Homogeneous enzymatic reactions in ionic liquids with poly(ethylene glycol)-modified subtilisin

Article abstract of DOI:10.1039/b608920h

Subtilisin Carlsberg was covalently modified with comb-shaped poly(ethylene glycol) (PM₁₃). PM₁₃-modified subtilisin (PM ₁₃-Sub) was readily solubilized in three different ionic liquids (ILs), i.e., [Emim][Tf₂N], [C₂OC₁mim][Tf ₂N] and [C₂OHmim][Tf₂N]. Analysis of homogeneous enzymatic reactions in the ILs revealed that PM₁₃-Sub exhibited excellent catalytic performance while the native enzyme suspended in ILs showed no activity. Hydrophobicity of ILs slightly affected enzyme activity, and the relatively hydrophobic IL [Emim][Tf₂N] was the preferred medium for enzymatic reactions, similar to enzymatic reactions in conventional organic solvents. Enzyme activity was much higher in [Emim][Tf₂N] than in conventional organic solvents, and excellent activity was associated with unique properties of ILs such as hydrophobicity and high polarity. Furthermore, PM₁₃-Sub showed good stability in [Emim][Tf ₂N], and maintained 80% of its initial activity after 60 h. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b608920h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Homogeneous enzymatic reactions in ionic liquids with poly(ethylene glycol)-
modified subtilisin
Kazunori Nakashima, Tatsuo Maruyama, Noriho Kamiya and Masahiro Goto*
Received 23rd June 2006, Accepted 12th July 2006
First published as an Advance Article on the web 7th August 2006
DOI: 10.1039/b608920h
Subtilisin Carlsberg was covalently modified with comb-shaped poly(ethylene glycol) (PM13).
PM13-modified subtilisin (PM13-Sub) was readily solubilized in three different ionic liquids (ILs), i.e.,
[Emim][Tf2N], [C2OC1mim][Tf2N] and [C2OHmim][Tf2N]. Analysis of homogeneous enzymatic
reactions in the ILs revealed that PM13-Sub exhibited excellent catalytic performance while the native
enzyme suspended in ILs showed no activity. Hydrophobicity of ILs slightly affected enzyme activity,
and the relatively hydrophobic IL [Emim][Tf2N] was the preferred medium for enzymatic reactions,
similar to enzymatic reactions in conventional organic solvents. Enzyme activity was much higher in
[Emim][Tf2N] than in conventional organic solvents, and excellent activity was associated with unique
properties of ILs such as hydrophobicity and high polarity. Furthermore, PM13-Sub showed good
stability in [Emim][Tf2N], and maintained 80% of its initial activity after 60 h.
of suspended enzymes in ILs is usually modest, several attempts
have been made to enhance activity of enzymes dispersed in ILs,
Introduction
including addition of water to ILs18,20 or immobilization of the
enzyme on a solid support.30 When considering the industrial use
of enzymes as heterogeneous catalysts in ILs, low solubility of
enzymes is not so serious. However, insolubility of enzymes limits
further applications for ILs. Development of universal strategies
for solubilization of biocatalysts in ILs would break new ground.
