Head-group size or hydrophilicity of surfactants: The major regulator of lipase activity in cationic water-in-oil microemulsions

Article abstract of DOI:10.1002/chem.200500244

To determine the crucial role of surfactant head-group size in micellar enzymology, the activity of Chromobacterium Viscosum (CV) lipase was estimated in cationic water-in-oil (w/o) microemulsions of three different series of surfactants with varied fiead-group size and hydrophilicity. The different series were prepared by subsequent replacement of three methyl groups of cetyltrimethylammonium bromide (CTAB) with hydroxyethyl (1-3, series I), methoxyethyl (4-6, series II), and n-propyl (7-9, series III) groups. The hydrophilicity at the polar head was gradually reduced from series I to series III. Interestingly, the lipase activity was found to be marked-ly higher for series II surfactants relative to their more hydrophilic analogues in series I. Moreover, the activity remained almost comparable for complementary analogues of both series I and III, though the hydrophilicity was drastically different. Noticeably, the head-group area per surfactant is almost similar for comparable surfactants of both series I and III, but distinctly higher in case of series II surfactants. Thus the lipase activity was largely regulated by the surfactant head-group size, which plays the dominant role over the hydrophilicity. The increase in head-group size presumably allows the enzyme to attain a flexible conformation as well as increase in the local concentration of enzyme and substrate, leading to the higher efficiency of lipase. The lipase showed its best activity in the microemulsion of 6 probably because of its highest head-group size. Furthermore, the observed activity in 6 is 2-3-fold and 8-fold higher than sodium bis(2-ethyl-1-hexyl)sulfosuccinate (AOT) and CTAB-based microemulsions, respectively, and in fact highest ever in any w/o microemulsions.

Full text of DOI:10.1002/chem.200500244

FULL PAPER
Chem. Eur. J. 2005, 11, 4881 – 4889
DOI: 10.1002/chem.200500244
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4881

Head-Group Size or Hydrophilicity of Surfactants: The Major Regulator of
Lipase Activity in Cationic Water-in-Oil Microemulsions
Debapratim Das, Sangita Roy, Rajendra Narayan Mitra, Antara Dasgupta, and
Prasanta Kumar Das*[a]
Abstract: To determine the crucial role
of surfactant head-group size in micel-
lar enzymology, the activity of Chro-
mobacterium Viscosum (CV) lipase
was estimated in cationic water-in-oil
(w/o) microemulsions of three different
series of surfactants withvaried head-
group size and hydrophilicity. The dif-
ferent series were prepared by subse-
quent replacement of three methyl
groups of cetyltrimethylammonium
bromide (CTAB) with hydroxyethyl
(1–3, series I), methoxyethyl (4–6, ser-
ies II), and n-propyl (7–9, series III)
groups. The hydrophilicity at the polar
head was gradually reduced from se-
ries I to series III. Interestingly, the
lipase activity was found to be marked-
ly higher for series II surfactants rela-
tive to their more hydrophilic ana-
logues in series I. Moreover, the activi-
ty remained almost comparable for
complementary analogues of bothser-
ies I and III, though the hydrophilicity
was drastically different. Noticeably,
the head-group area per surfactant is
almost similar for comparable surfac-
tants of bothseries I and III, but dis-
tinctly higher in case of series II surfac-
tants. Thus the lipase activity was large-
ly regulated by the surfactant head-
group size, which plays the dominant
role over the hydrophilicity. The in-
crease in head-group size presumably
allows the enzyme to attain a flexible
conformation as well as increase in the
local concentration of enzyme and sub-
strate, leading to the higher efficiency
of lipase. The lipase showed its best ac-
tivity in the microemulsion of 6 proba-
bly because of its highest head-group
size. Furthermore, the observed activity
in 6 is 2–3-fold and 8-fold higher than
sodium bis(2-ethyl-1-hexyl)sulfosucci-
nate (AOT) and CTAB-based microe-
mulsions, respectively, and in fact high-
est ever in any w/o microemulsions.
Keywords: enzymes · hydrophilici-
ty
· lipases · microemulsions ·
surfactants
Introduction
cetyltrimethylammonium bromide (CTAB)/water/isooctane/
n-hexanol w/o microemulsions across W0 ([water]/[surfac-
tant])=12–44 was unchanged, presumably due to the unal-
tered interfacial water concentration, [H2O]i (28.1–31.8m).[10]
The reported [H2O]i is significantly lower than bulk water
concentration (55.5m) and held responsible for poor activity
of lipase in CTAB microemulsions. At the same time, while
determining the role of interfacial water with respect to the
superior activity of lipase in AOT-based microemulsions
(AOT=sodium bis(2-ethyl-1-hexyl)sulfosuccinate),[1,6] Sri-
lakshmi and Chaudhuri found a value of [H2O]i =27.9–
32m,[12] similar to that observed in CTAB systems.[10] Thus,
the correlation between the lipase activity and the [H2O]i is
far from straightforward and deserves further investigation
to determine the role of several other parameters. However,
the higher surface area occupied by the AOT-head group
may have crucial role on the superior activity of lipase in ad-
dition to the influence of [H2O]i.
