Effect of Confinement on the Properties of Sequestered Mixed Polar Solvents: Enzymatic Catalysis in Nonaqueous 1,4-Bis-2-ethylhexylsulfosuccinate Reverse Micelles

Article abstract of DOI:10.1002/cphc.201501190

The influence of different glycerol, N,N-dimethylformamide (DMF) and water mixtures encapsulated in 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/n-heptane reverse micelles (RMs) on the enzymatic hydrolysis of 2-naphthyl acetate by α-chymotrypsin is demonstrated. In the case of the mixtures with DMF and protic solvents it has been previously shown, using absorption, emission and dynamic light-scattering techniques, that solvents are segregated inside the polar core of the RMs. Protic solvents anchor to the AOT, whereas DMF locates to the polar core of the aggregate. Thus, DMF not only helps to solubilize the hydrophobic substrate, increasing its effective concentrations but surprisingly, it does not affect the enzyme activity. The importance of ensuring the presence of RMs, encapsulation of the polar solvents and the corrections by substrate partitioning in order to obtain reliable conclusions is highlighted. Moreover, the effect of a constrained environment on solvent–solvent interactions in homogenous media and its impact on the use of RMs as nanoreactors is stressed.

Full text of DOI:10.1002/cphc.201501190

DOI: 10.1002/cphc.201501190
Articles
Effect of Confinement on the Properties of Sequestered
Mixed Polar Solvents: Enzymatic Catalysis in Nonaqueous
1,4-Bis-2-ethylhexylsulfosuccinate Reverse Micelles
Andres M. Durantini,[b] R. Dario Falcone,[a] Juana J. Silber,[a] and N. Mariano Correa*[a]
The influence of different glycerol, N,N-dimethylformamide
(DMF) and water mixtures encapsulated in 1,4-bis-2-ethylhexyl-
sulfosuccinate (AOT)/n-heptane reverse micelles (RMs) on the
enzymatic hydrolysis of 2-naphthyl acetate by a-chymotrypsin
is demonstrated. In the case of the mixtures with DMF and
protic solvents it has been previously shown, using absorption,
emission and dynamic light-scattering techniques, that sol-
vents are segregated inside the polar core of the RMs. Protic
solvents anchor to the AOT, whereas DMF locates to the polar
core of the aggregate. Thus, DMF not only helps to solubilize
the hydrophobic substrate, increasing its effective concentra-
tions but surprisingly, it does not affect the enzyme activity.
The importance of ensuring the presence of RMs, encapsula-
tion of the polar solvents and the corrections by substrate par-
titioning in order to obtain reliable conclusions is highlighted.
Moreover, the effect of a constrained environment on solvent–
solvent interactions in homogenous media and its impact on
the use of RMs as nanoreactors is stressed.
1. Introduction
The majority of the chemical reactions that occur in biology
take place at the interface of cell organelles rather than in
a medium outside the cell. It is therefore important to under-
stand the interactions of proteins with those interfaces. Even if
reverse micelles (RMs) are an oversimplified model, the amphi-
pathic essence of biological membranes is preserved and im-
portant properties such as hydrogen bonding tendencies can
be studied.[1] RMs are formed by surfactant molecules dis-
solved in nonpolar solvents with their polar head groups
pointing inward and the hydrocarbon chains pointing out to
the solvent.[2–4] Sodium 1,4-bis-2-ethylhexylsulfocuccinate (AOT)
is one of the most investigated and used anionic surfactants
because it forms stable RMs without the addition of co-surfac-
tants.[2–5] AOT RMs can encapsulate different amounts of water,
expressed as W0 (W0 =[water]/[AOT]), depending on the non-
polar solvent used, addition of solutes, and temperature.[4,6,7]
The most common solvent used to be dispersed is water but
polar organic solvents, which have high dielectric constants
and low solubility in the nonpolar solvent, can also be seques-
tered by the surfactant forming the nonaqueous RMs.[8] Some
of these polar solvents include dimethylsulfoxide (DMSO), for-
mamide, N,N-dimethylformamide (DMF), N,N-dimethylaceta-
mide, ethylene glycol, propylene glycol, glycerol (GY) and ethyl
lactate.[8–10] Also, mixtures of polar solvents can remain encap-
sulated within the RM polar core; if that happens, their pure or
“bulk” solvent structure changes dramatically and segregation
of the solvents occurs. Nonaqueous RMs and those in polar
solvent mixtures have been shown to be useful as nanoreac-
tors.[8] Studies have shown that protic solvents anchor to the
polar side of the AOT/n-heptane RMs interface, whereas aprot-
ic solvents form the polar core that interacts with the sodium
counterions.[9–11] Specifically, in reference [11] we showed for
the first time segregation of GY molecules at the polar side of
the AOT RMs interface, whereas the DMF molecules remained
in the polar core. Those findings motivated us to investigate if
such solvent segregation might favor the outcome of chemical
reactions.
Studies performed in our group indicated that the size of
RMs not only depends on the quantity of polar solvent added,
but also on the interaction of the polar solvent with the RMs
interface.[10,12] This is an interesting feature of the RMs in the
context of nanoreactors, because not only the size but also the
polar solvent structure can be controlled upon encapsulation.
