Synthesis of the 'green apple ester' ethyl valerate in organic solvents by Candida rugosa lipase immobilized in MBGs in organic solvents: Effects of immobilization and reaction parameters

Article abstract of DOI:10.1016/j.molcatb.2009.11.015

Ethyl valerate, also known as the green apple flavor is well known for its wide applications in the areas of food, pharmaceuticals and cosmetics industries. Candida rugosa lipase was immobilized in microemulsion based organogels (MBGs) and used for ethyl valerate synthesis in organic solvents. Various immobilization and reaction parameters were scrutinized for enhancement of ester production. Among the immobilization parameters, sodium bis-2-(ethylhexyl) sulfosuccinate (AOT), n-heptane and gelatin were found to be the highest yielding combination. Cyclohexane was found to be the solvent of choice as the reaction medium while pH 7, 40 °C and 1:1.6 ratio of valeric acid to ethanol were the reaction parameters exhibiting highest ester formation. The organogels were highly stable in the solvents and were reused for nine cycles with meager loss of activity. Also, immobilized enzyme was thermostable at 50-70 °C for ten hours. Hence, MBGs verify to be a promising enzyme immobilization system for ester synthesis in organic solvents.

Full text of DOI:10.1016/j.molcatb.2009.11.015

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 31–38
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Synthesis of the ‘green apple ester’ ethyl valerate in organic solvents by
Candida rugosa lipase immobilized in MBGs in organic solvents:
Effects of immobilization and reaction parameters
Tripti Raghavendra, Divya Sayania, Datta Madamwar∗
BRD School of Biosciences, Sardar Patel Maidan, Satellite Campus, Vadtal Road, Post Box No. 39, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Ethyl valerate, also known as the green apple flavor is well known for its wide applications in the areas of
food, pharmaceuticals and cosmetics industries. Candida rugosa lipase was immobilized in microemulsion
basedorganogels (MBGs) andusedforethylvaleratesynthesisin organic solvents. Various immobilization
and reaction parameters were scrutinized for enhancement of ester production. Among the immobiliza-
tion parameters, sodium bis-2-(ethylhexyl) sulfosuccinate (AOT), n-heptane and gelatin were found to
be the highest yielding combination. Cyclohexane was found to be the solvent of choice as the reac-
tion medium while pH 7, 40 ◦C and 1:1.6 ratio of valeric acid to ethanol were the reaction parameters
exhibiting highest ester formation. The organogels were highly stable in the solvents and were reused
for nine cycles with meager loss of activity. Also, immobilized enzyme was thermostable at 50–70 ◦C for
ten hours. Hence, MBGs verify to be a promising enzyme immobilization system for ester synthesis in
organic solvents.
Received 11 October 2009
Received in revised form
25 November 2009
Accepted 27 November 2009
Available online 2 December 2009
Keywords:
Biocatalysis
Esterification
Reverse micelles
Reusability
Thermostability
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
attractive for industrial applications [13–15]. One of the numer-
ous applications of lipases is production of flavor esters. In recent
From the time enzymes were discovered, they have been a major
subject of intense research owing to their spectacular properties.
Enzymes, being highly specific and extremely enantio- and regio-
selective catalysts, are being used extensively in the industrial
production of bulk chemicals, pharmaceutical and agrochemical
intermediates and food ingredients [1,2]. Though water is the nat-
ural milieu for enzymes, non-aqueous enzymology emerged as the
cutting edge for a variety of reactions which are otherwise virtu-
ally impossible in the aqueous counterpart, along with elimination
of interfering side reactions [3,4] and thereafter, this mode of bio-
catalysis is being widely used in various processes [5,6].
Among the enzymes used in organic syntheses, the most
frequently used enzyme is lipase [7,8]. Biocatalysis involving
lipases has been well studied and understood in the past few
decades [9,10]. Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3)
are ubiquitous enzymes that are activated when adsorbed to
an oil–water interface [11,12]. Due to their widely diversified
enzymatic properties, in particular, the ability to perform enantio-
selective hydrolytic reactions and catalyze the formation of a wide
range of ester and amide bonds, microbial lipases have become very
times, flavors represent over a quarter of the world market for food
additives and it has been shown that consumers prefer foodstuff
that can be labeled as “natural” [16]. The tag “natural” and value
added features of these biochemically produced esters excelling
their chemical counterparts by having better odor and flavor [17]
have intensified research activities in this field [18].
