Specific ion effects of ionic liquids on enzyme activity and stability

Article abstract of DOI:10.1039/c1gc15140a

Catalytic performance of two model enzymes, Penicillium expansum lipase (PEL) and mushroom tyrosinase, was examined in aqueous solution with addition of 14 different ionic liquids (ILs) and has been found to correlate well with the IL's kosmotropic/chaotr

Full text of DOI:10.1039/c1gc15140a

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PAPER
Specific ion effects of ionic liquids on enzyme activity and stability
Jing-Qi Lai,† Zhuang Li,† Yan-Hong Lu¨ and Zhen Yang*
Received 5th February 2011, Accepted 12th April 2011
DOI: 10.1039/c1gc15140a
Catalytic performance of two model enzymes, Penicillium expansum lipase (PEL) and mushroom
tyrosinase, was examined in aqueous solution with addition of 14 different ionic liquids (ILs) and
has been found to correlate well with the IL’s kosmotropic/chaotropic properties, which are
assessed by the viscosity B coefficients (B+ for cations and B- for anions). The activity and stability
of PEL were similarly correlated with B+ values but not with B- of the ILs tested. PEL can be
activated and stabilized by addition of ILs (e.g., 5.2-fold activation and 1.4-fold stabilization in the
presence of 0.63 M [choline][Ac] and 0.27 M [NHMe3][MeSO3], respectively). Choline ILs
activated PEL but imidazolium ones deactivated it. The results indicate that the IL cations play a
crucial role in affecting the enzyme performance and that ammonium ILs composed of chaotropic
cations (favorably with H-bonding capability) and kosmotropic anions are favored for enzyme
catalysis. The Hofmeister effect of ILs on PEL was confirmed by the kinetic and thermostability
studies and structural analysis on tyrosinase. Our investigations on both enzymes have thus
demonstrated that ILs can affect the enzyme functioning through the Hofmeister effect, and the
mechanisms have been discussed in terms of the influence of the IL cations and anions on the
surface pH, active site conformation, and catalytic mechanism of each specific enzyme, following
the Hofmeister series.
composed of a kosmotropic anion and a chaotropic cation is
Introduction
usually found to favor enzyme functioning.5,6,10 Based on the
comparison of a limited number of ILs, a few enzymes have been
found to have their catalytic behavior in IL-containing aqueous
systems responding to the Hofmeister effect,5,6 while disobeying
examples have also appeared in literature.11,12
Ionic liquids have been regarded as a promising new type of
nonaqueous solvents for biocatalysis because of their unique
solvent properties and their ability to present excellent enzymatic
performance.1–4 One of the major attractions of utilizing ILs in
place of organic solvents is their ‘designer solvent’ property:
combinations of different cations and anions can make up
different ILs with different physical and chemical properties.
However, the prerequisite for taking this advantage depends
on our understanding of the effects of ILs on the enzymatic
performance. It has been recently proposed that in addition to
the factors including polarity, hydrophobicity, nucleophilicity,
and H-bond basicity, the Hofmeister ion effects may also offer
a reasonable explanation for this.5–7
It has long been realized that enzyme performance in aqueous
solution can be altered by different inorganic salts and ions
following a common order known as the Hofmeister series.8 It
is believed to be correlated with the kosmotropic/chaotropic
properties of the ions, which can be characterized by the
Jones–Dole viscosity B coefficients.9 The presence of a salt
Meanwhile, there has been a growing demand for developing
‘greener’ ILs.13,14 Ionic liquids based on alkylammonium as
the cation represent promising biocompatible ones which can
be prepared from natural sources.7 Indeed, Candida antarctica
lipase B has been found to dissolve in [Et3MeN][MeSO4] while
maintaining its activity and structure,15 so did cytochrome
c in [choline][H2PO4]16 and subtilisin in diethanolammonium
chloride ([DEA][Cl]).17 Among the ammonium ILs, the protic
ones, with a proton attached to the cation nitrogen, possess
special characteristics including the capability of H-bonding
as well as the ionic and hydrophobic characters. Although
protic ILs are currently under intense study in a variety of
applications,18 their utility in biocatalysis has not been much
assessed yet.
In this study, both activity and stability of two model enzymes,
Penicillium expansum lipase (PEL) and mushroom tyrosinase,
were investigated in aqueous solution with addition of 14
different ILs. The aims of this study were: (1) to systematically
explore the specific ion effect of ILs on enzyme performance
and to verify its generality; (2) to assess the feasibility of using
College of Life Sciences, Shenzhen University, Shenzhen, Guangdong,
China, 518060. E-mail: zyang@szu.edu.cn; Fax: +86 755 2653 4277;
Tel: +86 755 2653 4152
† Equal contribution.