Since the pioneering works of Martinek et al.[13] were pub-
lished more than 30 years ago, it has become known that, by
using water as the dispersed solvent, the catalytic activity of
different enzymes can be enhanced by the juxtaposed solvent
effects.[2,13–23] In particular, these effects have been well estab-
lished for a-chymotrypsin (a-CT), which, being a hydrophilic
and globular enzyme, usually remains incorporated within the
RMs.[14,20] Interestingly, the group of A. J. Wand has developed
a method to examine the structure of certain proteins in differ-
ent RMs with water as a polar solvent by using high-resolution
NMR spectroscopy.[24,25] In their efforts to encapsulate proteins
with high structural fidelity in AOT RMs under these particular
[a] Dr. R. D. Falcone, Dr. J. J. Silber, Dr. N. M. Correa
Departamento de Química, Universidad Nacional de Río Cuarto
Agencia Postal # 3. C.P. X5804BYA Río Cuarto (Argentina)
E-mail: mcorrea@exa.unrc.edu.ar
[b] Dr. A. M. Durantini
Department of Chemistry and Center
for Self-Assembled Chemical Structures
McGill University, 801 Sherbrooke Street West
Montreal, Quebec H3A 0B8 (Canada)
Supporting Information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cphc.201501190.
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Articles
experimental conditions, they showed that this surfactant has
a strong denaturing effect on most of the proteins investigat-
ed that were located in the water pool; however a-CT has not
been studied in these experiments. This represents an avenue
of investigation, as it seems that enzymes can function upon
confinement, even though NMR data shows changes in their
conformation. Conversely, there are no reports of NMR studies
on nonaqueous AOT RMs and enzymes that are located at the
polar side of the RMs interface and not in the polar core.
By contrast, the substrates for a-CT in a living organism are
normally esters or peptides with low water solubility and the
hydrolysis reaction take place within a hydrophobic domain. In
order to mimic the physiological situation, in vitro, it is impor-
tant to design a nanoreactor that can bring the hydrophobic
substrate to the reaction milieu, in which the enzyme is locat-
ed. Also, it should bring about an improvement in the enzyme
activity. For organic nonpolar substrates in homogeneous
media, the use solvents in which they have increased solubility,
such as DMF or DMSO, will be required. One problem is that
DMSO acts as denaturant for a-CT,[26] and in DMF the enzyme
has been shown to be severely inactivated at low water con-
tent.[27,28] A question therefore arises: it possible to create such
nanoreactors using RMs, water, and solvents such as DMF and
DMSO? Because of the unique properties of the AOT RMs
mentioned above, the answer is yes, and this article shows
how to create such an enzymatic reactor and also how to
handle the data in order to obtain the “true” enzymatic param-
eters that take into account the substrate partition between
the RMs and the external solvent pseudophase.[14,20] Surprising-
ly, there are no reports in the literature that demonstrate enzy-
matic reactions using binary polar solvent mixtures for confine-
ment in RMs.
concentration inside the RM and the consequent enhancement
in the reaction rate. For the first time, we show that the use of
a denaturant solvent in a confined environment increases the
enzyme activity.
Our goal, motivated by our pioneering studies on of solvent
segregation inside AOT RMs,[11] was to ascertain if other nano-
reactors can be designed such that an enzymatic deactivating
solvent in homogeneous solution is used in a confined envi-
ronment. We sought to determine if there is an improvement
in the substrate solubility within the RMs without affecting
enzyme performance. To this end, two binary (GY/water and
DMF/water) and one ternary (GY/DMF/water) solvent mixtures
were investigated for the a-CT-catalyzed hydrolysis of 2-NA in
homogenous and AOT/n-heptane RMs systems. These are in-
teresting systems because GY is a polyol that stabilizes biologi-
cal molecules such as proteins and enzymes in water.[29–32]
DMF, in which 2-NA is very soluble, is of particular interest due
to the lack of a hydrogen atom that is capable of engaging in
hydrogen-bond interactions and because its bulk structure is
altered in aqueous mixtures.[33] Although DMF is one of the
most important aprotic organic solvents used in a wide variety
of chemical applications, its use severely decreases the enzyme
activity in homogeneous solution.[28] The use of the ternary
mixture is justified by our previous results that showed a favor-
able effect if GY was added to the aqueous pool of the RMs.[20]
We therefore expected that the presence of GY would improve
the catalytic activity of a-CT in the presence of DMF. It should
be considered that water is always necessary for the hydrolysis
reaction to occur. The proposed mechanism of the reaction
and the kinetic equations are detailed in the Supporting Infor-
mation.
In summary, we show that everything that happens in ho-
mogeneous solution has to be reconsidered because of the
fascinating effect of confinement.