However, high costs and tough purification procedures ren-
der the enzymes economically unattractive. Moreover, reactions
involving fats and oils require high temperatures for long durations
meant for their (fats) solubility in the medium, which may result
in enzyme denaturation. Hence, an enzyme has to fulfil important
requisites such as high turnover, solvent tolerance, reusability and
thermostability if it has to be used for bulk production processes. A
possible solution to this problem is its recovery and reuse by immo-
bilizing in/on a solid support which makes it cost effective [19].
Various modes of immobilization of the enzymes have been used
till date including adsorption, ionic binding, covalent attachment,
entrapment and encapsulation [20–22]. One of the well-explored
methods in non-aqueous biocatalysis is immobilization of enzymes
in microemulsion based organogels (MBGs). Structure of MBGs has
been very well established as an extensive, rigid, interconnected
network of gelatin/water rods stabilized by a monolayer of sur-
factants, in co-existence with a population of conventional w/o
microemulsion droplets [23,24]. Though MBGs have been used for
production of a variety of flavor esters [25–27], very scanty infor-
∗
Corresponding author. Tel.: +91 2692 229380; fax: +91 2692 231042.
E-mail addresses: tpt11@yahoo.co.uk (T. Raghavendra),
datta madamwar@yahoo.com (D. Madamwar).
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.11.015

32
T. Raghavendra et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 31–38
mation is available on production of ethyl valerate, especially by
immobilized enzyme [28–30]. Ethyl valerate, also known as green
apple flavor is an important ester used as a constituent in many
fruity flavors such as plum, apricot, peach, strawberry and pear
aromas [31].
of the reaction mixture was withdrawn every 24 h and immedi-
ately analyzed by gas chromatography for accumulation of ethyl
valerate.
2.1.4. Effect of solvents on esterification
In this study, ethyl valerate has been synthesized by conden-
sation of ethanol and valeric acid catalyzed by MBGs containing
Candida rugosa lipase (CRL) in small-scale model studies. The
immobilization parameters i.e. surfactants, organic solvents and
gelling agents were assessed for preparation of MBGs. Effect of reac-
tion parameters such as water/surfactant mole ratio (also known
as Wo), pH of buffer, substrate concentration, organic solvents and
temperature on the esterification reaction was investigated. Fur-
ther, the MBGs were subjected to reusability and thermostability
studies.
To study the interactive effect of the two solvents, one used for
MBG preparation and another as reaction medium on esterification,
AOT based organogels containing lipase (5790 U/mg of solid) were
used to carry out esterification in various organic solvents. Solvents
used for MBG preparation were isooctane, n-hexane and n-heptane
while those used as reaction medium were isooctane, n-hexane,
n-heptane, cyclohexane, acetone, acetonitrile and DMSO.
2.1.5. Effect of Wo on esterification
Reverse micellar solutions of Wo ranging from 10 to 100
were prepared in AOT/Buffered lipase/isooctane and gelatin sys-
tem while cyclohexane was used as reaction medium. The reaction
was performed under standard conditions as mentioned in Section
2.1.3.
2. Materials and methods
2.1. Materials
Sodium bis-2-(ethylhexyl) sulfosuccinate (AOT), Tween 80 and
n-hexanol were obtained from Fluka (Switzerland). Cetyl trimethyl
ammonium bromide (CTAB) and Triton X-100 were procured from
Hi-Media (India). Ethyl valerate and valeric acid were purchased
from Fluka (Switzerland). C. rugosa lipase (triacylglycerol acyl
hydrolase, E.C. 3.1.1.3, type VII) was supplied by Sigma (Germany),
with the total activity of 965 U/mg of solid. Agarose was from
Bangalore Genei (India), Carboxyl methyl cellulose (CMC) of high
viscosity and agar-agar were acquired from Hi-media (India). All
other organic solvents used were of HPLC/GC grade.