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ammonium ILs, especially the protic ones, in biocatalysis; and
(3) to propose a few useful guidelines for designing green and
biocompatible ILs based on the above results. The two selected
model enzymes belong to different classes. PEL is a hydrolase
effective for catalyzing hydrolytic and transesterification reac-
tions including biodiesel production, being much more active in
the IL [BMIm][PF6] than in commonly used organic solvents;19
while mushroom tyrosinase is an oxidoreductase catalyzing
the hydroxylation of monophenols to o-diphenols and their
subsequent oxidation to o-quinones, and its action in both
aqueous solution, organic solvents, and ionic liquids has been
extensively investigated.12,20,21
Stability tests for PEL
The PEL solution in IL-containing phosphate buffer (50 mM,
pH 6.5, final IL content 4.17%, w/v) was incubated at 35 C,
◦
and 0.2 ml sample was taken periodically for activity assay at
◦
37 C as noted above. The time-dependent loss in activity was
used to calculate the half-life for each enzyme solution.
Activity assays for tyrosinase
The activity of tyrosinase was measured spectrophotometrically
by following the oxidation of L-DOPA to dopachrome.20 The
purified enzyme (0.05 ml) was added to a mixture containing
1.2 ml phosphate buffer (50 mM, pH 6.0) and 0.8 ml L-DOPA
(10◦mM, prepared in the same buffer) to start the reaction at
30 C. For the reactions to study the IL effects, the phosphate
buffer was prepared with addition of a specified amount of each
IL. After pH measurement, the buffer solution was readjusted
to pH 6.0 before being used for the activity tests. For Km, Vmax
measurements, substrate solutions (air-saturated) with various
concentrations of L-DOPA (0–9.8 mM) were prepared in the
IL-containing phosphate buffer, and the apparent Km and Vmax
values were obtained from the Lineweaver–Burk plots.
Experimental
Materials
Penicillium expansum lipase was kindly donated by Shenzhen
Leveking Bioengineering Co. Ltd., China. All 14 ionic liquids
(99%, HPLC) were purchased from ShangHai Cheng Jie Chem-
ical Co. Ltd. p-Nitrophenyl palmitate (pNPP), p-nitrophenol
(pNP), and 3,4-dihydroxyl-phenylalanine (L-DOPA) were pur-
chased from Sigma–Aldrich China Inc. All other reagents used
were of analytical grade from local manufacturers in China.
Stability tests for tyrosinase
Purified tyrosinase solution was mixed with a phosphate buffer
(50 mM, pH 6.0) containing 10% (w/v) of each IL in a ratio of
1 : 1 (final IL content 5.0%, w/v), and the mixture was incubated
at a specified temperature (30 ◦C, 40 ◦C, 50 ◦C). Periodically,
0.1 ml sample was taken for activity assay at 30 ◦C as noted
above. The half-life for each enzyme solution was determined,
and the first-order deactivation rate constants were obtained by
fitting the thermoinactivation data to the classical first-order
deactivation model.
Enzyme preparation
PEL solution was obtained by solubilizing the enzyme powders
(3.75 g) in 15 ml of the sodium phosphate b◦uffer (50 mM,
pH 6.5) with magnetic stirring for 30 min at 4 C followed by
centrifugation.19 Mushroom tyrosinase was purified by extrac-
tion of fresh mushrooms into the phosphate buffer (50 mM, pH
6.0) and subsequent concentration with (NH4)2SO4, following by
gel filtration on a HiPrep 16/60 Sephacryl S-200 HR prepacked
column (eluted with 50 mM, pH 6.0 phosphate buffer), ion-
exchange chromatography on a DEAE Sephadex column (eluted
with a linear gradient of NaCl (0–1.0 M) in the phosphate
buffer), and gel filtration chromatography again. The purity of
the enzyme was confirmed by SDS-PAGE.
Fluorescence spectroscopy
Fluorescence measurements for tyrosinase were carried out
using a Hitachi F4500 spectrofluorimeter. Enzyme samples
were prepared by dissolving the purified tyrosinase solution
in the phosphate buffer (50 mM, pH 6.0) in the presence of
different ILs (0–0.5 M IL, final pH 6.0, final protein content
0.09 mg ml-1). The denatured enzyme sample was prepared by
incubating the enzyme solution in a boiling water bath for 10 min
and then cooling down to room temperature (no activity was
detectable after sucha treatment). Thefluorescence was recorded
for emission from 290–450 nm with excitation at 280 nm. A
blank medium without enzyme was subtracted to discount the
influence of the fluorescence of the ILs and buffer components
on the enzyme fluorescence spectrum.
Activity assays for PEL
The activity of PEL to catalyze the hydrolysis of pNPP to
pNP was assayed following the procedures described in19 with
minor modification. The enzyme (0.2 ml) was added to the
reaction mixture containing 2.07 ml sodium phosphate buffer
(50 mM, pH 6.5, with addition of 2% (w/v) Triton X-100)
and 0.23 ml pNPP solution (20 mM in isopropanol) to start
the reaction (at 37 ◦C), the formation of pNP was monitored
spectrophotometrically, and the molar extinction coefficient for
pNP under each condition was determined as described in ref.