The first studies performed on the catalytic behavior of a-CT
in RM-containing media have been done using water as the
polar solvent encapsulated in the core of the aggregates.[14]
However, in the last 10 years the properties of the enzyme
have also been studied in other solvents, such as GY and
DMSO, as well as their binary mixtures with water.[8,20,26] Fal-
cone et al.[20] investigated the kinetics of the enzymatic hydrol-
ysis of 2-naphthyl acetate (2-NA) by a-CT in AOT RMs designed
with GY and GY/water (38%v/v) mixtures /AOT/n-heptane. It
was demonstrated that addition of GY to the RMs results in en-
hanced catalytic efficiency of a-CT. GY addition decreases the
conformational mobility of a-CT, thus increasing enzyme stabil-
ity and activity.[20]
Recently, Moyano et al.[26] have studied the kinetics of the
enzymatic hydrolysis of N-benzoyl-l-tyrosine p-nitroanilide by
a-CT in homogenous media and in DMSO/water/AOT/n-hep-
tane RMs, at different DMSO/water content. In homogeneous
media, as expected, DMSO acts as a denaturant of the protein
and a dramatic decrease in the enzymatic activity was found at
higher DMSO contents. However, upon DMSO/water mixture
confinement, the hydrogen-bond interactions between DMSO
and water are disrupted; water remains at the polar side of the
AOT RMs interface where the enzyme is located, and DMSO
molecules migrate to the polar micelle core and help solubilize
the substrate. The overall result is an increase in the effective
2. Results and Discussion
In this work, we studied reactions in the following mixtures:
GY/water (40:60, v/v), DMF/water (20:80, v/v) and GY/DMF/
water (20:20:60, v/v/v), in homogeneous media (this refers to
the solvent mixture not encapsulated inside the RMs) and en-
capsulated in AOT/n-heptane RMs. In a confined environment,
results were obtained at WT values [WT =([GY]+[DMF]+
[water])/[AOT]=WGY +WDMF +Wwater] of around 4 to 6 because
this represents the best set of conditions found for the enzy-
matic reaction. At lower WT values the reaction is very slow,
and at higher WT, the RMs become unstable in the ternary mix-
ture.
The reactions were followed spectrophotometrically by the
increase at the maximum of the product 2-naphthol (2-NT) ab-
sorption band (lmax =327 nm) at 25.0Æ0.58C. Absorbance was
recorded on a Hewlett–Packard 8453 UV/Vis spectrophotome-
ter equipped with a thermostated cell (volume 3 mL, path
length 1 cm). The UV/Vis spectroscopic analysis shows that the
a-CT-catalyzed hydrolysis of 2-NA in all the mixtures and in
water, produced 2-NT in quantitative yield [Eq. (1)]:
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RMs and DMF molecules remain in the RM core, engaging in
electrostatic interactions with sodium counterions as was pre-
viously found for the individual solvents.[12]
The results summarized in Table 1 also demonstrate that the
presence of the enzyme does not affect the size of the differ-
ent RMs systems, because the droplets are essentially the same
size with and without the enzyme.
As the DMF/water and GY/DMF/water mixtures were never
previously studied inside AOT RMs, the following question
needs answering: Is this surfactant actually forming RMs with
those polar solvents? That is, before proceeding with the ki-
netic studies it is necessary to evaluate if the mixtures are ef-
fectively sequestered by AOT in order to form RMs. As has
been discussed in the literature,[8] GY, DMF and water and their
mixtures have very low solubility in n-heptane. Thus, the vari-
ous mixtures of polar solvents cannot be dissolved in this or-
ganic pseudophase. It is often reported in the literature that
because a transparent solution is obtained when polar solvents
are mixed with the surfactant, RMs are created. This is abso-
lutely incorrect because an RM medium needs to organize its
components into the interface and the polar core.[8] Thus,
when a new system is investigated it is mandatory to effective-
ly demonstrate that the polar solvents are encapsulated by the
surfactants creating organized systems with their unique prop-
erties. Furthermore, a-CT has a diameter of about 4 nm,[34,35]
which generates doubt about whether the encapsulation of a-
CT affect the diameter of the differently organized systems.
As shown in several studies, dynamic light scattering (DLS) is
a good technique for investigating these types of assemblies
and, it accurately reveals whether the solvents are encapsulat-
ed by the surfactants. Moreover, this technique can also esti-
mate the shape of the droplets and if they interact with each
other. In all the systems investigated, the droplet size value in-
creases with a linearly with WT (results not shown), indicating
that the different polar solvent mixtures are effectively encap-
sulated in the RMs.[4,6–10,36,37]
a-CT was analyzed by using fluorescence emission spectros-
copy, by monitoring the emission band of the tryptophan resi-
due centered at lem =350 nm. The spectrum of the enzyme
dissolved in solution containing 8m urea showed that its con-
formational structure was altered because of the denaturing
effect of the solute. In this sense if any of the polar solvents
used here are denaturing agents for the enzyme, the emission
band should appear around lem ꢀ350 nm. The results obtained
with the polar solvent blends are shown in Figure 1 at lexc
ꢀ280 nm. As is apparent, the emission profile for a-CT is not
altered in any mixture studied in homogeneous and AOT RMs.
The diameters and polydispersities of the RMs are listed in
Table 1 at a given value, WT =6. Table 1 shows that the diame-
ter increases with the presence of GY and remains almost con-
stant as DMF content is varied. Also, the sizes of GY/water and
GY/DMF/water/AOT/n-heptane RMs are very similar. According
to these results, when the binary or ternary mixtures are dis-
solved, GY and water are bound by hydrogen-bond interac-
tions with the AOT polar head group at the interface of the
Figure 1. Normalized emission spectra of a-CT in different media.
lexc =280 nm; [a-CT]=5”10À6 m, [AOT]=0.1m.
It seems that encapsulation of the enzyme in the AOT RM
media does not significantly alter the tertiary structure of a-CT.
Next, we discuss the hydrolysis reaction in homogenous so-
lution in which DMF and GY were used as a part of the polar
component in order to improve the 2-NA solubility in water.
Does DMF have any effect on the enzymatic hydrolysis? We
have shown (Figure 1) that it does not affect the enzyme terti-
ary structure, but what about the a-CT activity?
