Synergetic activation of lipase by an amino acid with Alkyl-PEG sulfate ionic liquid

Article abstract of DOI:10.1246/cl.130171

The synergetic effect of amino acids and an ionic liquid, 1-butyl-2,3-dimethylimidazolium α-cetylpolyoxyethylene(10) ether sulfate (IL1), as coating materials on lipase was discovered: coating on lipase PS using a certain amino acid with IL1 was found to

Full text of DOI:10.1246/cl.130171

doi:10.1246/cl.130171
Published on the web May 15, 2013
663
Synergetic Activation of Lipase by an Amino Acid with Alkyl-PEG Sulfate Ionic Liquid
Kazuhide Yoshiyama, Yoshikazu Abe, Shuichi Hayase, Toshiki Nokami, and Toshiyuki Itoh*
Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University,
4-101 Koyama-minami, Tottori 680-8552
(Received March 1, 2013; CL-130171; E-mail: titoh@chem.tottori-u.ac.jp)
IL1-coated lipase PS
The synergetic effect of amino acids and an ionic liquid,
OH
OAc
1-butyl-2,3-dimethylimidazolium ¡-cetylpolyoxyethylene(10)
ether sulfate (IL1), as coating materials on lipase was
discovered: coating on lipase PS using a certain amino acid
with IL1 was found to be very effective for accelerating the
lipase-catalyzed transesterification of secondary alcohols while
maintaining perfect enantioselectivity.
Vinyl acetate
R2
( )-1
R1
R2
(R)-2
R1
i-Pr2O, toluene, or ILs
coating IL
O
N
N
O
S
O
O
C16H33
O
C4H9
H3C
10
CH3
IL1
O
S
Lipases are among the most widely used enzymes appli-
cable for various substrates; however, the reaction rate and
enantioselectivity depend significantly on both the substrates
and reaction media: the reaction rates are generally dependent on
the reaction media, and very slow or poorly enantioselective
reactions are sometimes obtained.1
N
N
O
O
C16H33
10
O
C4H9
N
H
O
CH3
D-ProMe
Figure 1. Ionic liquid-coated lipase PS-catalyzed reaction
system.5,9
We have been investigating enzymatic reactions in the ionic
liquid (IL) solvent system and have established that various
types of ILs are applicable as solvents for biochemical
reactions.2-4 We further developed a powerful method for the
activation of lipase protein through coating with an ionic liquid:
Burkholderia cepacia lipase coated with the ionic liquid,
1-butyl-2,3-dimethylimidazolium ¡-cetylpolyoxyethylene(10)
ether sulfate (IL1),5,6 (IL1-PS), displayed excellent reactivity
for many substrates in conventional organic solvents5 and ionic
liquids.7
Luo and co-workers reported the preparation of chiral
imidazolium salts derived from proline.8 Inspired by their work,
we prepared two types of chiral pyrrolidine-substituted imida-
zolium ¡-cetylpolyoxyethylene(10) ether sulfate, and found that
(R)-3-butyl-2-methyl-1-(pyrrolidin-2-ylmethyl)-1H-imidazol-3-
ium ¡-cetylpolyoxyethylene(10) ether sulfate (D-ProMe) de-
rived from D-proline worked as an excellent activating agent
for lipase PS (Figure 1).9 An extraordinary acceleration was
accomplished with perfect enantioselectivity for the D-ProMe-
PS-catalyzed reaction, and a reaction 58 times faster (vs. lipase
PS) was recorded.9 However, more simple coating materials
need to be developed for the activation of lipase. We herein
report the synergetic activation of amino acids and IL1 as a
coating material for lipase.
Amino acids have been used as stabilizers of enzymes
during the purification process. For example, commercial lipase
PS contains ca. 20 wt % glycine as an essential stabilizer during
the preparation of the lipase protein through the lyophilization
process.5b We investigated the role of glycine and established
that it worked only as a stabilizer of the enzyme and had
no influence on the reactivity of lipase PS.5b Since it was
anticipated that a chiral amino acid may modify the enantiose-
lectivity of an enzymatic reaction, we next prepared amino acid-
coated lipase PS and investigated its properties in the trans-
esterification of («)-1-phenylethanol (1a) as a model substrate in
the presence of vinyl acetate as an acyl donor in the i-Pr2O
solvent system (Figure 2).10 As shown in Figure 2, the coating
of lipase PS with amino acids neither accelerated the reaction
nor modified its enantioselectivity, although coating lipase with
L-aspartic acid (Asp) and L-cysteine (Cys) caused a significant
reduction thereof. To our delight, we discovered very interesting
synergetic activation of an enzyme with an amino acid and IL1.
A significant acceleration was obtained by coating lipase PS
with a combination of the amino acid and IL1, which was
prepared by treating glycine-free PS with 100 mol equiv of
L-amino acid and IL1: 100- to 300-fold acceleration was found
compared to the native or amino acid-coated lipase PS. It was
found that the combination of IL1 and L-proline (Pro) was
particularly effective in activating the lipase PS: 330-fold
acceleration was accomplished by using L-proline and IL1-
coated PS.
Since it was already established that glycine worked only as
a stabilizer of the enzyme and had no influence on the reactivity
of lipase PS, we next prepared amino acid-coated PS samples by
using commercial lipase PS, which included 20 wt % of glycine,
in the presence of IL1 just after removal of the supporting celite,
and investigated their properties for the activation of lipase PS.
The coating effect between L-amino acids (Rate1 in
Figure 3) and D-amino acids (Rate2 in Figure 3) was com-
pared:10 («)-(E)-4-phenylbut-3-en-2-ol (1b)5b was used as a
model substrate, because the reaction rate of lipase PS for this
alcohol was not satisfactory, while the enantioselectivity was
perfect. As shown in Figure 3, different activation effect levels
were found for cysteine-, proline-, tyrosine-, and methionine-
coated enzymes. Interestingly, coating with nonnatural D-amino
acids generally led to slightly greater acceleration than coating
with natural L-amino acids. However, the difference was not
significant compared to that between D-ProMe and L-ProMe:
D-ProMe gave rise to double the acceleration of L-ProMe.9
To gain further insight into the synergetic activation of
lipase PS with amino acids and IL1, we conducted kinetic
Chem. Lett. 2013, 42, 663-665
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