There are at least two strategies for solubilizing enzymes in
ILs. One involves the introduction of functional groups into ILs
that show high affinity for protein molecules such as hydroxyl,
ether, and amide groups. Some ILs are known to solubilize
enzymes through hydrogen bonding interactions; however, these
ILs often induce conformational changes in proteins, resulting in
inactivation of enzymes.31,32 Another strategy for solubilization
of enzymes is to modify them with a compound which has an
affinity to ILs, and shows good solubility in ILs. Crown ether
and poly(ethylene glycol) (PEG), both of which are composed
of ethylene glycol units, dissolve well in ILs. By using the
inherent properties of this family of compounds, we successfully
demonstrated activation of lipases in ILs by physical complexation
with PEG,33 and quantitative extraction of cytochrome c from
an aqueous phase into ILs using crown ether.34 These results
prompted us to conduct chemical modification of enzymes with
PEG for solubilization in ILs. However, modification with only
a few PEG chains was insufficient to achieve high solubility, and
to activate enzymes. Most recently, we succeeded in solubilizing
subtilisin Carlsberg in ILs by chemical modification with comb-
shaped PEG (PM13), which is a promising modifier for introducing
a large number of PEG chains into a protein molecule.35
Ionic liquids (ILs) are novel and functional solvents with unique
properties such as negligible volatility, thermal stability, and
relatively high polarity.1–3 One of the most attractive features of
ILs is their solvent property which can be controlled by changing
combinations of cations and anions or by appending functional
groups to constituent ions.4,5 Thus, a “tailor-made solvent” can
be chosen for a specific reaction. For example, ILs composed of
hydrophobic ions and that are immiscible with water would offer
a dual nature, i.e., polarity and hydrophobicity.6,7
Some researchers have recently become interested in using ILs as
non-aqueous media for biotransformation.8–10 Using hydrophobic
ILs as reaction media in biocatalysis often enhances the efficiency
of synthetic reactions by suppressing hydrolytic side reactions.
Moreover, ILs can dissolve a variety of polar compounds which
are not usually dissolved in non-polar organic solvents. Fol-
lowing the pioneering studies by some groups,11–13 many works
have been actively conducted on biocatalytic reactions in ILs
with lipases,14–17 proteases,18–20 oxidoreductases,21–23 and whole-
cell biocatalysts.24,25 Compared to conventional organic solvents,
ILs provide a comfortable environment for enzymes,19,26,27 and
some ILs are less harmful to cell membranes,25 resulting in high
biocatalytic performance. The possible use of ILs as novel reaction
media for biocatalysis has been demonstrated over the past 5 years,
showing relatively high enzyme activity and good regio- and
enantioselectivity.16,28,29
Since enzymes are biomacromolecules composed of polypeptide
chains with a hydrophilic nature, native enzymes are usually
insoluble in ILs. Therefore, enzymatic reactions in ILs are basically
limited to the use of suspended enzymes. Because catalytic activity
In the present study, subtilisin was modified with comb-shaped
PM13 (Fig. 1), and its catalytic behavior in ILs was investigated.
We discussed effects of molecular structures of PEG derivatives
on catalytic activity, and investigated correlations between enzyme
activities and solvent properties of reaction media.
Department of Applied Chemistry, Graduate School of Engineering, and
Center for Future Chemistry, Kyushu University, 744 Moto-oka, Fukuoka,
819-0395, Japan. E-mail: mgototcm@mbox.nc.kyushu-u.ac.jp; Fax: +81-
(0)92-802-2810; Tel: +81-(0)92-802-2806
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Fig. 1 Schematic illustration of modification of subtilisin with comb-
shaped PEG (PM13).
Results and discussion
Characterization and solubility of PM13-modified subtilisin
Fig. 2 Chemical structures and abbreviations of ILs and PEG derivatives.
Proteins dissolve in aqueous solutions by significant contributions
from hydrogen bonding of water molecules. However, in ILs, there
are little interactions between protein molecules and IL molecules,
resulting in extremely low solubility of enzymes. Alterations of
chemical structures of ILs would be effective for solubilizing
enzymes; however, inactivation of enzymes due to denaturation
of proteins by surrounding ionic solvents has been reported.31,32
Alternatively, modification of enzymes with PEG is a rational
strategy for solubilization of enzymes in ILs because PEG dissolves
well in ILs. Some researchers attempted to improve catalytic
performance of enzymes by covalent modification with PEG;17,36
however, chemical modification with single-chained PEG did not
result in sufficient solubility and activity of enzymes. We assume
that this was due to insufficient numbers of PEG chains per
enzyme. To test this hypothesis, we used a highly-branched PEG
derivative, called comb-shaped PEG, as the enzyme modifier to
introduce sufficient quantities of PEG chains onto the surface of
a model enzyme, subtilisin Carlsberg.