Table 1. Apparent hydrodynamic diameter (dapp
) and polydispersity
values of the different RM systems with and without a-CT.[a]
Media
dapp
Polydispersity
[%]
[nm]
water/AOT/n-heptane
5.6
7
8
3
5
2
2
8
7
water/AOT/n-heptane+a-CT
5.7
The hydrolysis rates of the enzymatic reaction were followed
by absorption spectroscopy at the product absorption band in
every media. The best mechanism to explain the experimental
data is the classical Michaelis–Menten mechanism. The kinetic
parameters were determined by fitting the experimental data
as described in the Supporting Information. Accordingly, the
catalytic rate constant (kcat), the Michaelis–Menten constant
(KM) and the catalytic efficiency (kcat/KM) were calculated.
GY/water (40:60, v/v)/AOT/n-heptane
GY/water (40:60, v/v)/AOT/n-heptane+a-CT
DMF/water (20:80, v/v)/AOT/n-heptane
DMF/water (20:80, v/v)/AOT/n-heptane+a-CT
20.1
20.1
6.5
6.8
GY/DMF/water (20:20:60, v/v/v)/AOT/n-heptane 18.9
GY/DMF/water (20:20:60, v/v/v)/AOT/n-hepta-
ne+a-CT
18.2
[a] W0 =6, WT =6; [a-CT]=5”10À6 m; T=258C.
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2.1. Reaction in Homogeneous Media: DMF/Water and GY/
DMF/Water Mixtures
Table 3. Kp and KPmixture=nÀheptane values for the different RM systems and
homogenous mixtures.[a]
Medium
Kp
KPmixture=nÀheptane
Typical absorption spectra for hydrolysis of 2-NA at different
reaction times in GY/DMF/water (20:20:60, v/v/v) are shown in
Figure S2 (in the Supporting Information), and the formation
of the product 2-NT is evident [Eq. (1)]. Similar results were ob-
tained in DMF/water (results not shown).
(”10À2
)
RMs
water/AOT/n-heptane
GY/water (38:62, v/v)/AOT/n-heptane
0.92Æ0.05
5.65Æ0.73
–
–
–
–
DMF/water (20:80, v/v)/AOT/n-heptane 20.68Æ2.30
A plot of v0/[a-CT] versus the analytical concentration of 2-
NA ([2-NA]T) is shown in Figure S3A. Figure S3B shows a typical
result obtained by treating the data as a Lineweaver–Burk plot
[Eq. (S6)], the linearity of which indicates that, under the condi-
tions used, the Michaelis–Menten mechanism applies in the
polar solvent mixtures as has been previously found for water
and GY/water mixtures.[20,38] Although kinetic constants could
be obtained from the linear fit using the Equation S6, through-
out this work we determined the kinetic parameters kcat and
KM using a nonlinear fit according to Equation S4. The results
are shown in Table 2, which gives the values obtained for the
GY/DMF/water (20:20:60, v/v/v)/AOT/
n-heptane
15.7Æ0.79
Homogeneous media
water[b]
GY/water (38:62, v/v)[b]
DMF/water (20:80, v/v)
GY/DMF/water (20:20:60, v/v/v)
–
–
–
–
0.36Æ0.02
0.66Æ0.03
4.03Æ0.03
3.01Æ0.15
[a] [2-NA]=1”10À3 m, [a-CT]=5”10À6 m. [b] Values obtained from refer-
ence[20].
Information. If DMF is present in the water mixture the Kp
values increase considerably, that is, 2-NA is more soluble in
the water mixture than in n-heptane. However, a striking de-
crease of the a-CT efficiency was found when DMF was added,
even though the solubility of the substrate increases markedly.
rate constants in DMF/water (20:80, v/v) and GY/DMF/water
exp mixture
cat
(20:20:60, v/v/v) mixtures, where (k
Þ
is the experimental
catalytic constant and (KMexp
Þ
is the experimental Michae-
mixture
lis–Menten constant. Also, the results from reference [20] ob-
tained in water and in a GY/water (32:68, v/v) mixture are in-
cluded for comparison. Table 2 shows that the catalytic effi-
exp
cat
ciency of the enzyme, given by the ratio (k /KeMxp mixture, is
Þ
2.2. Reactions in AOT/n-Heptane RMs
much smaller for reactions occurring in binary or ternary DMF-
containing mixtures in comparison with the GY/water mixture.
A dramatic decrease in the catalytic efficiency value can be ob-
served if only DMF and water is used, as previously shown for
other enzymatic reactions.[27,28] The increase of the enzymatic
catalytic efficiency found for the GY/water mixture compared
with the DMF/water and GY/DMF/water mixtures is due to
The hydrolysis reaction was studied in the following RMs sys-
tems: water/AOT/n-heptane, GY/water (40:60, v/v)/AOT/n-hep-
tane, DMF/water (20:80, v/v)/AOT/n-heptane, and GY/DMF/
water (20:20:60, v/v/v)/AOT/n-heptane. It is important to recall
that all the studies were performed at the maximum WT value
that can be achieved for GY/DMF/water (20:20:60, v/v/v)/AOT/
n-heptane RMs, that is WT =6.