Enzymology in self-organized aggregates suchas water-in-
oil (w/o) microemulsions has been an area of interest for
several decades, because of its potential biotechnological ap-
plications.[1–4] Lipases, a class of surface-active enzymes,[1]
are widely exploited in diverse transformations in w/o mi-
croemulsions.[1,5–7] The catalytic efficiencies of these enzymes
are believed to be dependent primarily on the local concen-
trations of water and other ions present in the vicinity of the
enzyme.[8–11] To this end, Das and Chaudhuri showed that
the activity of Chromobacterium Viscosum (CV) lipase in
[a] D. Das, S. Roy, R. N. Mitra, A. Dasgupta, Dr. P. K. Das
Department of Biological Chemistry
Indian Association for the Cultivation of Science
Jadavpur, Kolkata 700032 (India)
In our previous study,[11] the hydrolytic ability of lipase
was dramatically enhanced in cationic w/o microemulsions
Fax :(+91)33-247-32805
E-mail: bcpkd@iacs.res.in
4882
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DOI: 10.1002/chem.200500244
Chem. Eur. J. 2005, 11, 4881 – 4889

Enzymology in Organized Aggregates
FULL PAPER
by the introduction of hydroxyethyl groups at the surfactant
polar head. Hydroxyethyl groups were introduced with the
aim of increasing the local concentration of water at the in-
terface.[11] The hydroxyethyl groups not only improved the
hydrophilicity, but at the same time also increased the head-
group size and consequently the surface area per head
group at the interface. The increase in the surface area with
hydroxyethyl groups may provide enough space to attain
flexible secondary conformation[13] of enzyme and also may
allow a larger population of substrate and enzyme molecules
at the interface,[14] leading to the enhancement in lipase ac-
tivity.[15]
At this point, the issue remains unresolved whether the
surfactant head-group size or the local concentration of
water at the interface plays the dominant role in regulating
the lipaseꢁs efficiency. A considerable amount of work has
been reported toward investigating the effect of the head-
group size and the hydrophilicity on numerous physical
properties[16,17] and also different self-assembly-mediated
chemical transformations.[18] The role of head-group size in
modulating the enzyme activity is mostly a neglected param-
eter in micellar enzymology. To the best of our knowledge,
no such attempt has been made to correlate the catalytic ac-
tivity of the lipase with the head-group size and its hydro-
philicity in reverse micelles.
Results and Discussion
In deciphering the role of head-group size and hydrophilici-
ty (the ability to increase the [H2O]i) on the efficiency of
lipase, we have synthesized three series of surfactants. The
hydrophilicity and size of the surfactant head groups in all
the series have been methodically varied through the re-
placement of the methyl groups at the CTAB polar head by
hydroxyethyl, methoxyethyl, and n-propyl groups (series I–
III, respectively).
The aqueous critical micelle concentration (cmc) of the
surfactants (1–9) has been measured tensiometrically using
the ring method (Table 1). The cmcꢁs were determined from
Table 1. The critical micellar concentration (cmc), area minimum (Amin
)
of surfactants 1–9, isosbestic point, and the molar extinction coefficients
(e) of p-nitrophenol/p-nitrophenolate couple in the w/o microemulsions
of surfactants 1–9.
Surfactant
cmc [m][a]
Amin [nm2]
Isosbestic
e [mꢀ1 cmꢀ1
]
point [nm]
1
2
3
4
5
6
7
8
9
2.02”10ꢀ4
1.53”10ꢀ4
0.36”10ꢀ4
2.03”10ꢀ4
1.22”10ꢀ4
0.73”10ꢀ4
6.12”10ꢀ4
2.04”10ꢀ4
1.38”10ꢀ4
1.18ꢁ0.02
1.42ꢁ0.02
2.30ꢁ0.03
2.12ꢁ0.04
2.53ꢁ0.03
2.90ꢁ0.05
1.23ꢁ0.01
1.40ꢁ0.02
2.20ꢁ0.04
340.0
338.0
339.8
339.2
337.0
338.6
338.0
339.4
338.2
4350
4250
4277
4529
4735
4420
4656
4509
4558
In the present study, for the first time a possible correla-
tion between the catalytic efficiency of lipase in cationic w/o
microemulsions with the head-group size of surfactant is de-
lineated. The catalytic activity of lipase was estimated in the
w/o microemulsions of cationic surfactants (1–9) withvary-
ing head-group size and hydrophilicity. Interestingly, the ef-
[a] Values were determined tensiometrically at 258C.
plots of surface tension versus logc (log[surfactant]). The
cmc values obtained for eachseries were in good agreement
with the fact that with increase in the head-group size the
cmc of the surfactant decreases.[19] The minimum area per
surfactant head group at the micellar interface, Amin in nm2,
was calculated from the following equations [Eqs. (1) and
(2)], as obtained from literature.[16,19]
1
dp
ð1Þ
ð2Þ
Gmax
¼
¼
lim
4:606 RT c!ccmc d log c
1018
NGmax
Amin
In these equations, p is the surface pressure calculated
from the equation p=gwaterꢀgsolution (g denotes the surface
tension), Gmax is the maximum surface excess concentration,
and N is the Avogadroꢁs number. The calculated Amin values
show a proportional dependence on the head-group size.
The surface area per head group for each series of surfac-
tants increased withincreasing ehad-group size and tihs
ficiency of CV lipase was found to be similar in case of com-
parable head-group size, but with different hydrophilicity.
The lipase activity distinctly increases with the head-group
size regardless of its hydrophilic nature. Through the varia-
tion in the head-group size, we found that the use of surfac-
tant 6 leads to the highest ever activity of CV-lipase in any
w/o microemulsions. The observed activity in w/o microe-
mulsion of 6 is almost 2–3-fold higher than that in AOT-
based systems and eight times higher than that found in
CTAB-based w/o microemulsions.
[16]
trend is in accordance withthe literature.