19. To study the IL effects, a specified amount of each IL was
added to the phosphate buffer (50 mM, pH 6.5, containing 2%
(w/v) Triton X-100) to reach a final concentration of 0–15%
(w/v), and the pH of each such solution was measured. These
buffer solutions were readjusted to pH 6.5 with HCl or NaOH
before being used for the activity tests. All the experiments
throughout this study were at least duplicated, being subject
to less than 5% error for each data point.
Results and discussion
Kosmotropicity/chaotropicity of the IL cations and anions used
in this study
All the 14 ILs and the available viscosity B coefficients of their
cations and anions are listed in Table 1. The kosmotropicity of
-
the IL anions used in this study varied in the order of H2PO4
>
Ac- > MeSO3- ~ MeSO4- > NO3 ; the first three are kosmotropes
-
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Table 1 Fourteen ionic liquids used in this study
a
a
IL No.
Ionic liquid
B+
B-
IL-1
IL-2
IL-3
IL-4
IL-5
IL-6
IL-7
IL-8
IL-9
IL-10
IL-11
IL-12
IL-13
IL-14
[MMIm][MeSO4]
[EMIm][MeSO4]
[BMIm][MeSO4]
[choline][H2PO4]
[choline][Ac]
[choline][MeSO3]
[choline][NO3]
[NMe4][Ac]
1,3-dimethylimidazolium methylsulfate
1-ethyl-3-methylimidazolium methylsulfate
1-butyl-3-methylimidazolium methylsulfate
choline dihydrophosphate
choline acetate
cholate methylsulfonate
+0.49b
+0.61b
+0.34
+0.246
+0.127
-0.043
+0.246
+0.246
+0.127
+0.127
+0.34
choline nitrate
tetramethylammonium acetate
tetrabutyllammonium acetate
tetrabutyllammonium methylsulfonate
trimethylammonium methylsulfonate
trimethylammonium dihydrophosphate
triethylammonium dihydrophosphate
tributylammonium dihydrophosphate
+0.123
+1.275
+1.275
+0.117
+0.117
+0.385
[NBu4][Ac]
[NBu4][MeSO3]
[NHMe3][MeSO3]
[NHMe3][H2PO4]
[NHEt3][H2PO4]
[NHBu3][H2PO4]
+0.34
+0.34
a B+ and B- are the viscosity B coefficients of the cation and anion, respectively, obtained from ref. 23 except those labeled with ‘b’. b The B+ values
for the two imidazolium cations were obtained as estimated by Zhao22 based on the linear relationship between the viscosity B coefficients and ion
hydration radii.
while the last one is a chaotrope. With respect to the IL cations,
most of their viscosity B coefficients are not available. But based
on the earlier studies on the hydration behavior of quaternary
ammonium salts, more hydrophobic ammonium cations (with
longer alkyl chains) experience hydrophobic hydration and are
considered as more kosmotropic, and the same is true to the
imidazolium cations.22 The positive B coefficients for the limited
number of ammonium cations listed in23 can be taken as a good
support for this. Two typical examples24,25 have clearly illustrated
this cation effect, confirming that consistent with the effect
of inorganic cations, proteins are also stabilized by chaotropic
cations and destabilized by kosmotropic ones from ILs.
It is worth mentioning that the pH of the aqueous buffer can
be significantly affected by addition of ILs. For instance, pH
lowered by more than 1 unit was observed in the presence of
[choline][H2PO4] and particularly the last 4 protic ILs listed in
Table 1 (all 5% w/v in the 50 mM, pH 6.5 phosphate buffer).
This may to some extent be connected to the Hofmeister series
but can be better understood in view of the acidity/basicity
of the used ILs. Therefore, in order to avoid the IL-induced
interference in the buffer pH, for the later reactions the IL-
containing phosphate buffers were all readjusted to the required
pH prior to use.
Effect of ILs on the activity and stability of PEL
Fig. 1A shows the activities of PEL in aqueous solu-
tion with addition of all the 14 ILs (4.14% w/v), which
Fig. 1 Activity (A) and stability (B) of PEL in phosphate buffer
(50 mM, pH 6.5) containing different ILs (4.14% and 4.17%, w/v,
varied in a very broad range: The enzyme can be ac-
tivated up to 316.4% (by [NHMe3][MeSO3]) and deacti-
vated down to 36.4% (by [BMIm][MeSO4]) of the rate ob-
tained by the enzyme in the IL-free solution. The ILs that
can stimulate the enzyme activity include [NHMe3][MeSO3],
[NMe4][Ac], [choline][Ac], [choline][MeSO3], [choline][NO3],
and [NHMe3][H2PO4]. A closer look at the graph can tell
us that these activity data follow the Hofmeister series very
well. In the presence of the ILs holding the same anions, the
enzyme activity decreased in the order of [MMIm][MeSO4] >
[EMIm][MeSO4] > [BMIm][MeSO4], [NMe4][Ac] > [NBu4][Ac],
[NHMe3][MeSO3] > [NBu4][MeSO3], and [NHMe3][H2PO4] >
respectively). The relative activity (%) refers to the percentage of the
initial reaction rate obtained by the enzyme in the IL-containing aqueous
solution relative to the one obtained in the IL-free system (control,
7.21 mM min-1).