Sub was a highly crosslinked enzyme with a molecular weight
ranging from 20 000 to around 1 000 000 Da. Thirdly, we examined
solubility of subtilisin in ILs. As expected, native subtilisin did
not dissolve in the typical IL [Emim][Tf2N]. Functionalized ILs
[C2OC1mim][Tf2N] and [C2OHmim][Tf2N], which have hydrogen
bonding abilities are expected to solubilize enzymes. Nevertheless,
native subtilisin did not dissolve in either of these ILs. In
contrast, PM13-Sub was readily solubilized in all ILs tested to
the concentration of at least 40 mg mL−1 (containing 2 mg mL−1
of subtilisin). It is noteworthy that PM13-Sub was dissolved in
pure ILs with no addition of water. Solubilization of the enzyme
in these ILs is attributed to the high density of PEG chains on the
protein surface, although we could not estimate the exact number
of PEG chains attached per enzyme.
Enzymatic reactions in ILs
Comb-shaped PEG, PM13, is a branched PEG derivative, and
has multivalent reactive sites (carboxylic acid anhydride) in its
backbone which can react with amino groups in a protein molecule
(Fig. 2). Degree of modification of amino groups in the protein
molecule and protein content of PM13-Sub were determined by
the TNBS method and the BCA assay, respectively. Based on
these examinations, we found that about 50% of amino groups in
subtilisin were modified with carboxyl groups of PM13, and that
the resultant PM13-Sub conjugate contained 5 wt% of subtilisin.
Since modification of enzymes often induces loss of activity,
we first examined enzyme activity of PM13-Sub by hydrolysis of
p-nitrophenyl butyrate in water. The specific activity of PM13-
Sub was comparable to that of native subtilisin, suggesting that
decline of enzyme activity during the modification process was
negligible. Secondly, we investigated the size of PM13-Sub using
size-exclusion chromatography equipped with multi-angle light
scattering (SEC-MALS). Since PM13 has multivalent reactive sites
for protein molecules, it is possible to crosslink enzymes via PM13 as
a crosslinker. The SEC-MALS measurements revealed that PM13-
Several groups have reported that enzymes dissolved in ILs signifi-
cantly lose their original activities.12,31,37 It is likely that ILs that can
solubilize enzymes also interact with protein molecules, and cause
structural changes, resulting in inactivation of enzymes. Although
we succeeded in solubilization of subtilisin in ILs, the solubilized
enzyme may not exhibit maximal activity. Enzyme activity of
PM13-Sub solubilized in [Emim][Tf2N] was thus investigated by
transesterification of N-acetyl-L-phenylalanine ethyl ester with 1-
butanol as a model reaction. Subtilisin Carlsberg has been widely
studied in enzymatic reactions in non-aqueous media owing to
its comparatively high tolerance for organic solvents and its high
catalytic activity.38,39 However, in ILs, native subtilisin showed no
activity (Fig. 3). On the other hand, PM13-Sub exhibited remark-
ably high catalytic performance in ILs. It is worth mentioning that
PM13-Sub allowed homogeneous enzymatic reactions in ILs. A
large quantity of PEG chains covering an enzyme surface could
enhance solubility of the enzyme, and protect the enzyme in
ILs. Hydrophilic PEG chains may also contribute to retention
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Table 1 Water content of ILs in dried and water saturated conditions
(20 ◦C)
Water content (ppm)
Dried
Water saturated
[Emim][Tf2N]
[C2OC1mim][Tf2N]
[C2OHmim][Tf2N]
495
830
1640
18 200
24 700
84 500
Fig. 3 Time-courses of transesterifications of N–Ac–L–Phe–OEt with
1-butanol catalyzed by native subtilisin and PM13-Sub in [Emim][Tf2N].
Reaction conditions: N–Ac–L–Phe–OEt (100 mM) and 1-butanol
(500 mM), and subtilisin or PM13-Sub were stirred in 1 mL of [Emim][Tf2N]
at 40 ◦C. Each reaction was performed under these conditions unless stated
otherwise.
of essential water, which is required for enzyme activity.40 We
found that modification with PM13 was quite effective not only
for solubilization, but also for enhancing enzyme activity in ILs.