The pseudoternary phase diagrams of the surfactants (1–
9) with n-hexanol (1:2, w/w)/water/isooctane systems at
258C are presented in Figure 1. These three diagrams
(Figure 1) have been the subject of a systematic phase be-
havior study to determine the water solubilization region
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P. K. Das et al.
yl-containing surfactants (4–6) than the corresponding hy-
droxyethyl-substituted analogues (1–3); possibly because of
the replacement of hydrogen-bond acceptor atoms of latter
analogues with methyl ether linkages. The isotropic area is
further lowered (Figure 1) for n-propyl-substituted ana-
logues (7–9), perhaps due to the complete absence of any
hydroxyl moiety at the head group. Thus, the head-group
hydrophilicity of the surfactant plays an important function
in the molecular packing of organized aggregates.
Toward investigating the role of head-group hydrophilicity
and size, the catalytic activity of CV lipase was examined in
the w/o microemulsions of 0.05m surfactant (1–9)/isooctane/
n-hexanol/water at pH 6.0 (20 mm phosphate), z ([alcohol]/
[surfactant])=4.8 and 258C across a varying range of W0 (at
which isotropic solutions are formed). The second-order
rate constant, k2 (Figure 2) for the lipase-catalyzed hydroly-
Figure 2. Variation of the second order rate constant (k2) for the lipase-
catalyzed hydrolysis of p-nitrophenyl-n-hexanoate in different cationic
w/o microemulsions formed at z=4.8, 258C, and pH 6.0 (20 mm phos-
phate). [surfactant]=50 mm, [enzyme]=1.02”10ꢀ6 gmLꢀ1
3 mm. The experimental errors are given in the text.
, [substrate]=
sis of p-nitrophenyl-n-hexanoate was found to be independ-
ent of W0 for all the surfactants. In accordance with our pre-
vious report,[11] the activity of lipase increased (Figure 2)
from 1–3 by ~65% (324ꢁ13, 461ꢁ10, and 540ꢁ
8 cm3 gꢀ1 sꢀ1 for 1–3, respectively) withsequential increase
in the number of hydroxyethyl moiety at the polar head
group (series I). Under the similar experimental conditions,
the k2 was found to be 308ꢁ12 cm3 gꢀ1 sꢀ1 in case of CTAB
and 161ꢁ9 cm3 gꢀ1 sꢀ1 when z=16 (most commonly used so-
lution compositions of CTAB w/o microemulsion).[11] Intro-
duction of hydroxyethyl group(s), possessing significant hyd-
rating ability due to the presence of hydrogen-bond donor
and acceptor atoms at the polar head of surfactants (1–3),
possibly increases the [H2O]i.[11,20] At the same time, the
area per head group (Amin) of surfactants also increases
(Table 1) from 1–3 (series I), due to the sequential substitu-
tion of hydroxyethyl groups. Thus, the influence of either of
the parameters towards observed enhancement in lipase ac-
tivity from 1–3 (Figure 2) is yet uncertain.
Figure 1. Pseudoternary phase diagrams of the quaternary systems of
(1–9)/n-hexanol(1:2 w/w)/water/isooctane at 258C. Scale magnitudes are
reduced by 1/100thof the plot.
with varying head-group size and hydrophilicity. In each
phase diagram, complementary analogues of series I–III
were merged together to observe the effect of variation in
head-group hydrophilicity. The diagrams show that the pres-
ence of hydroxyethyl group(s) increases the hydrophilicity
of the surfactant head, since the isotropic regions for the
amphiphiles 1–3 are higher relative to the corresponding
members of the other two series. The high isotropic area in
the w/o microemulsion region for 1–3 is probably the result
of high water uptake by the hydroxyethyl groups due to the
presence of hydrogen-bond donor and acceptor atoms. The
isotropic area is distinctly lower in the case of methoxyeth-
To this end, we reduced the hydrophilicity of surfactant
ꢀ
head groups (1–3) by protecting the OH groups witha
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Enzymology in Organized Aggregates
FULL PAPER
methyl ether linkage to obtain the methoxyethyl analogues
(4–6, series II) of the series I surfactants. Replacement of
hydroxyethyl by methoxyethyl groups (series II) expectedly
leads to a decline in the hydrophilicity at polar head, due to
the removal of the hydrogen-bond acceptor atoms (H). The
catalytic activity of lipase was measured in the w/o microe-
mulsions of series II surfactants (4–6) keeping all other ex-
perimental conditions identical. The k2 was found to be fur-
ther increased by ~35–65% (530ꢁ9, 618ꢁ6, and 770ꢁ
8 cm3 gꢀ1 sꢀ1 for 4, 5, and 6, respectively) compared to their
hydroxyethyl analogues 1–3 (Figure 2). The hydrolytic effi-
ciency of lipase was also found to improve withsequential
increment in the number of methoxyethyl groups at the
polar head of the surfactants 4–6 similar to that observed in
series I (Figure 2). The decrease in the hydrophilicity at
polar head of surfactant did not reduce the activity of
lipase; instead it was increased (Figure 2) in spite of lower-
ing the hydrophilicity. At this point, a distinct enhancement
in the head-group size of series II surfactants compared to
their corresponding surfactants of series I is evident from
their Amin values (Table 1). Similarly, the head-group area is
also increased with the sequential increase in the number of
methoxyethyl groups at the polar head of surfactants from
4–6. Thus, the increase in the head-group size of surfactants
may be correlated with the enhanced efficiency of the
lipase, while head-group hydrophilicity may not have signifi-
cant influence on the catalytic activity of lipase in cationic
w/o microemulsions.
lipase activity in 7–9 was found (Figure 2) to be similar to
their complementary analogues of series I (1–3). The calcu-
lated Amin values (Table 1) indicate that the surface area per
head group also does not vary for the corresponding surfac-
tants of series I and III. Although the head-group hydrophi-
licity is distinctly different between series I and III surfac-
tants, the almost unchanged lipase activity in comparable
surfactants of bothseries is presumably attributed to their
similar head-group size. Thus, a possible correlation can be
established for the first time that the efficiency of lipase is
steadily dependent on the head-group size of surfactant in
cationic w/o microemulsions. Moreover, the dominating role
of head-group size in modulating the efficiency of enzyme in
organized solution is also delineated.