[NHEt3][H2PO4] > [NHBu3][H2PO4]; while for the ILs hold-
ing the same cations, [choline][Ac] > [choline][MeSO3] >
[choline][NO3], [NBu4][Ac] > [NBu4][MeSO3]. When incu-
bated in the phosphate buffer containing different ILs
(4.17% w/v), the enzyme also exhibited variant half-lives
as shown in Fig. 1B. The stability order resembles the
activity one, except for the choline ILs showing the op-
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posite trend. The enzyme can be stabilized by a few ILs
including [NHMe3][MeSO3], [choline][MeSO3], and [NMe4]-
[Ac], which are also listed among the ILs that can activate the
enzyme.
IL anion facilitate enzyme functioning. Moreover, the activation
by all the choline ILs and depression by all the imidazolium
ILs have exemplified the IL cation’s dominating effect, and
it is reasonable to assume that imidazolium cations are much
more kosmotropic than the choline cation. Unfortunately, the
viscosity B coefficients for these two types of cations are not
available yet.
The correlation between the enzyme performance and the
Hofmeister series can be better illustrated by plotting the activity
and stability data against the viscosity B coefficients of the
anions (B-) and cations (B+) of the ILs (Fig. 2). Interestingly,
the two plotting sets resemble each other again: Both activity
and stability show no obvious relationship with B- (Panels A
and C) but are well correlated with B+ (Panels B and D). This
seems to give an indication of a more severe impact from the
IL cations instead of from the anions, which is in contrast to
the situation generally observed in the study of the effect of
inorganic salts on enzyme performance.6
Therefore, our results have clearly substantiated that the PEL
activity and stability in aqueous solution can be affected by
addition of various ILs, following the Hofmeister series, which
is in line with the impact of inorganic salts and ions on enzymes
-
in aqueous solution. The anion, H2PO4 , seems to be anomalous
in this case, because both the enzyme activity and stability were
unexpectedly low in the presence of all the ammonium ILs
holding it as the anion, in spite of its highly positive viscosity B
coefficient (+0.34). In fact, the B coefficient of an ion does not
always agree well with its kosmotropicity,22 and H2PO4- has once
been considered as a borderline ion.26 Besides, this anion may
be further ionized in the buffer solution to produce a fraction
a HPO42-, which may also contribute to some extent to the
observed effect. The fairly good activity and stability obtained
in the presence of all the choline ILs can be attributed to the
hydroxylated choline cation. Recently we have pointed out that
ILs with hydroxylated cations (such as choline) facilitate the
enzyme action because they hold the H2O-mimicking property
and H-bonding functionality, so as to help the enzyme resume
its flexible and active conformation,6,7 and this idea has been
supported by previous experiments.16,27,28 Our observation that
In order to confirm the influences of both the IL cations and
anions, two sets of ILs (3 imidazolium ILs holding the same
-
anion, MeSO4 , and 3 ammonium ILs holding the same cation,
choline) were further investigated (Fig. 3). The enzyme activity
was depressed gradually with addition of all the 3 imidazolium
ILs. But comparatively, the more kosmotropic cation elicited a
lower reaction rate, in the order of BMIm+ < EMIm+ < MMIm+
(Fig. 3A). On the other hand, the enzyme was activated by all
the 3 choline ILs, and under each IL level the reaction rate
was higher in the presence of a more kosmotropic anion, i.e.,
in the order of Ac- > MeSO3 > NO3 (Fig. 3B). The tests on
these two sets of ILs have thus confirmed our notion that a high
chaotropicity in the IL cation and a high kosmotropicity in the
-
-
Fig. 2 Enzyme activity (top, A and B) and stability (bottom, C and D) of PEL (data from Fig. 1) as a function of the viscosity B coefficients of the
anions (B-) and cations (B+) of the ILs used in this study. The dashed lines are drawn to direct the variation trend.