664
IL1-PS
L-Pro+IL1
PS
2500"
IL1-PS
2000"
1500"
1000"
500"
0"
>200
100
PS
Control"
(Lipase"
PS)"
E2
L6His"
L6Pro"
L6Met"
L6Trp"
E1
L6Phe"
L6Val"
L6Leu"
Rate2
Rate1
L6Ala"
L6Asn"
L6Gln"
L-amino acid or L-
amino acid + IL1-
coated Lipase PS
L6Asp"
L6Glu"
L6Cys"
L6Tyr"
OAc
OH
Vinyl acetate
(R)-2a
(
)-1a
i-Pr2O, 35°C
Figure 2. Effect of coating on lipase PS only with an amino acid (Rate1 and E1) or with both of an amino acid and IL1 (Rate2 and E2)
on transesterification of («)-1-phenylethanol (1a) using vinyl acetate as acyl donor. Enantioselectivity was evaluated by the E value.11
Rate2 of the control is the result of IL1-coated PS.
1600
1400
1200
D
D
D
L
D
1000
800
600
400
200
D
D
L
D
L
L
D
L
D
L
L
D
D
L
L
L
L
0
Rate2
Rate1
D- or L-amino
OH
acid+IL1-coated
Lipase PS
OAc
Vinyl acetate
(R)-2b
(
)-1b
i-Pr2O, 35°C
Figure 3. Effect of coating on lipase PS with L-amino acids or D-amino acids with IL1 on transesterification of («)-4-phenylbut-3-en-
2-ol (1b). Rate1 = L-amino acid with IL1. Rate2 = D-amino acid with IL1. Perfect enantioselectivity (E >200) was obtained for all
reactions.
Table 1. Results of transesterification of (R)- or (S)-4-phenyl-
but-3-en-2-ol (1b) using four types of enzymes
experiments on three types of IL1-coated lipase PS samples
using (R)- and (S)-(E)-4-phenylbut-3-en-2-ol (1b) as substrates;
the results are shown in Table 1. A significant increase in the
Coating
a
¹1
Substrate
Vmax
Km/M
Kcat/min
Kcat/Km
K
cat value was observed for the (R)-isomer (37-fold acceleration
material
¹3
¹1
¹3
¹2
was obtained for IL1 + D-Pro compared to the control), while
only a small acceleration was obtained for the (S)-isomer (7-fold
acceleration was recorded for IL1 + D-Pro compared to the
control) (Table 1). On the other hand, the Km values were
increased slightly for both enantiomers compared to those of PS
(none) (Table 1), though it was difficult to identify the origin of
these small differences.
We reported earlier that the Kcat value of the IL1-coated
lipase PS-catalyzed reactions was increased compared to those
of native lipase PS.5b Modified Km values were also observed
between enantiomers of the substrate alcohol when lipase PS
(R)-1b
None
(control)
IL1
1.9 © 10 1.3 © 10
7.5 © 10
5.9 © 10
¹2
¹1
¹1
¹1
¹1
¹2
¹1
¹1
¹3
(R)-1b
8.7 © 10 3.1 © 10
3.5 © 10
4.0 © 10
2.8 © 10
2.0 © 10
1.1
1.1
1.2
¹1
(R)-1b IL1 + L-Pro 1.0 © 10 3.5 © 10
¹2
(R)-1b IL1 + D-Pro 7.0 © 10 2.3 © 10
¹4
¹2
(S)-1b
None
(control)
IL1
5.0 © 10 1.4 © 10
1.5 © 10
¹2
¹1
¹2
¹2
¹1
¹1
¹1
(S)-1b
5.8 © 10 1.7
2.3 © 10
9.9 © 10
1.4 © 10
1.3 © 10
2.3 © 10
1.5 © 10
¹2
¹1
¹1
(S)-1b IL1 + L-Pro 2.5 © 10 4.3 © 10
¹2
(S)-1b IL1 + D-Pro 3.5 © 10 9.2 © 10
aM min¹1 mg-enzyme
.
¹1
Chem. Lett. 2013, 42, 663-665
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