At this point an obvious question arises: how does the
head-group size affect the catalytic efficiency of lipase in
cationic w/o microemulsions? It is a well-known fact that
the head-group structure of an amphiphile has an important
role towards the molecular packing of organized self-assem-
blies.[16,17,21] An increase in the head-group size leads to in-
crease in the interfacial area,[16,19] and accordingly the space
between two head groups is also enhanced. The augmented
space between two head groups presumably allows the
lipase to solubilize itself smoothly at the interfacial region.
Consequentially the enzyme may attain a more flexible sec-
ondary conformation (Scheme 1), leading to its higher effi-
ciency. Furthermore, the possibility of increasing concentra-
tion of bothenzyme and substrate is also improved at the
enhanced interfacial region[14] (Scheme 1), probably because
of alteration in partition coefficients.[6,9,22] As it was found
that the lipase activity is larger in series II surfactants (4–6)
relative to corresponding surfactants of series I and III, we
can explain this from the present study exclusively on the
basis of the head-group area (Table 1). Although the differ-
ences in Amin value did not appear to be significant in some
cases, it is probably sufficient enoughto affect the enzyme
activity. At the same time, surfactants of different series
withcomparable ehad-group area ( 3, 4, and 9, Table 1)
In the preceding paragraph, lipase activity was measured
in series II surfactants, whereby the head-group hydrophilic-
ity was partially reduced withconcurrent increase in the
area per head group relative to series I surfactants. At this
stage, to examine the straightforward correlation between
the head-group size and lipase activity, series III surfactants
(7–9) were synthesized in which the hydroxyethyl groups of
series I were replaced by n-propyl groups. The hydrophilicity
at polar head, due to the presence of hydroxyl moieties in
series I, was completely removed in series III by substituting
ꢀ
the OH functionalities with
methyl groups and keeping the
head-group
size
similar
(Table 1) for comparable sur-
factants of bothseries. The cat-
alytic efficiency of lipase was
measured in the w/o micro-
emulsions of 7–9 under the
same experimental conditions
as mentioned above. As the
number of propyl group in-
creases at the polar head of
the surfactants 7–9, th ek2
again increased (365ꢁ7, 425ꢁ
7, and 565ꢁ8 cm3 gꢀ1 sꢀ1, re-
spectively) witehahd-group
size (Table 1) similar to the
trend observed in series I and
II (Figure 2). Interestingly, the
Scheme 1. Pictorial representation of the interfacial region of w/o microemulsions prepared with varying head-
group size. The increase in head-group size leads to smooth occupancy of lipase and increases in the local con-
centration of the enzyme and substrate.
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P. K. Das et al.
showed similar activity (Figure 2) under equivalent solution
compositions (z=4.8). Therefore, the increase in the surfac-
tant head-group size plays a crucial role in modulating the
lipase efficiency, presumably by rendering a flexible confor-
mation to the enzyme and increasing the local concentration
of the reactants (Scheme 1) at the augmented interface.[13–16]
One of the aims of our research is to improve the lipaseꢁs
efficiency in cationic w/o microemulsions.[11] To this end, be-
sides the molecular architecture of surfactant, solution com-
positions also play a role in regulating the enzyme activity.
Till now we have prepared the w/o microemulsions using
the solution composition surfactant, (0.05m)/water/isooc-
tane/n-hexanol at z=4.8. At this stage the competitive in-
hibition by alcohols on the lipase activity has also been iter-
ated several times in the literature.[11,23] Hence, to enhance
the lipaseꢁs efficiency in cationic w/o microemulsions it is es-
sential to prepare the microemulsions without or with a
minimum amount of alcohol (co-surfactant). To reduce the
inhibitory action of n-hexanol, we prepared the cationic w/o
microemulsions of 0.05m 1–9/water (pH 6.0)/isooctane with
the minimum amount of n-hexanol needed to form isotropic
solutions (z values for eachsurfactant are given on top of
the activity bar in Figure 3). Noticeably, the n-hexanol con-
creasing head-group size of series II and III surfactants. The
observed k2 values for 4–6 (Figure 3) is 2–2.5-fold higher
than the corresponding more hydrophilic analogues 1–3 in
series I and also compared to AOT/isooctane/water micro-
emulsions.[1,6] Moreover, this activity is 4- and 8-fold higher
than that found in CTAB w/o microemulsions at z=4.8 and
16, respectively. Thus, overall we can conclude that the
head-group size of surfactant is the major regulator of lipase
activity profile in cationic w/o microemulsions.
