, 36, 3.
2. J. D. Cui, L. M. Sun, L. L. Li, Appl. Biochem. Biotechnol.
2
013, 170, 1827.
3. Y. L. Khmelnitsky, S. H. Welch, D. S. Clark, J. S. Dordick,
J. Am. Chem. Soc. 1994, 116, 2647.
4. V. M. Paradkar, J. S. Dordick, J. Am. Chem. Soc. 1994,
116, 5009.
5. M. T. Ru, J. S. Dordick, J. A. Reimer, D. S. Clark, Bio-
technol. Bioeng. 1999, 63, 233.
6. M. T. Ru, K. C. Wu, J. P. Lindsay, J. S. Dordick,
J. A. Reimer, D. S. Clark, Biotechnol. Bioeng. 2001, 75, 187.
7. A. L. Serdakowski, J. S. Dordick, Trends Biotechnol. 2008,
Conclusion
In summary, I have prepared benzoate surfactants for
enhancing the activity of BSLPL in organic solvent and
demonstrated that the benzoate-surfactant-coated BSLPL
displayed dramatically enhanced activity in organic solvent.
In particular, the turnover number of benzoate-surfactant-
coated BSLPL in organic solvent was even superior to its
aqueous counterpart. Therefore, it is expected that the
benzoate-surfactant-coated BSLPL should find use as the
powerful catalyst for synthetic reactions in organic solvent.
2
6, 48.
1
1
8. J. K. Lee, M.-J. Kim, J. Organomet. Chem. 2002, 67, 6845.
9. T. Itoh, Y. Matsushita, Y. Abe, S. Han, S. Wada, S. Hayase,
M. Kawatsura, S. Takai, M. Morimoto, Y. Hirose, Chem.
Eur. J. 2006, 12, 9228.
20. Y. Abe, K. Yoshiyama, Y. Yagi, S. Hayase, M. Kawatsura,
T. Itoh, Green Chem. 2010, 12, 1976.
1. K. Yoshiyama, Y. Abe, S. Hayase, T. Nokami, T. Itoh, Chem.
Lett. 2013, 42, 663.
Acknowledgments. Author is grateful to the National
Research Foundation of Korea (2015R1D1A1A01059851)
for financial support of this work.
2
2
2. T. Itoh, Chem. Rev. 2017, 117, 10567.
2
3. Y. Inada, K. Takahashi, T. Yoshimoto, A. Ajima,
A. Matsushima, Y. Saito, Trends Biotechnol. 1986, 4, 190.
4. Y. Mine, K. Fukunaga, N. Maruoka, K. Nakao, Y. Sugimura,
J. Biosci. Bioeng. 2000, 90, 631.
References
2
2
2
2
2
2
3
3
1
. K. Drauz, H. Groeger, O. May, Enzyme Catalysis in Organic
Synthesis, 2nd ed., Vol. 1, Wiley-VCH, Weinheim, 2012.
. K. Faber, Biotransformations in Organic Chemistry, Springer,
Berlin, 2000.
5. Y. Mine, K. Fukunaga, M. Yoshimoto, K. Nakao,
Y. Sugimura, J. Biosci. Bioeng. 2001, 92, 539.
6. Y. Mine, K. Fukunaga, M. Yoshimoto, K. Nakao,
Y. Sugimura, Biotechnol. Lett. 2003, 25, 1863.
7. Y. Mine, K. Fukunaga, K. Samejima, M. Yoshimoto,
K. Nakao, Y. Sugimura, Carbohydr. Res. 2004, 339, 493.
8. H. Kim, Y. K. Choi, J. Lee, E. Lee, M.-J. Kim, Angew.
Chem. Int. Ed. 2011, 50, 10944.
9. K. Kim, E. Lee, C. Kim, J. Park, M.-J. Kim, Org. Biomol.
Chem. 2017, 15, 8836.
2
3
4
. A. M. Klibanov, Acc. Chem. Res. 1990, 23, 114.
. C.-S. Chen, C. J. Sih, Angew. Chem., Int. Ed. Engl. 1989,
2
8, 695.
5
6
7
8
. U. T. Bornscheuer, R. J. Kazlauskas, Hydrolase in Organic
Synthesis, Wiley-VCH, Weinheim, 1999, p. 25.
. I. Svensson, P. Adlercreutz, B. Mattiasson, Appl. Microbiol.
Biotechnol. 1990, 33, 255.
. J.-H. Xu, Y. Kato, Y. Asano, Biotechnol. Bioeng. 2001,
0. J. Lee, Y. Oh, Y. K. Choi, E. Choi, K. Kim, J. Park, M.-
J. Kim, ACS Catal. 2015, 5, 683.
1. H. S. Lee, Y. Oh, M.-J. Kim, W. Im, J. Phys. Chem. B 2018,
7
3, 493.
. L. He, J. Sun, Y. Xu, Z. Sun, C. Zheng, Prep. Biochem. Bio-
technol. 2008, 38, 376. 02325501
1
22, 10659.
Bull. Korean Chem. Soc. 2019, Vol. 40, 1093–1097
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