exp mixture
mixture
both an increase in (k
Þ
and a reduction in (KMexp
Þ
cat
values. The results demonstrate that DMF has a negative effect
on the enzyme activity, affecting dramatically its hydration
level or conformational structure but without denaturing the
enzyme (Figure 1).[28]
We have previously studied this reaction in water/AOT/n-
heptane and GY/water (38:62%v/v)/AOT/n-heptane RMs, albeit
at WT =13.2.[20] Therefore, in order to compare these results
with the one obtained here, the kinetic parameters were inves-
tigated at W0 =6 and WT =6. Knowing the location and proper-
ties of each of the polar solvents both alone and in the mix-
tures within AOT RMs, a systematic study of the hydrolysis re-
action of the substrate 2-NA was made for each system.[8,9,20]
From the data in Figures S4A, S5A, S6A and S7A it is possi-
ble to observe how the initial rate increases upon increasing
the substrate concentration at constant enzyme concentration
([a-CT]=5”10À6 m) for all the systems studied. As in homoge-
neous media, if the substrate concentration is high, v0 is essen-
tially independent of the substrate concentration. Figures S4B,
S5B, S6B and S7B indicate that the reaction mechanism inside
the RMs follows the Michaelis–Menten kinetics as straight lines
are obtained in the Lineweaver–Burk plots.
In contrast, it was observed that the 2-NA solubility increas-
es markedly if DMF is present in the binary and ternary mix-
tures. Table 3 shows the Kp values for 2-NA in the different mix-
tures investigated. Absorption spectroscopy was used to
obtain these data and details are provided in the Supporting
Table 2. Summary of experimental kinetics parameters of the enzymatic
reactions in homogeneous media.[a]
exp mixture
cat
[”10À2 sÀ1
mixture
exp
cat
[mÀ1 sÀ1
]
mixture
Medium
(k
Þ
(KeMxp
Þ
(k /KeMxp
Þ
]
[”10À3 m]
water[b]
GY/water
(38:62, v/v)[b]
GY/DMF/water
(20:20:60, v/v/v)
DMF/water
(20:80, v/v)
2.10Æ0.21
8.80Æ0.88
0.57Æ0.06
0.31Æ0.03
36.00
284.00
Typical results obtained on the effect of varying the AOT
concentration on the relationship between v0/[a-CT] and the
analytical concentration of 2-NA at WT =6 are shown in
Figure 2 for GY/DMF/water (20:20:60, v/v/v)/AOT/n-heptane
RMs. This data shows that the v0/[a-CT] ratio is lower at higher
concentrations of AOT.
0.37Æ0.04
0.21Æ0.04
0.64Æ0.04
3.32Æ0.02
5.78
0.62
[a] [a-CT]=5”10À6 m. [b] Values obtained from reference [20].
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consideration in order to obtain the enzymatic kinetic parame-
ters in the RM media. This correction factor is not always to be
found among the vast literature on enzymatic catalysis. The
values of (KeMxp
Þ
were corrected by the partitioning of 2-NA
mic
(Kp) with Equation (2).[14,20,40] Kp values were obtained using the
procedure described in the Supporting Information and are
gathered in Table 3.
À
Á
mic
KeMxp
mic
ðKcMorr
Þ
¼
À
Á
ð2Þ
1 þ Kp½AOTŠ
These results are listed in Table 5 from which it can be seen
that the corrected catalytic efficiency values, (k /KMcorr
exp
cat
Þ
mic, are
larger than the efficiency obtained for those uncorrected,
(k /KeMxp
Þ
mic. Furthermore, the values of (k /KcMorr
Þ
obtained
exp
cat
exp
cat
mic
for the encapsulated mixtures are larger than those obtained
for the reaction that occurs in RMs encapsulating only water.
Note that the largest (k /KMcorr
Figure 2. Effect of AOT concentration on the relationship between the initial
rate of reaction (per enzyme) and the analytical concentration of 2-NA in re-
verse micelles of GY/DMF/water (20:20:60, v/v/v)/AOT/n-heptane at WT =6.
[a-CT]=5”10À6 m.
exp
cat
mic
Þ
values correspond to the
mixture with DMF, which is interesting because the exact op-
posite result was found in homogenous media (Table 2).
Table 5. Summary of experimental kinetics parameters of the enzymatic
reaction in different RMs systems.