Since co-surfactant plays a significant role in the packing
of the microemulsions, it is necessary to verify whether this
head-group size effect on lipase activity profile is dependent
on the co-surfactant architecture. Accordingly, the activity
of enzyme was studied (Figure 4) in the microemulsions of
Figure 4. Effect of varying co-surfactant on the second-order rate con-
stant for lipase-catalyzed hydrolysis of p-nitrophenyl-n-hexanoate in dif-
ferent cationic w/o microemulsions at z=9.6, 25 C, and pH 6.0 (20 mm
phosphate). [surfactant]=50 mm. [enzyme]=1.02”10ꢀ6 gmLꢀ1
strate]=3 mm.
.
[sub-
1, 4, and 7 (comparable analogues of eachseries) with n-bu-
tanol, n-hexanol, and n-octanol as the co-surfactant at z=
9.6 (solution compositions at which each surfactant can form
w/o microemulsions at same z value withany co-surfactant).
Irrespective of the co-surfactants, the efficiency of lipase
(Figure 4) increased withhead-group size from 1 to 4. Al-
though 1 and 7 are of comparable head-group size (Table 1),
the k2 was found to be lower in case of 1, possibly because
of the different inhibiting effect of alcohols at such high z=
9.6. However, for eachof the surfactants the lipase activity
increased with increasing alcohol chain length, and was
highest in the case of n-octanol.
Figure 3. Variation of the second-order rate constant (k2) for the lipase-
catalyzed hydrolysis of p-nitrophenyl-n-hexanoate in different cationic
w/o microemulsions of surfactants using least amount of n-hexanol re-
quired at 258C and pH 6.0 (20 mm phosphate). [surfactant]=50 mm.
[enzyme]=1.02”10ꢀ6 gmLꢀ1. [substrate]=3 mm. The experimental errors
are given in the text. The W0 ranges where activity was measured are
40–56, 40–60, 56, 44–60, 32–44, 36–52, 32–44, 12–16, 8–20, 28–32, and 12
for CTAB, 1–9, and AOT respectively.
tent could be reduced in case of series II (4–6) and III (7–9)
surfactants, which are distinctly less hydrophilic at the inter-
face relative to series I analogues (1–3). As expected, the ac-
tivity of lipase was found to increase (Figure 3) by ~15–
70% (820ꢁ15, 920ꢁ13, 1290ꢁ12, 420ꢁ8, 500ꢁ10, and
715ꢁ10 cm3 gꢀ1 sꢀ1 for 4–9, respectively) withdecreasing al-
cohol content in the microemulsions of 4–9. Here also the
observed k2 values show similar trend of head-group-size de-
pendence, as the activity of lipase enhanced with the in-
To ascertain whether or not the observed head-group size
effect is substrate specific, p-nitrophenylalkanoates of vari-
ous chain length (C4 to C16) were hydrolyzed (Figure 5) by
CV lipase in the cationic w/o microemulsions of 2, 5, and 8
(corresponding surfactants of eachseries) keeping all other
experimental conditions identical. As it can be seen in
Figure 5, surfactants 2 and 8, withcomparable head-group
size (Table 1), had very similar activity profiles across the
range of substrates. Whereas regardless of the substrate
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Chem. Eur. J. 2005, 11, 4881 – 4889

Enzymology in Organized Aggregates
FULL PAPER
group area per surfactant is similar for comparable surfac-
tants of both series I and III, but distinctly higher in case of
series II surfactants. Thus, the hydrophilicity of the surfac-
tant head group does not have much influence over the
enzyme efficiency, while the head-group size was found to
largely regulate the activity. The increase in the head-group
size presumably provides greater space for the enzyme to
attain a flexible conformation and also increases the local
concentration of enzyme and substrate molecules at the in-
terface, leading to the higher efficiency of lipase. The overall
results showed for the first time that the hydrolytic activity
of lipase steadily increased with the head-group size of sur-
factant irrespective of its hydrophilicity. The observed k2
value in microemulsions of 6 is 2–3-fold higher than that
found in AOT-based systems and 8-fold higher compared to
the CTAB/water/isooctane-n-hexanol (z=16) system; it is
also the highest ever in any w/o microemulsions.
Figure 5. Effect of the substrate chain length on the second-order rate
constant (k2) for the lipase-catalyzed hydrolysis of p-nitrophenylalka-
noates in different cationic w/o microemulsion systems at 25C and
pH 6.0 (20 mm phosphate). [surfactant]=50 mm. [enzyme]=1.02”
10ꢀ6 gmLꢀ1. [substrate]=3 mm. Th eW0 ranges where activity was mea-
sured are 56, 36–52, 32–44, and 8–20 for 2, 5, 6, and 8, respectively.
Experimental Section
Materials: Chromobacterium viscosum Lipase (EC 3.1.1.3, Type XII) was
purchased from Sigma and was used as received. Analytical grade CTAB
from Spectrochem (India) was recrystallized three times from methanol/
diethyl ether; the recrystallized CTAB was without minima in its surface
tension plot. HPLC grade isooctane, n-butanol, n-hexanol, n-octanol, sol-
vents, and all other reagents used in the syntheses were obtained from
SRL (India) and were of the highest analytical grade. Amberlyst A-26
bromide ion exchange resin from Lancaster was used to convert the
chain length, surfactant 5 exhibited markedly higher activity
(Figure 5), presumably due to its larger head-group size
(Table 1). Furthermore, irrespective of the surfactants, the
highest activity was found for p-nitrophenyloctanoate (C8),
and bell-shaped activity profiles were obtained in all cases.