Next, we examined the effects of the molecular structure of a
modifier on enzyme activity in ILs using three PEG derivatives,
i.e., single-chained PEG1, double-chained PEG2 and comb-shaped
PM13 (Fig. 2). In terms of solubility, while PEG1-Sub was insoluble
in [Emim][Tf2N], PEG2-Sub was solubilized in [Emim][Tf2N] as
was observed with PM13-Sub. Amounts of PEG chains introduced
into the enzymes were almost equal in PEG1-Sub and PEG2-Sub
(about 4 to 5 chains). This indicated that solubility of the modified
enzymes might be affected by the shape of PEG rather than by the
amount of PEG attached to the enzyme. With regard to enzyme
activity, the higher enzyme activity was obtained with PM13-Sub
although PEG2-Sub was also solubilized in [Emim][Tf2N] (Fig. 4).
We suppose that coverage of the protein surface with abundant
PEG chains is necessary for achievement of high enzyme activity
in ILs.
Fig. 5 Transesterification activity of PM13-Sub in three ILs with different
structures of the imidazolium cation.
time-courses of transesterification catalyzed by PM13-Sub in the
three ILs. As the ILs became more hydrophilic, activity of PM13-
Sub decreased. This tendency might be associated with stripping
of essential water from the protein microenvironment to the bulk
solvent, leading to protein denaturation. Since all reactions were
performed in dry ILs, water molecules adsorbed to the protein
surface would partition more easily into hydrophilic ILs than
into hydrophobic ones. PM13-Sub would show a lower activity
in the more hydrophilic IL [C2OHmim][Tf2N]. This tendency was
consistent with enzymatic reactions in organic media. In addition,
[C2OC1mim][Tf2N] and [C2OHmim][Tf2N], which possess coordi-
nating and hydrogen bonding abilities, may have directly affected
enzymes. The results suggested that hydrophobic IL appeared to
be suitable for homogeneous enzymatic reaction in ILs using PEG-
modified enzymes. Thus [Emim][Tf2N] was used in the following
experiments.
We compared enzyme activities in ILs with those in organic
solvents commonly used in non-aqueous enzymatic reactions.
We selected toluene, dichloromethane, methyl tert-butyl ether
(MTBE), tetrahydrofuran (THF), dimethylsulfoxide (DMSO),
and acetonitrile as organic solvents. Since PM13-Sub did not
show sufficient solubility in MTBE, the reaction was conducted
heterogeneously in MTBE.
Transesterification efficiencies in [Emim][Tf2N] and organic
solvents, and polarities of reaction media are shown in Table 2.
Markedly high activity was observed in [Emim][Tf2N], while
no enzyme activity was shown in hydrophilic organic solvents
such as THF, DMSO, and acetonitrile. Toluene seemed to be
a relatively suitable medium for PM13-Sub. Absence of activity
in MTBE might be due to insolubility of PM13-Sub in the
reaction medium. We hypothesized that the excellent catalytic
activity in [Emim][Tf2N] was ascribed to the high polarity and
the hydrophobicity of the IL. Based on solvatochromism of
Reichardt’s dye, polarity of [Emim][Tf2N] was higher than that
of the organic solvents tested, and was very close to those of
short-chain alcohols such as ethanol. The transition state of this
Fig. 4 Effects of molecular structures of PEG derivatives on catalytic
activity with modified subtilisin.
Effects of solvents on catalytic activity
We prepared three different ILs with the same Tf2N− anion,
i.e., [Emim][Tf2N], [C2OC1mim][Tf2N], and [C2OHmim][Tf2N]
(Fig. 2), all of which were water-immiscible. Table 1 shows the
water content of these ILs in dried and water-saturated conditions.