2.1.6. Effect of buffer pH on esterification
It has been shown that the water present within reverse micelles
exhibits different properties as compared to bulk water [33]. This
may result in a different pH of the buffer inside the water pool of
reverse micelles when compared to pH of the same solution mea-
sured in bulk. Hence, to determine the optimum pH required for
esterification using MBGs, lipase (5790 U/mg of solid) was dissolved
in five buffers of different pH: acetate buffer—pH 5, sodium phos-
phate buffer—pH 6, 7 and 8, Tris–HCl buffer—pH 8.8. All the buffers
were of 0.1 M strength. The buffers containing lipase were immo-
bilized in AOT based organogels and monitored for esterification
reaction.
2.1.1. Preparation of reverse micelles using different surfactants
Thermostable reverse micellar solutions were prepared accord-
ing to the procedure of Dandavate and Madamwar [32]. Anionic,
cationic and non-ionic reverse micellar solutions were prepared
using AOT, CTAB, Triton X-100 and Tween 80 respectively. The
organic solvents used were isooctane, n-hexane and n-heptane and
the aqueous phase consisted of lipase (CRL) dissolved in buffer of
pH 7.2 (except for pH studies). Water/surfactant mole ratio (Wo)
and ratio of surfactant to co-surfactant, abbreviated as Po (in case
of cationic and non-ionic surfactants) were determined for each
reverse micellar solution.
2.1.7. Effect of temperature on esterification
Effect of temperature was studied by carrying out the reaction
at five different temperatures (25, 30, 37, 40 and 45 ◦C). The reac-
tion system consisted of MBGs of AOT/buffered lipase (5790 U/mg
of solid)/isooctane in cyclohexane. A control was kept at each tem-
perature with free lipase as the catalyst.
2.1.8. Effect of substrate on esterification
2.1.2. Preparation of w/o based microemulsion gels using various
gelling agents
For determination of effect of substrates on esterification, the
concentration of one substrate was kept constant while the other
was varied. The concentration of the varied substrate correspond-
ing to complete utilization of both substrates was then kept
constant and the other (kept constant in first set) was varied. In
first set of reactions, ethanol concentration was kept constant at
0.1 M while varying valeric acid concentration from 0.02 to 0.09 M,
and in second set of reactions, ethanol concentration was varied
from 0.04 to 0.2 M while keeping valeric acid concentration con-
stant at 0.06 M. The reaction system comprised AOT/buffered lipase
(5790 U/mg of solid)/isooctane in cylcohexane.
Microemulsion based organogels were prepared following the
procedure used by Dandavate and Madamwar [32] with slight
modifications, as follows. Gelatin obtained from porcine skin was
dissolved in double distilled water, autoclaved and cooled down to
55 ◦C. To 1 mL of thermodynamically stable reverse micellar solu-
tion, 1.5 mL gelatin (of concentrations ranging from 10 to 20%)
maintained at 55 ◦C in a hot water bath was added and the mixture
was vortexed vigorously for 2–5 min. The resulting mixture was
cooled to 25 ◦C, poured into plastic petriplates and kept for air dry-
ing overnight. The dried gels were cut into small pieces and used
for esterification reaction. In a similar fashion, AOT based MBGs
of agarose, CMC and agar-agar (1–10% concentration each) were
prepared using various concentrations of the gelling agents.
2.1.9. Reusability of the MBG preparation
After completion of a reaction cycle, the MBGs prepared using
three different solvents viz. isooctane, n-hexane and n-heptane
were washed with solvent 2–3 times for complete removal of
substrates and product. They were air dried and reused for ester
production. After 3–4 runs, when a sharp decline in percent-
age esterification was observed, the MBGs were treated with dry
reverse micellar solution of 1 M AOT overnight for removal of excess
water (byproduct of esterification reaction); given 2–3 solvent
washes for complete removal of AOT and used for esterification.
2.1.3. Esterification reaction conditions
The reaction was carried out in 250 mL glass stoppered flasks.