Variations in the number of methylene groups in the hydro-
phobic domain of the substrates alter the hydrophilic–lipo-
philic balance (HLB) of the molecules. The HLB probably
reaches an optimal value at C8, at which the interfacial lo-
calization of the molecules becomes highest for isooctane/n-
hexanol bulk oil composition. Accordingly, the local concen-
tration of C8 substrate possibly increases at the interface
and consequently the lipase activity increases. Throughout
the investigation the best lipase activity was observed either
in the microemulsions of 6 or by using C8 substrate in re-
spective series of surfactants and substrates. In continuation
of our efforts to improve the lipase activity in w/o micro-
emulsions, we measured the enzymeꢁs efficiency in 6/water/
isooctane-n-hexanol (z=2.9, W0 =32–44) using p-nitrophe-
nyloctanoate as the substrate. The observed k2 value (1748ꢁ
24 cm3 gꢀ1 sꢀ1) is more than three times higher than that
found in the widely used AOT-based system and in fact the
highest ever activity of lipase found in any w/o microemul-
sions.
1
iodide salts to their corresponding bromide forms. H NMR spectra were
recorded on an Avance 300 MHz (Bruker) spectrometer. Chemical shifts
are reported in ppm, by using TMS for 1H NMR as internal standard.
Mass spectrometric (MS) data were acquired by electrospray ionization
(ESI) techniques on a Q-TOF Micro-Quadruple mass spectrometer, Mi-
cromass, UK. The p-nitrophenylalkanoate substrates were synthesized
conventionally from an equimolar solution of the corresponding acid and
p-nitrophenol in dichloromethane with an equivalent amount of N,N-di-
cyclohexylcarbodiimide (DCC) and catalytic amount of 4-N,N-dimethyl-
aminopyridine (DMAP). The syntheses of different surfactants are listed
below.
Synthesis of N-hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium
bromide (1) and N-hexadecyl-N,N-bis(2-hydroxyethyl)-N-methylammoni-
um bromide (2): Both the amphiphiles were prepared following the pro-
cedure mentioned in a recently published protocol.[11, 17a] Briefly, 1-bro-
mohexadecane and the corresponding amines (N,N-dimethylethanola-
mine for 1 and N-methyldiethanolamine for 2) were taken in the molar
ratio 1.2:1 in 30% methanol/acetonitrile and refluxed. After 24 h, the sol-
vent was evaporated on a rotary evaporator and pure products were ob-
tained by crystallization of the reaction mixture from methanol/ethyl ace-
tate. The yields were 87% and 80% for 1 and 2, respectively.
1
Data for 1: H NMR (300 MHz, CDCl3): d=4.14 (br, 2H), 3.69 (br, 2H),
3.48–3.45 (br, 2H), 3.41 (s, 6H), 1.77 (br, 2H), 1.33–1.18 (m, 26H),
0.86 ppm (t, J=6.9 Hz, 3H); elemental analysis calcd (%) for
C20H44BrNO: C 60.89, H 11.24, N 3.55; found: C 60.85, H 11.16, N 3.39;
MS (ESI): m/z calcd for C20H44NO (the 48 ammonium ion, 100%):
314.34; found: 314.2605 [M+].
Conclusion
Data for 2: 1H NMR (300 MHz, CDCl3) : d=4.13 (br, 4H), 3.71 (br,
4H), 3.49 (br, 2H), 3.31 (s, 3H), 1.75 (br, 2H), 1.36–1.18 (m, 26H),
0.88 ppm (t, J=6.9 Hz, 3H); elemental analysis calcd (%) for
C21H46BrNO2: C 59.42, H 10.92, N 3.30; found: C 59.45, H 10.86, N 3.08;
MS (ESI): m/z calcd for C21H46NO2 (the 48 ammonium ion, 100%):
344.60; found: 344.4506 [M+].
The regulatory role of the surfactant head-group size on the
lipase activity is distinct throughout the investigation. The
activity was found to increase markedly for series II surfac-
tants relative to their more hydrophilic analogues in series I.
At the same time the activity in case of series III surfactants
remained almost comparable to that with series I, in spite of
large differences in their hydrophilicity. Notably, the head-
Synthesis of N-hexadecyl-N,N,N-tris(2-hydroxyethyl)ammonium bromide
(3): Aqueous solution of NaOH (2.72 g, 0.068 mol, in 25 mL doubly dis-
Chem. Eur. J. 2005, 11, 4881 – 4889
www.chemeurj.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4887

P. K. Das et al.
tilled water) was added dropwise to a mixture of 2-bromoethanol (6.5 g,
0.081 mol) and hexadecylamine (5 g, 0.027 mol) under reflux. After 24 h
of refluxing, the reaction mixture was extracted with chloroform (3”
50 mL). Chloroform was removed on a rotary evaporator followed by
drying under vacuum. The residue was then crystallized from methanol/
ethyl acetate and filtered. The resulting mixture showed three spots (with
Rf =0.55, 0.4, and 0) on thin-layer chromatography (TLC) by using 25:75
(v/v) methanol/chloroform as the TLC developing solvents. The dried
product (with Rf =0.55) was purified from the white solid obtained from
crystallization by column chromatography in a 230–400 mesh silica gel
column with 7% methanol/chloroform. The yield was 40%. 1H NMR
(300 MHz, CDCl3): d=4.08 (br, 6H), 3.49–3.35 (br, 6H), 3.23–3.18 (br,
2H), 1.83 (br, 2H), 1.34–1.25 (m; 26H), 0.88 ppm (t, J=6.9 Hz, 3H); ele-
mental analysis calcd (%) for C22H48BrNO3: C 58.15, H 10.57, N 3.08;
found: C 58.13, H 10.64, N, 3.08; MS (ESI): m/z calcd for C22H48NO3 (the
48 ammonium ion, 100%): 374.62; found: 374.2458.
with methyl iodide in methanol for 2 h and then methanol was evaporat-
ed on a rotary evaporator and the material was taken in ethyl acetate
and washed with sodium thiosulfate solution followed by brine wash. The
organic part was concentrated to get the iodide form of surfactant 8. Th e
iodides were then passed through Amberlyst A-26 bromide ion exchange
column to get the corresponding bromides 7 and 8. These salts were crys-
tallized from MeOH/diethyl ether three times to get the pure com-
pounds.