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an enzyme-substrate tetrahedral intermediate. The His residue
functions as a general base to remove the serine proton, thus
making the Ser more nucleophilic, and the positive charge that
forms on the His residue is stabilized by the negatively charged
-
Glu/Asp. The detrimental effect of H2PO4 to PEL observed
in this study can thus be explained by its strong H-bonding
interactions with Glu/Asp and His, hence weakening the ability
of these critical amino acid residues in assisting the catalysis
at the active site. Based on the structural studies on Candida
rugosa lipase, the active site of the enzyme is shielded from the
solvent by a part of the polypeptide chain, the flap, which is
hydrophilic on the side exposed to the aqueous milieu outside
and hydrophobic on the side facing the protein, interacting with
the hydrophobic residues surrounding the active site. The lipase
molecule experiences transition between two conformations,
active or inactive, depending on whether this flap is open or
not, respectively.31,32
The lipase used in this study was produced from a special
strain, PF898, of Penicillium expansum. With 258 amino acid
residues and a molecular mass of 28 kDa, this lipase has a
sequence homology of 39–49% as compared to other lipases
from fungi and a high percentage of hydrophobic residues
(51.4%) in the N-terminal region.33,34 It is reasonable to suspect
that this enzyme holds a similar active site structure and catalytic
mechanism as noted above, although the details have not been
reported yet.
In this study, both activity and stability of PEL were highly
sensitive to the choice of IL cations (Panels B and D of
Fig. 2). A few enzymes have been found to be less active35–37
and less stable25,27 in the presence of ILs holding cations
with larger and longer hydrophobic alkyl chains, indicative of
higher kosmotropicity and hydrophobicity. PEL provides one
more example to support this. The depressing effect caused
by these IL cations can be attributed to:6 (1) their strong ion-
pairing interactions with the kosmotropic moieties of the enzyme
molecule, such as the carboxylic groups, on the enzyme surface,
according to the “law of matching water affinity”,38 which states
that a cation and an anion with equal water affinity, i.e., both
kosmotropic or both chaotropic, are expected to have a strong
propensity to ion-pair with each other in aqueous solution;
and (2) their strong hydrophobic interactions with the inner
hydrophobic moieties of the enzyme molecule, leading to the
disruption of the enzyme’s native conformation.
There are some more reasons specific for PEL to account for
its sensitive response to the IL cations. An IL cation with a long
alkyl chain is hydrophobic and kosmotropic as stated before. It
tends to open the flap that covers the active site and to enter the
active site for its favorable interactions with the hydrophobic
moieties inside, thus resulting in a serious distortion in the
enzyme’s active site conformation. The molecular dynamics
simulations conducted by Kla¨hn et al.39 have also suggested the
diffusion of the long alkyl chain of IL cations (such as BMIm+)
into the active site of Candida antarctica lipase B (CALB), which
may obstruct the substrate molecules to get access to the active
site. Once inside, the IL cation also has a high tendency of
interacting with the critical Glu (or Asp) of the catalytic triad,
in view of the “law of matching water affinity”.38 This surely
disables the stabilizing role played by this amino acid residue in
the catalytic triad, as described earlier. As a result, a reduction
Fig. 3 Initial rate of the PEL-catalyzed hydrolysis reaction as a function
of the IL concentration: (A) 3 imidazolium ILs with MeSO4 as the
anion; (B) 3 ammonium ILs with choline as the cation. The relative
activity (%) refers to the percentage of the initial reaction rate obtained
by the enzyme in the IL-containing aqueous solution relative to the one
obtained in the IL-free system (11.0 mM min-1).
-
PEL was activated by the 3 choline ILs but deactivated by the
3 imidazolium ones (Fig. 3) can also be taken as a support
for this. The superior activating and stabilizing effect presented
by the protic IL [NHMe3][MeSO3] can be attributed to both
the chaotropicity of the IL cation (in combination with a
kosmotropic anion) and to its H-bond donating ability.
In our recent study of the Hofmeister effects on alkaline
phosphatase, we have concluded that while the effect of inorganic
salts and ions on the enzyme stability is general, their effect on
activity is enzyme-specific.29 A brief review of what is known
about the structure and mechanism of lipases can help us
understand better the specific effect of ILs on the PEL’s activity
and stability.
Lipases are carboxyl-esterases acting on hydrolysis or transes-
terification of long-chain acylglycerides, following a mechanism
similar to the well-established acyl-enzyme mechanism for serine
proteases.30 At the active site of the free enzyme, the three critical
amino acid residues (Ser, His, and an acid residue Glu or Asp) are
linked in the form of Glu/Asp-His-Ser by H-bonding, and both
Glu/Asp and His are in their deprotonated states. The first step
in lipase catalysis involves the nucleophilic attack of the hydroxyl
group of Ser to the carbonyl carbon on the ester substrate to form
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in the enzyme activity is expected. Moreover, the opening of
the flap triggers the exposure of the active site to the solvent,
thus making the enzyme more vulnerable to the surrounding
environments and in turn a lower stability. In contrast, a more
chaotropic IL cation may help to protect the enzyme in its closed
conformation, thus making it more stable. Even if accessing the
active site, this cation has a low affinity for the critical Glu (or
Asp) but can help to stabilize the negatively charged enzyme-
substrate tetrahedral intermediate. This will lower the activation
energy and hence lead to a higher enzyme activity. The above
discussion can thus explain well why PEL was both active and
stable in the presence of a chaotropic IL cation and both inactive
and unstable with a kosmotropic one (Panel B and D of Fig. 2).