The dependence on the surfactant concentration can be at-
tributed to three factors:[14,38] 1) changes in the size, shape and
microscopic properties of the RMs; 2) changes in the proper-
ties of the enzyme; and 3) the substrate distribution between
different RM pseudophases. As Table 1 shows that the droplet
sizes are the same with or without a-CT, factor 1 can be dis-
carded. As discussed before, a-CT remains totally associated to
the RMs. Besides, the emission spectra do not show denatura-
tion of the enzyme (Figure 1), a fact which allows us also to
discard factor 2. In addition, as shown by the experimental ki-
netic parameters obtained at different [AOT] (Table 4), the
mic
exp
cat
[m À1 sÀ1
mic
exp
cat
mic
RM
ðKcMorr
Þ
(k /KcMorr
Þ
(k /KeMxp
Þ
[”10À2 m]
]
[m À1 sÀ1
]
water/AOT/n-heptane
GY/water (40:60, v/v)/AOT/n-
heptane
0.30Æ0.01 0.9
0.70Æ0.02 2.2
0.6
1.5
DMF/water (20:80, v/v)/AOT/n- 0.29Æ0.02 4.5
1.4
1.0
heptane
GY/DMF/water (20:20:60, v/v/
v)/AOT/n-heptane
0.07Æ0.02 2.9
values of the experimental catalytic constant within the RMs,
exp
cat
(k
Þ
mic, are practically constant at all the surfactant concentra-
Herein, the correction made by the substrate partition
allows a comparison to be made of the kinetic parameters
among different RMs media, independent of the external sol-
vent and the polar solvent mixtures encapsulated in the polar
core of the RMs. Nevertheless, it is not possible to compare
the data with homogeneous media. To do that, it is necessary
to know the substrate partition between the different solvent
mixtures and the external solvent used (n-heptane) but in the
absence of the surfactant. In other words, in order to make
a significant comparison of the kinetic parameter values ob-
tained for the AOT RMs with the values obtained in homoge-
neous medium it is necessary to express the values in terms of
a common thermodynamic substrate activity scale and not in
terms of substrate concentrations.[20,26,41] The simplest way to
do that is to compare the rate constants, taking as the refer-
ence the external solvent of the RMs (n-heptane). The catalytic
tions used. The same results were obtained for other mixtures
studied. It is known that by varying of the AOT concentration,
but keeping W constant, the shape and the size of the RMs are
maintained.[4,39] Thus, no variation in kcat with [AOT] was ex-
pected. On the contrary, there is an increase in the experimen-
tal Michaelis–Menten constant in the RMs (KMexp
mic
Þ
(Table 4 and
Figure S8), which reflects the distribution of the substrate be-
tween the micelles and the external solvent. The differences in
the kinetic profiles shown in Figure 2 can be attributed to par-
titioning of the substrate between the micellar pseudophase
and the external solvent. This distribution has to be taken into
Table 4. Summary of experimental kinetic parameters of the enzymatic
reaction in GY/DMF/water (20:20:60, v/v/v)/AOT/n-heptane RMs at differ-
ent [AOT].[a]
efficiency of the enzymatic reaction obtained in the bulk
exp mic
cat
[”10À2 sÀ1
mic
exp
cat
mic
[AOT] [m]
(k
Þ
(KeMxp
Þ
(k /KeMxp
Þ
exp
solvents, (k /KeMxp mixture, must be corrected by substrate parti-
Þ
cat
]
[”10À2 m]
[m À1 sÀ1
]
tioning between the polar mixtures and n-heptane
(KmP ixture=nÀheptaneÞ, which were calculated (Supporting Informa-
tion) and gathered in Table 3. This correction is expressed as
Equation (3):
0.05
0.10
0.20
0.40
0.22Æ0.02
0.20Æ0.01
0.21Æ0.01
0.19Æ0.01
0.11Æ0.02
0.20Æ0.01
0.30Æ0.02
0.64Æ0.03
2.0
1.0
0.7
0.3
ðKcMorr
Þ
¼ ðKeMxp
Þ
mixture=KmP ixture=nÀheptane
mixture
[a] WT =6; [a-CT]=5”10À6 m; T=258C.
ð3Þ
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Table 3 list the KPmixture=nÀheptane values obtained for the polar
solvent mixtures and pure water in homogenous media. These
values indicate the ability of the DMF to solubilize the sub-
strate with the distribution constant much larger for the DMF/
water and GY/DMF/water mixtures than for pure water and
also larger compared to the GY/water mixture.
with the results observed in homogenous media, if the
enzyme was interacting with DMF molecules, its enzymatic ac-
tivity would be dramatically reduced. We estimate that the
polar core of the aggregate is where DMF interacts with the
surfactant’s sodium counterions. Thus, the reaction occurs at
the RM–polar solvent interface rich in GY and water molecules
and, a synergistic contribution to the activity and efficiency of
a-CT was observed for the reaction in confined media.
Table 6 summarizes the values of the corrected catalytic
efficiencies (kecaxtp/KMcorr
Þ
obtained using Equation (3). The
exp
k
/KcMorr values for the DMF/water (20:80, v/v)/AOT/n-heptane
cat
and GY/DMF/water (20:20:60, v/v/v)/AOT/n-heptane RMs are
3. Conclusions
We have shown that although DMF is a polar solvent that in-
hibits the catalytic activity of a-CT in homogeneous DMF/
water and GY/DMF/water mixtures, upon encapsulation in AOT
RMs, the situation changes. DMF is excluded from the interface
at which the reaction occurs, but helps to solubilize the sub-
strate, thus increasing the rate of the hydrolysis reaction. This
seems to be always the case if a mixture of protic and aprotic
solvents is encapsulated in RMs of AOT. Hence, the aprotic sol-
vent—in which the substrate is usually more soluble, even if it
is not favorable for the enzyme activity—upon encapsulation
is excluded from the reaction site, and an important overall in-
crease in the reaction rate is observed. At present, we also
have preliminary results that show that ionic liquids that inhib-
it completely the enzyme performance in homogeneous
media, are surprisingly good upon encapsulation with water in
RMs.
Table 6. Summary of experimental kinetics parameters of the enzymatic
reaction in the different homogeneous mixtures and RMs systems.
exp
cat
[sÀ1 mÀ1
]
Medium
kecaxtp/KM
[sÀ1 mÀ1
k
/KcMorr
]
Homogeneous media
water[a]
36.0
284.0
0.62
5.80
0.10
1.80
0.025
0.12
GY/water (38:62, v/v)[a]
DMF/water (20:80, v/v)
GY/DMF/water (20:20:60, v/v/v)
RM
water/AOT/n-heptane
0.90
2.20
4.50
2.90
GY/water (40:60, v/v)/AOT/n-heptane
DMF/water (20:80, v/v)/AOT/n-heptane
GY/DMF/water (20:20:60, v/v/v)/AOT/n-heptane
[a] Values obtained from reference [20].