Data for 7: 1H NMR (300 MHz, CDCl3): d=3.48–3.40 (br, 4H), 3.32 (s,
6H), 1.76–1.56 (br, 4H), 1.18–1.11 (br, 26H), 1.03–0.97 (t, J=7.2 Hz,
3H), 0.83–0.79 ppm (t, J=6.82 Hz, 3H); elemental analysis calcd (%) for
C21H46BrN: C 64.26, H 11.81, N 3.57; found: C 64.35, H 11.60, N 3.49;
MS (ESI): m/z calcd for C21H46N (the 48 ammonium ion, 100%): 312.36;
found: 312.4034 [M+].
Data for 8: 1H NMR (300 MHz, CDCl3): d=3.47–3.41 (br, 6H), 3.34 (s,
3H), 1.81–1.69 (br, 6H), 1.25 (br, 26H), 1.09–1.04 (t, J=7.23 Hz, 6H),
0.90–0.85 ppm (t, J=6.81 Hz, 3H; elemental analysis calcd (%) for
C23H50BrN: C 65.69; H 11.98; N 3.33; found: C 65.85, H 11.80, N 3.42;
MS (ESI): m/z calcd for C23H50N (the 48 ammonium ion, 100%): 340.9;
found: 340.4234 [M+].
Synthesis of N-hexadecyl-N-(2-methoxyethyl)-N,N-dimethylammonium
bromide (4), N-hexadecyl-N,N-bis(2-methoxyethyl)-N-methylammonium
bromide (5), and N-hexadecyl-N,N,N-tris(2-methoxyethyl)ammonium
bromide (6): Compounds 4–6 were prepared from compounds 1–3, re-
spectively. The corresponding starting materials were treated with excess
methyl iodide in dry DMF in presence of sodium hydride (2, 4, and
6 equiv for 4–6 respectively) under a nitrogen atmosphere for 48 h. After
completion of the reaction, water was added to the reaction mixtures,
which were then extracted with chloroform. The organic layer was
washed with aqueous sodium thiosulfate solution followed by brine, con-
centrated on a rotary evaporator, and finally under reduced pressure.
The dried material was washed with dry diethyl ether and then passed
through Amberlyst A-26 bromide ion exchange column to get the bro-
mide form of the compound. The white solids were then washed with dry
diethyl ether three times to get the pure forms of compounds 4, 5, and 6.
Data for 4: 1H NMR (300 MHz, CDCl3): d=3.94–3.9 (br, 2H), 3.87 (br,
2H), 3.56–3.53 (br, 2H), 3.45 (s, 6H), 3.33 (s, 3H), 1.79–1.74 (br, 2H),
1.25 (br, 26H), 0.88–0.84 ppm (t, J=6.48 Hz, 3H); elemental analysis
calcd (%) for C21H46BrNO: C 61.74, H 11.35, N 3.43; found: C 61.40, H
11.50, N 3.33; MS (ESI): m/z calcd for C21H46NO (the 48 ammonium ion,
100%): 328.36; found: 328.4758 [M+].
Synthesis of N-hexadecyl-N,N,N-tripropylammonium bromide (9): Am-
phiphile 9 was synthesized by refluxing n-hexadecyl bromide (1.2 equiv)
and tripropylamine (1 equiv) in 30% methanol/acetonitrile. After 48 h
the solvents were removed on a rotary evaporator and the mixture was
then crystallized from methanol/diethyl ether three times to get the pure
ammonium salt 9. 1H NMR (300 MHz, CDCl3): d=3.31–3.29 (br, 8H),
1.74–1.60 (br, 8H), 1.17 (br, 26H), 1.01–0.96 (t, J=7.11 Hz, 9H), 0.79–
0.77 (t, J=6.6 Hz, 3H); elemental analysis calcd (%) for C25H54BrN: C
66.93, H 12.13, N 3.12; found: C 66.59, H 11.93, N 3.03; MS (ESI): m/z
calcd for C25H54N (the 48 ammonium ion, 100%): 368.43; found:
368.4215 [M+].
Critical micelle concentration and minimum surface area: Unless other-
wise mentioned, the aqueous critical micelle concentration (cmc) values
of the surfactants were determined by measuring surface tension with a
temperature-controlled Krüss tensiometer by means of the ring method
at 25ꢁ0.18C. The cmc values are listed in Table 1. The accuracy of meas-
urements in duplicate experiments was withinꢁ2%.
Preparationof microemulsions (phase behavior) : The mixture of surfac-
tants, n-hexanol, and water were titrated with isooctane to prepare the
microemulsions. A constant mass ratio (1:2) of the surfactant and n-hexa-
nol was dissolved in water, forming solutions of different concentrations
taken in different screw-topped test tubes and stirred until the solutions
became clear. Isooctane was then added to these solutions in measured
quantities at 258C until just turbid or phase separation. The pseudoterna-
ry phase diagrams of the different surfactants (1–9) are presented in
Figure 1. The isotropy/turbidity of the solutions were checked by the
naked eye, which means the measured phase boundaries are of fair accu-
racy.