On the other hand, IL anions can also affect the enzyme
performance by following the Hofmeister series. Because of
its strong interaction with water, a kosmotropic anion is used
to competing effectively for the water molecules originally
associated with the enzyme molecule,40 and hence may render the
flap that covers the enzyme’s active site to be more open, taking
the hydrophilic character of its upper side into account. This
may cause the formation of the enzyme’s active conformation,
leading to, on the one hand more substrate molecules to be
available to the enzyme’s active site and hence a higher enzyme
activity, and on the other hand more exposure of the active site
to the solvent and thus a lower enzyme stability. This can then
account for the opposite responses of the activity and stability
upon the addition of the choline ILs (Fig. 1).
Fig. 4 Initial rates of tyrosinase-catalyzed oxidation of L-DOPA in the
presence of different ILs (5.85%, w/v) relative to the rate obtained in
the IL-free buffer solution (which was 0.104 mM min-1 and taken as 100
for comparison).
the same cation (such as choline+ and NBu4 ), the correlation
+
between the activity data and the kosmotropicity of the anion
was somewhat opposite to what we expected. The fact that the
activity responded well upon the choice of the IL cation but not
upon that of the anion may be taken as a support again to our
earlier statement that the cation of an IL plays a more dominant
role than its anion in affecting the enzyme performance. Another
feature that is very different from the result obtained by PEL is
that the tyrosinase activity is relatively high in the presence of all
-
the ILs holding H2PO4 as the anion, suggesting that although
However, a chaotropic anion has not only a low affinity
for water but also a high polarizability, preferring to bind to
the protein/water interface and to interact with the chaotropic
cationic moieties (such as amino groups) on the enzyme surface.6
This will cause more H+ ions to be accumulated on the surface
of the enzyme, leading to a reduction in the surface pH.29,41
Bostro¨m et al.42 have developed a modified ion-specific double-
layer model to demonstrate that the surface pH of a protein is
dependent on the salt concentration and on the ionic species
following the Hofmeister series. A low surface pH would force
the Glu (or Asp) and His at the active site to be protonated,
thereby weakening their ability to assist the Ser residue in the
nucleophilic attack to the carbonyl group of the substrate. As
a result, the enzyme activity is reduced. The adsorption of
the chaotropic anion on the enzyme surface and its strong
interaction with the enzyme can also render the enzyme structure
to be distorted, thus leading to a lower stability.
detrimental to PEL, this anion is favored by tyrosinase.
In the interest of achieving some insights into the IL effects,
a further investigation was carried out to assess the influence of
the 3 imidazolium ILs on both the kinetics and thermostability
of the enzyme. The kinetic studies reveal that for each IL tested,
upon an increase in the IL content the Km and Vmax values
of the enzyme increased and decreased, respectively, resulting
in a reduction in Vmax/Km. But at any fixed IL levels within
the tested range, the Vmax is altered following the sequence of
[MMIm][MeSO4] > [EMIm][MeSO4] > [BMIm][MeSO4] (Fig.
5), again consistent with the Hofmeister cation effect, whereas
Effect of ILs on the activity and stability of mushroom tyrosinase
In order to verify whether the above results are applicable to
other enzymes, we extended our study to a different type of
enzyme, mushroom tyrosinase. The screening of its activity in
the presence of all the 14 ILs (Fig. 4) shows some similar
results (although none of the ILs tested can stimulate the
tyrosinase activity): in the presence of the ILs holding the
same anion, the initial rate of the tyrosinase-catalyzed oxidation
reaction showed a decreasing order of [MMIm][MeSO4] >
[EMIm][MeSO4] > [BMIm][MeSO4], [NMe4][Ac] > [NBu4][Ac],
and [NHMe3][H2PO4] > [NHEt3][H2PO4]. This suggests that the
variation of tyrosinase activity also followed the kosmotrop-
icity/chaotropicity of the IL cations. But for the ILs holding
Fig. 5 Vmax obtained by mushroom tyrosinase in phosphate buffer
containing 3 imidazolium ILs with different concentrations (the protein
content in the reaction system was 6.58 mg ml-1).