Altogether, our results highlight the importance of RMs as
nanoreactor media for enzymatic reactions, in which the
enzyme not only retains but increases its activity in compari-
son with homogeneous media, even in the presence of sol-
vents such as DMF or DMSO, which act as inhibitors. We hope
to stimulate the scientific community to use these fascinating
and unique nanoreactors to perform different types of chemi-
cal reactions, including with enzymes other than a-CT and for
nanoparticle synthesis.
larger than for the water/AOT/n-heptane RMs and are of the
same order of magnitude as for the GY/water encapsulated
mixture. A comparison of the RM media with homogenous
exp
media for the same mixtures shows that the k /KcMorr value is
cat
24 times larger if the ternary mixture is encapsulated and
180 times larger if the binary mixture is encapsulated (Table 6).
exp
The lowest k /KcMorr value in homogenous media corresponds
cat
to the mixture with DMF, which is consistent with the dehy-
drating and conformational effect that this solvent has on the
enzyme. However, and in contrast to what happens in homo-
geneous medium, in the RM media the greatest catalytic effi-
ciency is achieved for the encapsulated DMF-containing mix-
ture.
Experimental Section
General
AOT (>99% purity, Sigma–Aldrich) was used as received and was
kept under vacuum over P2O5 to minimize water absorption. The
absence of acidic impurities was confirmed using the solvato-
chormism of 1-methyl-8-oxyquinolinium betaine.[43]
These data can be explained by considering what we learn
about the behavior of polar solvent mixtures upon encapsula-
tion by using different techniques such as electronic spectros-
copy and DLS.[9–12] Hence, for reactions occurring in different
systems, the solvent within the micelle acquires a particular lo-
cation such that the stability of the enzyme is always favored.
GY increases the catalytic efficiency of the enzyme by reducing
its conformational mobility. GY has also been found to readily
form a polyol hydrogen-bond network that fixes the structure
of the protein in a more rigid matrix.[20] Also, water is essential
for the enzyme to perform its function as a hydrolase.[14] For
this reason, for encapsulation of only the binary GY/DMF mix-
ture, the reaction does not occur. Water and GY are placed at
the polar side of the AOT RM interface and we hypothesize
that the enzyme is located this place of the AOT RMs interface,
surrounded by the solvents. Otherwise, and in concordance
a-CT (molecular weight 24800, from bovine pancreas), and 2-NA
(Sigma–Aldrich) were purchased from Sigma–Aldrich and used as
received. n-heptane (Sintorgan HPLC), GY and DMF (both fluores-
cence spectroscopy quality, from Merck) were used without purifi-
cation. Ultrapure water was used throughout and prepared to
a
specific resistivity of >18 MWcm (Labonco equipment
model 90901-01). The solvent mixtures used were GY/DMF/water
(20:20:60, v/v/v), DMF/water (20:80, v/v) and GY/water (40:60, v/v).
The pH of the bulk solutions (water or mixtures) was maintained at
8.7 by using phosphate buffer (20 mm). The pH cannot be mea-
sured inside the polar core of the RM aggregate.[43] A meaningful
approximation to the pH within the aqueous pseudophase of the
RMs can be made using pure AOT with sufficient buffering capacity
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Articles
in the bulk solutions (i.e., such that the observed rates are inde-
pendent of the buffer concentrations).[44] In this sense, the value of
the pH inside the polar core is referenced to the homogeneous
buffer solution, and is termed pHext. Nevertheless, as the concept
of pH inside RMs is a matter of discussion,[43,45] we measured the ki-
netics with no buffer solution and the results were the same, that
is, the kinetic parameters do not depend on the presence of
buffer.
sured. Afterwards, this mixture (5 mL) was added to n-heptane
(5 mL). After shaking the mixture for about 5 min, the n-heptane
phase was extracted and its absorbance measured. The
Kmp ixture=nÀheptane was determined according to Equation (4):
KPmixture=nÀheptane ¼ ½ðA0ÀAÞ=AŠ VÀ1 Vmixture
ð4Þ
org
where A0 is the absorbance of the 2-NA in n-heptane before the
extraction with the mixture, A is the absorbance in the same phase
after extraction with the mixture, Vmixture is the volume of the phase
mixture and Vorg is the volume of the organic phase.
DLS experiments were performed at a fixed surfactant concentra-
tion of 0.1m and therefore the RM solutions are not at infinite dilu-
tion. Thus, it is appropriate to introduce an apparent hydrodynam-
ic diameter (dapp), in order to make the comparison with the
system studied here. A similar approach has been used previous-
ly.[46] The dapp values of the different RM systems were determined
by DLS on a Malvern 4700 instrument with a goniometer and an
argon ion laser operating at l=488 nm. Cleanliness of the cuvettes
used for measurements was of crucial importance for obtaining re-
liable and reproducible data.[47] Cuvettes were washed with etha-
nol, and then with doubly distilled water, and dried with acetone.