1
Data for 5: H NMR (300 MHz, CDCl3): d=3.94–3.84 (br, 4H), 3.80 (br,
4H), 3.55–3.46 (br, 2H), 3.42 (s, 3H), 3.33 (s, 6H), 1.69 (br, 2H), 1.26
(br, 26H), 0.83–0.78 ppm (t, J=6.96 Hz, 3H); elemental analysis calcd
(%) for C23H50BrNO2: C 61.04, H 11.14, N 3.10; found: C 60.88, H 11.09,
N 3.20; MS (ESI): m/z calcd for C23H50NO2 (the 48 ammonium ion,
100%): 372.38; found: 372.4155 [M+].
Data for 6: 1H NMR (300 MHz, CDCl3): d=3.95–3.9 (br, 6H), 3.87 (br,
6H), 3.54–3.52 (br, 2H), 3.38 (s, 9H), 1.7 (br, 2H), 1.25 (br, 26H), 0.88–
0.86 ppm (t, J=6.92 Hz, 3H); elemental analysis calcd (%) for
C25H54BrNO3: C 60.46, H 10.96, N 2.82; found: C 60.30, H 10.88, N 2.96;
MS (ESI): m/z calcd for C25H54NO3 (the 48 ammonium ion, 100%):
416.41; found: 416.5907 [M+].
Activity of interfacially solubilized lipase: The second-order rate constant
(k2) in lipase-catalyzed hydrolysis of p-nitrophenyl-n-hexanoate in cation-
ic w/o microemulsions was determined spectrophotometrically (on a Shi-
madzu 1700 spectrophotometer) at the isosbestic points as described pre-
viously.[6,10,11,22] In a typical experiment, the aqueous enzyme stock solu-
tion (4.5 mL, 0.34 mgmLꢀ1) and the substrate (10 mL, from 0.45m stock
solution in isooctane) were added to the w/o microemulsion (1.5 mL)
previously prepared withthe desired surfactant concentration and pH
(pH refers to the pH of the aqueous buffer solutions used for preparing
the w/o microemulsions; pH within the water pool of w/o microemulsions
did not vary significantly, <1 unit),[6,24] in a cuvette to attain the particu-
lar W0 and reactant concentrations. Gentle shaking produced clarification
of the microemulsion within 1 min. The initial linear rate of increase in
absorbance, that is, the absorbance of the liberated p-nitrophenol, was
then recorded at the isosbestic points (liso). The overall concentrations of
lipase and p-nitrophenyl-n-hexanoate were 1.02”10ꢀ6 gcmꢀ3 and 3”
10ꢀ3 m, respectively. Although the lipase was essentially confined to the
dispersed water droplets (at the oil/water interface), for simplicity, the
concentration of reactants were referred to the overall concentration to
avoid the complexity of the volume fraction of water droplet in the w/o
Synthesis of N-hexadecyl-N,N-dimethyl-N-propylammonium bromide (7)
and
N-hexadecyl-N-methyl-N,N-dipropylammonium
bromide
(8):
Hexadecylamine(1eq) and n-propyl bromide (1.2 equiv) were refluxed in
30% MeOH/MeCN solvent mixture for 36 h, and then the white solid
was crystallized from MeOH/ethyl acetate three times. The desired com-
pound with Rf =0.5 (in 10% MeOH/CHCl3) was isolated by column
chromatography on a 60–120 mesh silica gel column using MeOH/CHCl3
as the mobile phase. The ammonium salt thus obtained was basified with
ammonia and then extracted with diethyl ether; the ether layer was
washed with brine to neutrality and concentrated. The secondary amine
(N-propyl-N-hexadecylamine) thus obtained was divided in two parts and
the first part was quarternized with excess methyl iodide in presence of
K2CO3 (2.2 equiv) and a pinch of [18]crown-6 ether in dry DMF for 2 h.
The reaction mixture was extracted with ethyl acetate and washed with
5% aqueous sodium thiosulfate solution and brine. It was then concen-
trated to get the iodide form of surfactant 7. Another part of the secon-
dary amine was again refluxed with n-propyl bromide (1.2 equiv). The
corresponding tertiary amine was isolated following the similar procedure
mentioned above. The tertiary amine thus obtained was then quaternized
4888
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 4881 – 4889

Enzymology in Organized Aggregates
FULL PAPER
microemulsions and the partitioning coefficient of the substrate.[6,9,22]
Moreover, for the sake of consistency with the previous observations, we
measured the second-order rate constant (k2) instead of first-order Mi-
chaelis–Menten catalytic constant (Kcat), since the initial rate of lipase-
catalyzed hydrolysis of p-nitrophenyl alkanoate were observed to be first
order withrespect to the substrate concentration. [6,10,11,22] Similar observa-
tions of second-order rate constant have also been found in micelle-medi-
ated organic transformations in chemical systems.[18c–e] The isosbestic
points (liso) and the molar extinction coefficients (e) at liso of the p-nitro-
phenol/p-nitrophenolate couple in w/o microemulsions of different sur-
factants (1–9)/water/isooctane/n-hexanol were determined spectrophoto-
metrically (Table 1).
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Received: March4, 2005
Published online: June 24, 2005
Chem. Eur. J. 2005, 11, 4881 – 4889
www.chemeurj.org
ꢀ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4889