The reaction mixture consisted of 20 mL of solvent and equimo-
lar concentration (0.1 M) of valeric acid and ethanol (except for
substrate effect studies). To initiate the reaction, the MBG pieces
were added to the reaction mixture and kept on orbital shaker at
37 ◦C (except for temperature effect studies) and 150 rpm. 500 ␮L

T. Raghavendra et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 31–38
33
2.1.10. Thermostability studies
in a turbid solution indicating dissociation of reverse micelles. The
stable reverse micelles thus produced were immobilized in gelling
agents for further studies.
Thermostability studies were carried out for free as well as
immobilized lipase in order to evaluate the innate stability of
the enzyme and the additional stability offered by immobilization
against thermal denaturation. The analysis was done by preincu-
bating free and immobilized enzyme (5790 U/mg of solid each) at
50, 60 and 70 ◦C for 1–10 h in cyclohexane. After incubation, sub-
strates were added to the flasks and esterification was carried out
under standard conditions.
3.2. Esterification using MBGs prepared by various gelling agents
Enzymes are relatively expensive, and therefore discarding
them after a single use is not economical. Another problem is their
general instability towards heat, organic solvents, acids or bases,
etc. One method to circumvent these issues is to immobilize the
enzyme on a suitable supporting medium. Not only does this facili-
tate easier recovery and reuse of the enzyme, but also in many cases,
immobilized enzymes show a higher stability than free species [35].
For this reason, various gelling agents have been used to immobilize
them making them easily recoverable [36,37]. Four gelling agents
viz. gelatin, carboxy methyl cellulose (CMC), agarose and agar-agar
were used in different concentrations for preparation of AOT based
organogels. MBGs prepared using gelatin (14%) were uniformly
porous and thick. For agar-agar, agarose and CMC, 5% gelling agent
was sufficient for organogel preparation though the MBGs were
thin, translucent and non-porous. Also, concentrations above 5%
gelling agent either resulted in gels with phase separation or highly
viscous gels that could not be poured into the petriplates at all. The
esterification yield of gelatin MBG (∼99%) was many folds higher
than the yield obtained using CMC (25.55%) and agarose/agar-agar
(negligible).
2.2. Analytical procedure
After initiation of reaction, 500 ␮L sample of the reaction mix-
ture was withdrawn periodically every 24 h and immediately
analyzed by gas chromatography (PerkinElmer, Model Clarus 500,
USA) equipped with a flame ionization detector and 30 m TRX-R-20
(Crossband 80% dimethyl-20% diphenyl polysiloxane) capillary col-
umn. The carrier gas was nitrogen at a split flow rate of 90 mL/min.
The injector and detector temperatures were 250 and 280 ◦C
respectively and oven temperature was programmed to increase
from 100 to 160 ◦C at the rate of 20 ◦C/min, from 160 to 280 ◦C at
the rate of 2 ◦C/min and from 165 to 175 ◦C at the rate of 1 ◦C/min.
Ester identification and quantification were done by comparing the
retention time and peak area of the sample with standard. Pure
ethyl valerate was used as external standard.
Thus, different concentrations of gelatin were used for prepara-
tion of MBGs using CTAB, Triton X-100 and Tween 80. The physical
characteristics of the same are displayed in Table 2a. The finest
organogels thus obtained were further used for esterification. As
seen from Table 2b, AOT based organogels catalyzed the reaction
more efficiently than the others. Highest esterification was seen in
AOT/n-heptane organogels in n-heptane medium (97.82%) which
can be attributed to high Wo value for the same (Wo = 70). Being
anionic in nature, AOT does not require any co-surfactant for stabi-
lization, and therefore, it has been the surfactant of choice for many
investigators [38–40]. Thus, AOT based organogels using gelatin
were used for further experimentation. Gelatin has been the gelling
agent of choice for this purpose for many years [41–43].
3. Results and discussion
Ethyl valerate, which is well known for its fragrance, has been
synthesized biocatalytically by many investigators. However, many
of them have reported catalysis using whole cells [29,34] whilst a
few used immobilized enzyme [28] and others have tried both ways
[30].
The total yields obtained by immobilized enzyme [28,30] and
those from whole cells [30] have however not been very appre-
ciable. Hence, this study was focused mainly on immobilization of
purified enzyme for the same purpose of obtaining higher percent-
age conversions.