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Table 2 Thermostability of mushroom tyrosinase in the presence of
the three imidazolium ILs (5% w/v) as compared to the control in the
absence of any IL
Incubation
[MMIm]- [EMIm]- [BMIm]-
temperature Control [MeSO4] [MeSO4] [MeSO4]
Half life (min) 30 ◦
C
C
C
856.9
119.7
14.9
347.4
31.5
4.6
152.7
22.5
3.8
124.0
8.3
2.1
40 ◦
50 ◦
First-order
30 ◦C
1.0
4.0
44.9
2.0
22.0
n.a.
3.6
28.4
n.a.
4.2
deactivation 40 ◦
rate constant 50 ◦
(10-3 min-1)
C
C
n.a.a
n.a.
a ‘n.a.’ means ‘not applicable’, referring to the situations that signifi-
cantly deviated from the classical first-order kinetics, in which case the
deactivation rate constants were not determined.
no such correlation was found for either Km or Vmax/Km values.
Lou et al.37 have reported that when papain was used to catalyze
hydrolysis in aqueous buffer containing a series of imidazolium
ILs ([CnMIm][BF4], n = 2~6, 15% v/v), a reduction in Vmax, along
with an increase in Km, was also observed with a longer alkyl
chain attached on the IL cation.
Scheme 1 Catalytic cycle of the tyrosinase-catalyzed oxidation from
o-diphenol to o-quinone.
The thermostability data are listed in Table 2. At each
incubation temperature, the enzyme stability in the presence
of the 3 imidazolium ILs varied in the same order as above:
[MMIm][MeSO4] > [EMIm][MeSO4] > [BMIm][MeSO4], al-
though none of these 3 ILs showed the ability of stabilizing the
enzyme. In our previous study,12 tyrosinase in aqueous solution
has been found to be destabilized by the three imidazolium ILs
holding the same cation, namely [BMIm][PF6], [BMIm][BF4],
and [BMIm][MeSO4]. The stability order observed in this study
confirms this and reinforces the fact that a more kosmotropic
IL cation is less favorable for maintaining the enzyme stability.
The deviations from first-order deactivation kinetics at a higher
temperature (50 ◦C) and in the presence of a more kosmotropic
IL cation (BMIm+) suggest that under these conditions, the
enzyme might undertake a more complex mechanism for its
deactivation. It is actually not surprising to have observed
these deviations, simply because the first-order kinetics model is
oversimplified.
A closer look at the active site structure and catalytic
mechanism of tyrosinase is necessary for us to understand how
ILs affect the enzyme activity through the Hofmeister effect.
Mushroom tyrosinase is a metalloenzyme consisting of two
tetragonal Cu2+ atoms at the active site, as shown in Scheme 1.43,44
Vmax is dependent on the two rate constants (k2, k4) that govern
the formation of the quinone product, whereas Km is associated
not only with these two rate constants but also with the two
dissociation constants (K1, K3) that govern the nucleophilic
attack of the substrate o-diphenol on the active site Cu2+ ion.44
Cu2+ ion is a strong kosmotrope with a highly positive viscosity
B coefficient of +0.376.23 Based on the “law of matching water
affinity”,38 a kosmotropic anion has a strong power of ion-
pairing with this metal ion, thus stabilizing the ground state
of the enzyme and diminishing the ability of the copper ion in
accepting the nucleophilic attack from the substrate, o-diphenol.
As a result, the activation energy of the enzymatic reaction is
increased and the reaction rate is lowered. This can explain
the unexpected activity order obtained by the ILs holding
the same cation (Fig. 4): [choline][Ac] < [choline][MeSO3] <
[choline][NO3] and [NBu4][Ac] < [NBu4][MeSO3].
The above discussion can also be used to explain why
tyrosinase activity was always lower than the control when
conducting catalysis in the presence of the three imidazolium
-
ILs. This is simply because the IL anion, MeSO4 , is a strong
kosmotrope. In addition, a highly kosmotropic IL cation like
BMIm+ may compete with the similarly kosmotropic Cu2+ cation
at the enzyme’s active site for the substrate, o-diphenol, thereby
preventing the latter from nucleophilic attack to the Cu2+ ion for
proceeding the catalysis. This offers a plausible explanation for
the sensitive response of the tyrosinase activity to the IL cation
in the order of control > MMIm+ > EMIm+ > BMIm+. The
interaction between the imidazolium cation and the substrate
leads to a reduction in the two rate constants, k2 and k4, and an
increase in the two dissociation constants, K1 and K3. According
to the two equations listed in Scheme 1, Vmax is decreased whereas
the change in Km is either increased or decreased, depending
on the balance between the two rate constants and the two
dissociation constants. This can readily explain why it is the
V
max, rather than the Km, that varied by following the Hofmeister
effect of the IL cation (Fig. 5).