665
was coated with chiral imidazolium salts:9 the Km value of the
(S)-isomer was reduced compared to that of PS when D-ProMe-
PS was used as a catalyst, while the value was increased for the
(R)-isomer (double that of native PS).9 These results suggest that
the cationic part of the ionic liquid and amino acid might bind
with the lipase protein, causing a conformational change in the
enzyme and contributing to the difference in Km between the
enantiomers. The chiral imidazolium cation might affect the
enzyme reactivity strongly compared to amino acids when it
binds with the protein. We also assume that the ionic liquid may
bind with the enzyme protein and form an IL layer on the protein
surface,12 thus contributing to the increased flexibility of the
enzyme protein. Since our ionic liquid has amphiphilic proper-
ties, this also contributes to the concentration of the hydrophobic
substrate on the enzyme protein so that initial acceleration of the
rate might be realized.
In conclusion, we have discovered a synergetic activation of
the lipase protein through coating with a combination of certain
amino acids and 1-butyl-2,3-dimethylimidazolium ¡-cetylpoly-
oxyethylene(10) ether sulfate (IL1). Although the induced
activation is slightly less effective than that of the chiral
pyrrolidine-substituted imidazolium salt (D-ProMe),9 the present
method is much more practical for attaining sufficient activation
of lipase PS, because the coating material is just a mixture of an
amino acid, such as L- or D-proline, with a simple imidazolium
alkyl-PEG sulfate (Figure 3). Here, we suggest a possible way
to realize the activation of a lipase by using a very simple
methodology and hope that this may become an even more
promising approach after refinement of the molecular design of
the coating material. We believe that further investigation of the
scope and limitation of this salt-mediated activation of enzymes
will make it even more valuable.
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3
4
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5
The present work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (No. 23360369), and
also by a fund for Environmental Research from Tottori
Prefecture. The authors are grateful to Dr. Yoshihiko Hirose
of Amano Enzyme, Ltd., for providing the Celite-free lipase
PS.
6
7
IL1-PS is commercially available from Tokyo Chemical
Industry Co., LTD. TEL: +81-3-5640-8857, FAX: +81-3-
5640-8868.
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This paper is dedicated to Professor Teruaki
Mukaiyama in celebration of the 40th anniversary of the
Mukaiyama aldol reaction.
8
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References and Notes
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2
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10 For the details of lipase-catalyzed reaction, see Supporting
Information. Supporting Information is available electroni-
cally on the CSJ-Journal Web site, http://www.csj.jp/
journals/chem-lett/index.html.
11 C.-S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am.
Chem. Soc. 1982, 104, 7294.
12 The enzyme surface seemed to be covered with at least 6 or 7
IL1-ion pairs based on the results of MALDI-TOF-MS
experiments, though we have not yet succeeded in obtaining
reproducible results.
Chem. Lett. 2013, 42, 663-665
© 2013 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/