3.1. Preparation of stable reverse micelles using different
surfactants
3.3. Effect of organic solvents
It is crucial to introduce organic solvents for bioconversions of
lipophilic compounds for improving the poor solubility of the sub-
strates in water. Organic solvents produce various physicochemical
effects on enzyme molecules, and the effects differ depending
on the kinds of organic solvents and enzymes used. It has been
reported that suspension of enzymes in organic solvents results in
conformational changes and thereby the specificity of substrates
[44,45]. Among the various organic solvents used, there was no
esterification observed in acetonitrile which can be attributed to
its highly polar nature. It has been hypothesized that polar sol-
vents strip away the essential water bound to the enzyme by
participating in non-covalent solvent protein interaction result-
ing in their active conformational distortion. This is observed in
the form of lowered or complete absence of catalytic properties
of the enzyme. DMSO dissolved the organogels completely which
may have been due to loss of surfactant in this medium. Similar
effects with DMSO have been observed by Aguiar et al. [46] and
Dave and Madamwar [47]. Deleterious effects of other solvents such
as ethanoic acid and butanoic acid have also been reported [36,48].
It can be seen from Fig. 1a–c that all the organogels showed high
ester production in isooctane, n-heptane, n-hexane and cyclohex-
ane. They showed exceptionally higher yield at faster rates when
cyclohexane (Fig. 1a–c) was used as the medium. Highest yields
with all three MBGs were seen in shortest time period in this reac-
Due to amphipathic nature, surfactants behave as the interface
between the organic phase and the enzyme harboring buffer pro-
viding first level of protection to the enzyme entrapped within the
micelle. Thus, it is important to use a surfactant which is stable
in such conditions. All the four surfactants i.e. AOT, CTAB, Triton
X-100 and Tween 80 formed thermostable reverse micellar solu-
tions with the solvents. However, optimum value of Wo and Po (in
case of cationic and non-ionic surfactants) for stable micelle forma-
tion using different surfactants in the three solvents was dissimilar
(Table 1). Addition of buffer above this optimum Wo value resulted
Table 1
Optimum values of Wo (ratio of water to surfactant) and corresponding Po (ratio of
co-surfactant to surfactant) of reverse micellar solutions prepared using the surfac-
tants (0.1 M)-AOT, CTAB, Triton X-100 and Tween 80; solvents—isooctane, n-hexane
and n-heptane; co-surfactant-n-hexanol and 0.1 M sodium phosphate buffer.
Surfactant
Organic solvents
Isooctane
n-Hexane
Wo
n-Heptane
Wo
Po
–
7.84
11.76
15.68
Po
–
5.49
25.88
14.11
Wo
Po
AOT
CTAB
Triton X-100
Tween 80
60
30
50
40
50
60
30
40
70
90
50
50
–
10.98
15.68
18.04

34
T. Raghavendra et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 31–38
Table 2a
Hence, AOT/isooctane MBG was chosen for further experimenta-
tion in combination with cyclohexane as reaction medium.
Physical characteristics of organogels prepared using reverse micellar solutions
containing surfactants (0.1 M) AOT, CTAB, Triton X-100, Tween 80 and solvents
n-hexane, n-heptane and isooctane with various concentration of gelatin.
3.4. Effect of Wo on esterification
Surfactant
Organic
solvent
Gelatin (%)
Physical characteristics of
organogel
The water/surfactant mole ratio, given the symbol Wo is directly
proportional to the size (radius) of the dispersed water droplets.
Fig. 2 shows that ester formation increased with increase in Wo
from 10 to 60. Reverse micellar solutions of Wo higher than 60
were turbid and showed a slight decrease in esterification (Fig. 2).
Thus, optimum value of Wo for AOT/isooctane was 60 for maxi-
mum esterification. Turbidity above a certain Wo value indicated
that the reverse micelles which could not hold more water started
breaking and hence showed lowered values of esterification. Sim-
ilar observations have been made by Jenta et al. [42] and Soni and
Madamwar [48].