[Emim][Tf2N] is the most hydrophobic IL among the three types
of ILs. Transesterification activity of PM13-Sub was examined in
the three ILs in the absence of water (i.e. dry ILs). Fig. 5 depicts
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Table 2 Reichardt’s dye polarity (ENT) values for ILs and organic
solvents, and transesterification efficiency of PM13-Sub in these media
Initial reaction rate
[mmol/(h·g of enzyme)]
Polaritya/EN
T
[Emim][Tf2N]
Acetonitrile
DMSO
0.66
0.46
0.44
0.31
0.21
0.12
0.10
18.8
0
0
0
0
0
5.6
Dichloromethane
THF
MTBEb
Toluene
a Measurements of EN values for ILs were conducted according to the
T
literature.16 The values for organic solvents were taken from a recent
review.44 b The reaction mixture was heterogeneous.
transesterification reaction is supposed to be relatively polar,39
and it would be stabilized more effectively in a highly polar
environment provided by the IL, leading to acceleration of the
reaction. However, polar organic solvents often cause protein
denaturation by stripping essential water as observed in DMSO
and acetonitrile. On the other hand, [Emim][Tf2N] is highly
polar, yet hydrophobic, and thus would strip hardly any water
molecules bound to proteins, resulting in retention of catalytically
active conformations of enzymes. Possession of these two opposite
properties (i.e., high polarity and hydrophobicity) cannot be
attained in conventional organic solvents, but is possible in ILs
whose physico-chemical properties can be controlled by changing
molecular structures of ILs. The present study also demonstrated
that ILs had great potential as reaction media for enzyme-
catalyzed reactions. Details of high catalytic efficiency in ILs have
not yet been clarified. Further investigations including kinetics
studies and spectroscopic analyses are required to elucidate the
mechanisms of enzymatic activation in ILs.
Fig. 6 Effects of pH on enzyme activity in water and in IL: (A) hydrolysis
of p-nitrophenyl butyrate in water with different pHs catalyzed by native
subtilisin (B) transesterification of N–Ac–L–Phe–OEt with 1-butanol in
[Emim][Tf2N] catalyzed by PM13-Sub prepared from aqueous solutions
with different pHs. Buffer solutions: 10 mM phosphate (circles), 10 mM
tris-HCl (squares), and 10 mM borate (triangles).
maintained 80% of its initial activity after 60 h (Fig. 7). The half-
life time (t1/2) of PM13-Sub in [Emim][Tf2N] was about 194 h, which
was much higher than those of native enzymes in [Emim][Tf2N]
without corresponding substrates (a-chymotrypsin: t1/2 = 1.08 h,19
C. antarctica lipase B: t1/2 = 3.7 h26). Enzymes sometimes exhibit
improved thermal and chemical stabilities by immobilization or
crosslinking. Subtilisin was assumed to acquire improved stability
by crosslinking via comb-shaped PM13. In addition, substantial
coverage of the protein surface by PEG chains may play an
important role in stabilization.
pH Memory and stability of subtilisin in ILs
In organic solvents, catalytic activity of enzymes depends on
the pH of the last aqueous solution to which the enzyme
was exposed because ionizable groups in the enzyme acquire
corresponding ionization states, which remain in organic solvents.
This phenomenon is known as “pH memory”.41,42 The pH memory
of enzymes in ILs is of great interest, although it has been scarcely
discussed. Therefore, we examined influence of pH on catalytic
activities of subtilisin in water, and PM13-Sub in [Emim][Tf2N]
(Fig. 6). The maximum activity of native subtilisin around pH 8 in
water coincided with that in the literature.42 In [Emim][Tf2N], the
catalytic activity of PM13-Sub was remarkably influenced by the
pH of the aqueous solution from which the enzyme was prepared.
The highest activity was found at the same pH in the case of the
aqueous system. These results suggested that pH memory was also
evident in ILs as well as in organic solvents.
Fig. 7 Stability of PM13-Sub in [Emim][Tf2N] at 25 ◦C.
For practical use of ILs as reaction media for biotransfor-
mations, long-term stability of a biocatalyst in ILs is desirable.