Structural study of mushroom tyrosinase
Investigation on any possible conformational changes induced
by ILs may shed some light on the effect of ILs on enzyme
performance. Fluorescence spectroscopy can be used to follow
the changes in protein conformation, such as unfolding. This is
reflected by both the decrease in the maximal intensity of the
fluorescence (Imax) and the red-shift in the maximal emission
wavelength (lmax), both resulting from the increased polarity
experienced by the fluorophore residues (particularly Trp) of
the protein globule.45
Fig. 6 shows the fluorescence spectra of tyrosinase in phos-
phate buffer with addition of two sets of ILs holding the same
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Fig. 6 Fluorescence spectra of tyrosinase in phosphate buffer (50 mM, pH 6.0) in the absence and presence of different ILs (2.93%, w/v): (A) 3
-
imidazolium ILs with MeSO4 as the anion; (B) 4 ammonium ILs with choline as the cation. The data are normalized with respect to the maximal
fluorescence intensity obtained at 336 nm in the IL-free buffer solution.
anion (A) and cation (B), respectively. As compared to its
native form the fully denatured enzyme exhibited a red-shift
in lmax (from 336 nm to 340 nm) and a significant drop in
Imax (from 100% to 48.2%), confirming the unfolding of the
protein structure leading to the exposure of the tryptophan
residues to the solvent. Addition of both groups of the ILs
resulted in a noticeable reduction in Imax, which is also shown in
Fig. 7, and negligible shift in lmax. This suggests that although
the tryptophan residues were not very much exposed, a more
loosely packed conformation of the protein was triggered by
all the ILs tested, in concert with the reduced activity assayed
in the presence of these ILs (Fig. 4). Upon IL addition the
fluorescence intensity of the enzyme decreased following the
increase in the kosmotropicity of the IL cation (Fig. 7A) and the
decrease in the kosmotropicity of the IL anion (Fig. 7B). A severe
situation was observed in the presence of [choline][NO3], which
holds a chaotropic anion obviously detrimental to the protein
structure:6 A red-shift of 9 nm in lmax and a 99.5% reduction in
agrees with the activity data, particularly in the case of the
imidazolium ILs holding the same anion. Interestingly, the order
of the fluorescence intensity ([choline][Ac] ~ [choline][MeSO3] >
[choline][NO3], Fig. 7B) was inverse to the activity order
shown in Fig. 4, and in the presence of [choline][NO3] the
enzyme presented a relatively high activity while yielding a
poor fluorescence intensity, which is even weaker than the one
obtained by the enzyme totally denatured by heat. It has been
proposed that the folded state does not always induce enzyme
activation,46,47 and our results seem to agree with this.
Conclusions
This work has clearly demonstrated that specific ion effect of
ILs plays an important role in affecting enzymatic performance.
Both activity and stability of the two model enzymes in aqueous
solution are controlled directly or indirectly by the Hofmeister
effect of the ILs, as has been supported by the structural study
with fluorescence spectroscopy. Specific impacts of ILs may
be associated with the influence of their cations and anions
on the surface pH, active site conformation, and catalytic
mechanism of each specific enzyme, following the Hofmeister
I
max were caused when the concentration of this IL was increased
from 0 to 0.53 M. All these strongly substantiate that the impact
of ILs on the protein structure follows the Hofmeister series very
well.6 The IL-induced variation in the protein structure basically
Fig. 7 Effect of ILs and their concentrations on the maximal fluorescence intensity of tyrosinase (obtained at 336 nm), relative to the maximal
-
fluorescence intensity obtained by the enzyme at 336 nm in the IL-free buffer solution. (A) 3 imidazolium ILs with MeSO4 as the anion; (B) 4
ammonium ILs with choline as the cation.
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series. Ammonium ILs, especially those composed of chaotropic
cations and kosmotropic anions, offer potential advantages in
facilitating enzyme functioning. A few ammonium ILs such as
[NHMe3][MeSO3] (which is a protic IL) and [choline][Ac] (which
possesses a hydroxylated cation) are effective in promoting
enzyme activity and stability, and the plausible reason may be
related to both the H-bonding capability and chaotropicity of
these IL cations (in combination with a kosmotropic anion).
The IL cations may play a predominant role over their counter-
anions in affecting the enzyme behavior. By testing 79 ILs of
different types in their acetylcholinesterase (AchE) inhibition
screening assay, Arning et al.48 have already observed a more
profound effect elicited by the IL cations, in terms of their head
groups and side chains, whereas the vast majority of the anion
species exhibited no impact on AchE.
However, it has to be stated that the specific ion effect is
not the only factor controlling enzyme performance in the IL
systems, and that the behavior of an enzyme in IL-containing
aqueous solution may be somewhat different from that in an IL-
dominating system. As has been demonstrated,49 the catalytic
activity is highly dependent on the characteristics of the IL,
the structure of the substrate, and the nature of the enzyme
and the support. Further research has to be carried out in
order to advance our knowledge about biocatalysis in ILs,
thus promoting the development of green biotransformation
processes in such promising nonaqueous media.
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