Prior to use, the samples were filtered three times to avoid dust or
particles present in the original solution, using an Acrodisc syringe
filter with a 0.2 mm PTFE membrane (Sigma–Aldrich) for the RM
samples. Prior to data acquisition, the samples were allowed to
equilibrate in the DLS instrument for 10 min at 258C. To obtain
valid results from DLS measurements, knowledge of the system’s
refractive index and viscosity, in addition to well-defined conditions
is required. The refractive indices and viscosities for the AOT RMs
were assumed to be the same as neat n-heptane. Multiple samples
at each size were made, and 30 independent size measurements
were performed for each individual sample at the scattering angle
of 908. The instrument was calibrated before and during the
course of experiments, using several different size standards. Thus,
the magnitudes obtained by DLS measurements can be taken as
statistically meaningful for all the systems investigated. The algo-
rithm used was CONTIN and the DLS experiments showed that the
polydispersity of the AOT RMs was less than 5%.
Reaction in Mixed Solvent/AOT/n-Heptane RMs
A stock solution of AOT in n-heptane was prepared by by weighing
17.28 g of the surfactant dissolved in 100 mL to reach a concentra-
tion of 0.4 M. Stock solutions of a-CT in phosphate buffer (20 mm)
and 2-NA (1 x 10-2 M) in different mixed solvents and in pure water
were prepared. The values of WGY/water and WDMF/water in the mixtures
of polar solvents have two contributions [see Eqs. (5) and (6)] and
WT has three contributions [Eq. (7)].
WGY=water ¼ ð½GYŠ þ ½waterŠÞ=½AOTŠ ¼ WGY þ Wwater
ð5Þ
WDMF=water ¼ ð½DMFŠ þ ½waterŠÞ=½AOTŠ ¼ WDMF þ Wwater
ð6Þ
ð7Þ
WT ¼ ð½GYŠ þ ½DMFŠ þ ½waterŠÞ=½AOTŠ ¼ WGY þ WDMF þ Wwater
where WGY, WDMF and Wwater represent the values of the molar ratio
between each of the polar solvents and the AOT, respectively. In
the RM media, the water, GY/water, DMF/water and GY/DMF/water
molar ratios were expressed in terms of WGY, WDMF and Wwater, which
are equivalent to the molar ratio composition in homogeneous
media.
A stoppered cell was filled with AOT solution (3 mL) at the appro-
priate concentration. The desire Wwater value was achieved by
adding sufficient stock a-CT phosphate buffer solution with a mi-
crosyringe in order to obtain [a-CT]=5”10À6 m, and the mixture
was shaken until a clear solution was obtained. Once the desired
WGY and WDMF values were reached, the enzymatic reaction was ini-
tiated by adding the appropriate volume of the stock 2-NA solu-
tion in water to give the desired W0 values.
Methods
Mixed solvents were prepared by mixing the required volume of
solvent to obtain the required %v/v. A stock solution of 2-NA
(0.01m) in the different solvent mixtures and in pure water was
also prepared.
For kinetic measurements in homogeneous media, solutions of a-
CT (3 mL) in various mixed solvents and water [phosphate buffer
(20 mm), pH 8.7, [a-CT]=5”10À6 m] at the required molar ratio
were introduced into a thermostated cell. Then, the enzymatic re-
action was initiated by addition of different mL quantities of stock
solutions of 2-NA in order to have a total volume of 3 mL of solu-
tion at the desired 2-NA concentration. For example, for [2-NA]=
1”10À3 m, 30 mL of stock solution was added to the cell. The con-
Calculation of the 2-NA PArtition Constant in RM Media
The partitioning of 2-NA between the RMs and the organic solvent
pseudophases can be considered within the framework of the
pseudophase model.[4,14,49–51] This model considers the RMs to be
distinct pseudophases, the properties of which are independent of
the AOT concentration and are only determined by the value of
the characteristic parameter W. In this model, only two solubiliza-
tion sites are considered, that is, the external nonpolar solvent and
the RMs (i.e., all the surfactant molecules). Hence, the distribution
of 2-NA between the micelles and the external solvent pseudo-
phase defined in Equation (8) can be expressed in terms of the par-
tition constant Kp [Eq. (9)]:
centration of 2-NA was varied between 10À3 and 10À2
m
Calculation of the 2-NA Partition Constant in Homogeneous
Media
The partition constant values in homogeneous media,
Kmp ixture=nÀheptane, for the 2-NA distribution between DMF/water or
GY/DMF/water mixture and n-heptane were determined by the
hand-shaking method.[48] Solutions of 2-NA were prepared in differ-
ent solvent mixtures and the absorbance at labs =314 nm, the max-
imum absorbance wavelength of 2-NA in n-heptane, was mea-
½2-NAŠ Ð ½2-NAŠ
ð8Þ
ð9Þ
f
b
#
b
½2 À NAŠ
Kp
¼
½2 À NAŠ
f
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Acknowledgements
Financial support from the Consejo Nacional de Investigaciones
Científicas y TØcnicas (CONICET), Universidad Nacional de Río
Cuarto, Agencia Nacional de Promoción Científica y TØcnica and
Agencia Córdoba Ciencia is gratefully acknowledged. R.D.F.,
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Keywords: aggregates · enzyme catalysis · reverse micelles ·
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Manuscript received: December 22, 2015
Revised: February 10, 2016
Accepted Article published: February 19, 2016
Final Article published: March 16, 2016
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