Some groups demonstrated increased stability of enzymes in ILs
compared to organic media, and further enhancement of stability
by addition of a substrate.19,26 Nonetheless, native enzymes were
inactivated within a few hours in ILs. Here, we studied the stability
of PM13-Sub solubilized in [Emim][Tf2N]. We found that PM13-
Sub showed good stability for a long period in [Emim][Tf2N], and
Conclusions
We demonstrated solubilization of subtilisin in three types of ILs in
the absence of water by chemical modification with a comb-shaped
PEG, PM13. The PM13-modified subtilisin, PM13-Sub, solubilized
in [Emim][Tf2N], exhibited remarkably high activity and good
stability in the IL. A higher activity of PM13-Sub was observed
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in a more hydrophobic IL, and this activity was much greater
than those seen in conventional organic solvents. Since enzymes
are homogeneously dissolved in ILs in the present system, it is
possible to analyze microscopic protein structures and to evaluate
catalytic activities of enzymes in ILs by spectroscopic methods.
The solubilization strategy demonstrated in this work could be
applied to other enzymes.
(PM13-Sub) was obtained. Degree of modification of amino
groups in a subtilisin molecule was determined using 2,4,6-
trinitrobenzenesulfonic acid (TNBS),46 and protein content of
PM13-Sub was estimated by the bicinchoninic acid (BCA) assay.47
Subtilisin modified with two different PEG derivatives, i.e., single-
chained PEG1 and double-chained PEG2, was prepared in a
similar manner to PM13-Sub except for reaction temperature
(25 ◦C) for PEG1 modification.
Experimental
Materials
Size-exclusion chromatography with multi-angle light scattering
(SEC-MALS)
Subtilisin Carlsberg was purchased from Sigma (St. Louis,
MO), and was used without further purification. Comb-
shaped PEG (PM13; SUNBRIGHT AM-1510 K, Mw: 15 000
to 20 000) and o-methoxypoly(ethylene glycol)-succinate N-
succinimidyl ester (PEG1; SUNBRIGHT ME-050CS, Mw:
5 000) were obtained from NOF Co. (Tokyo, Japan); and
2,4-bis(o-methoxypolyethylene glycol)-6-chloro-s-triazine (PEG2,
Mw: 10,000) was from Seikagaku Co. (Tokyo, Japan). Ionic liquids
1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide
([Emim][Tf2N]), and its ether ([C2OC1mim][Tf2N]) and hy-
droxyl ([C2OHmim][Tf2N]) analogues were prepared as previously
described,6,43 and were dried for 48 h under vacuum. Molecular
structures and abbreviations of PEG derivatives and ILs used in
this study are shown in Fig. 2. All other chemicals were of reagent
grade.
To investigate the molecular weight of PM13-Sub, we used the
SEC-MALS system. Weight-averaged molecular weight (Mw) can
be determined by light scattering responses of eluted compounds
in various fractions separated by SEC. The SEC-MALS analysis
was performed using the JASCO HPLC system equipped with a
multi-angle light-scattering detector (DAWN EOS, Wyatt Tech-
nology Co.), an RI detector (RI-2031 Plus, JASCO) and a UV
detector (UV-970, JASCO). A Superdex 200 10/300 GL column
(Amersham Biosciences) was used for SEC separation with 10 mM
sodium phosphate buffer containing 0.1 M NaCl as eluent, at a
flow rate of 0.8 mL min−1. PM13-Sub was dissolved in the elution
buffer, and was injected into HPLC for analysis.
Preparation of the pH-adjusted enzyme
Water content and polarity evaluation of ILs
PM13-Sub (70 mg) was dissolved in deionized water (10 mL), and
was dialyzed twice against 2 L 10 mM phosphate buffer (pH 7.0),
followed by lyophilization for 48 h. A 15 mg portion of the dialyzed
PM13-Sub was dissolved again in 2 mL of each buffer solution
(phosphate, Tris-HCl or borate buffer) with different pHs, and
then lyophilized for 48 h to obtain pH-adjusted PM13-Sub.
Each dried IL was diluted with anhydrous methanol to reduce
viscosity, then its water content was measured using a Karl
Fischer moisture titrator (Mitubishi, CA-07 Moisturemeter).
Water contents of the dry ILs were calculated by subtracting that
of anhydrous methanol. Those of water saturated ILs were also
measured in the same manner after equilibration of ILs with water.
Polarity of ILs was evaluated by solvatochromism of
Reichardt’s dye.44 Reichardt’s dye (2,6-diphenyl-4-(2,4,6-
triphenylpyridinio)phenolate, 0.8 mg) was dissolved in each
IL (1 mL), and was centrifuged to remove insoluble particles.
Wavelength of the absorption maximum was measured using a
UV–vis spectrophotometer (JASCO V-570) at 25 ◦C, and was
converted to normalized polarity scales (ENT) ranging from 0.0 for
tetramethylsilane to 1.0 for water, using the following equations.
Enzyme activity assay in water, organic solvents, and ILs
The catalytic activity of subtilisin in water was examined by
hydrolysis of p-nitrophenyl butyrate in each buffer at 37 ◦C,
monitoring p-nitrophenol liberated during hydrolysis by subtilisin
with a UV–vis spectrophotometer at 420 nm. In ILs and organic
solvents, enzyme activities were evaluated in transesterification of
100 mM N-acetyl-L-phenylalanine ethylester (N–Ac–L–Phe–OEt)
28591
ET(solvent) [kcal/mol] =
kmax [nm]
◦
with 500 mM 1-butanol by 1 mg mL−1 of enzyme at 40 C. The
reaction was started by adding the enzyme into the reaction media
containing the substrates. Samples were periodically removed from
reaction mixtures, diluted 10-fold with acetonitrile, and injected
into a HPLC system (LC-10AT VP, Shimadzu) equipped with an
Inertsil ODS-3 column (4 × 250 mm, GL Science). A solution of
acetonitrile–5% acetic acid (80 : 20 v/v) was used as eluent, at a
flow rate of 1.0 mL min−1. Eluted compounds were monitored by
a UV–vis detector (SPD-M10A VP) at 257 nm.
In the enzyme stability test, PM13-Sub was solubilized in
[Emim][Tf2N], and was settled at 25 ◦C. A portion of the solution
was periodically taken out, and was mixed with the IL containing
substrates to initiate the reaction. The 100% activity corresponded
to initial activity at 0 h.
ET(solvent) − 30.7
ETN(solvent) =
32.4
Modification of subtilisin with PEG derivatives
Modification of subtilisin with comb-shaped PEG (PM13) was
performed according to Inada et al.45 To a subtilisin solution
(2 mg mL−1, 4 mL) in 0.1 M sodium borate buffer (pH 8.5),
crystalline PM13 (200 mg) was added, followed by stirring at
4
◦C in an ice-bath for 1 h. The reaction mixture was filtered
using an Ultra-4 membrane (MWCO: 30 000, Millipore), and was
washed three times with deionized water to remove unreacted
PM13. Following lyophilization for 48 h, PM13-modified subtilisin
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21 G. Hinckley, V. V. Mozhaev, C. Budde and Y. L. Khmelnitsky,
Biotechnol. Lett., 2002, 24, 2083–2087.
Acknowledgements
22 J. A. Laszlo and D. L. Compton, J. Mol. Catal. B: Enzym., 2002, 18,
109–120.
This work was supported by a Grant-in-Aid for the 21st Century
COE Program, “Functional Innovation of Molecular Informat-
ics” and Scientific Research (No. 18045027) from the Ministry
of Education, Culture, Sports, Science and Technology, Japan.
K. N. was supported by Research Fellowships of the Japan Society
for the Promotion of Science (JSPS) for Young Scientists. The
authorswouldliketothankProfessorAtsushiMaruyama(Kyushu
University) for the use of the SEC-MALS system and for his
helpful discussions.
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