AOT
Isooctane
n-Hexane
n-Heptane
<14
14
>14
<14
14
>14
<14
14
Thin, fragile, transparent
Porous, translucent, strong
Thick, opaque, non-porous
Thin, fragile, transparent
Porous, translucent
Thick, opaque, non-porous
Thin, fragile, transparent
Porous, translucent
>14
Thick, opaque, non-porous
CTAB
Isooctane
n-Hexane
<16
16
>16
<20
20
Thin, fragile, chalky
Porous, white, brittle
Thick, non-porous
Thin, chalky
Porous, white, brittle
Very thick, chalky,
non-porous
3.5. Effect of pH on esterification
>20
Water inside reverse micelles displays unusual properties when
compared to those of bulk water [33]. This had led many investi-
gators to analyze properties of reverse micellar water [51]. Thus,
in this study, the optimum pH required for esterification reaction
using lipase entrapped in MBGs was investigated for the same pur-
pose. High esterification values were observed at low pH values of
pH 5 (85.7%), pH 6 (97.3%) and pH 7 (98.91%). Further increase in
pH resulted in decrease in ester production to a mere 22.34% at pH
8.8. As is evident from Fig. 3, the immobilized lipase was stable and
showed highest esterification rates in acidic to neutral pH than in
alkaline pH. Similar results of esterification maxima at low pH have
been observed by others [51,52].
It has also been shown that at alkaline pH, there is a negative
potential that appears in the active site in C. rugosa lipase result-
ing in higher activity towards ester hydrolysis [53] and hence, the
vice-versa activity at low/acidic pH resulting in reverse reaction
(condensation to form ester) can be expected.
n-Heptane
<20
20
>20
Thin, chalky
Porous, white
Very thick, chalky,
non-porous
Triton X-100
Isooctane
n-Hexane
n-Heptane
<18
18
>18
<20
20
>20
<18
18
Thin, transparent, brittle
Thin, translucent, elastic
Thick, translucent
Thin, transparent, brittle
Translucent, elastic
Thick, opaque
Thin, transparent
Thin, translucent, elastic
Thick, translucent
>18
Tween 80
Isooctane
n-Hexane
n-Heptane
<16
16
>16
<16
16
>16
<16
16
Thin, very frail, transparent
Translucent, elastic
Thin, hard, non elastic
Thin, fragile, transparent
Translucent, elastic
Thin, hard
Thin, fragile, transparent
Translucent, elastic
Thin, hard, non elastic
3.6. Effect of temperature on esterification
>16
The activity of CRL (free and immobilized in MBGs) towards
the esterification reaction was monitored as a function of incuba-
tion temperature. Increase in temperature from 25 to 45 ◦C showed
increase in lipase activity (Fig. 4).
tion medium (Fig. 1a–c). This can be attributed to the extreme
hydrophobic nature of the solvent stabilizing the hydrated enzyme
structure. However, the free enzyme showed poor product for-
mation under similar conditions (Fig. 1a–c). Furthermore, it was
observed that better results were obtained when two different sol-
vents were used for the two purposes (one for MBG preparation and
the other as reaction medium) as compared to the reactions using
the same solvent for both purposes. Similar effects of these organic
solvents have been observed by Yesiloglu and Kilic [49], Gogoi et
al. [50] and Jenta et al. [42].
It was observed that at higher temperatures (>37 ◦C), increase
in incubation temperature led to significant increase of the reac-
tion’s initial rate resulting in high product formation at 37, 40 ◦C
as well as 45 ◦C whereas at lower temperatures (25 and 30 ◦C) very
low conversions were observed (Fig. 4). This suggests that at higher
temperatures, the conversion rate is controlled by reaction steps
and hence strongly increases with increase in temperature. How-
ever, at lower temperatures, the reaction rate is limited by mass
transport phenomena and not by the reaction steps which can be
attributed to the low increase in ester production with increase
in temperature [40]. Similar results have been observed by Dave
As Wo of MBG prepared in isooctane was lower (60) than n-
heptane (70), it follows that the AOT/isooctane system used less
lipase than the AOT/n-heptane system and contributed to equiva-
lent production (Higher the Wo value, more is the enzyme used).
Table 2b
Ester synthesis using AOT, CTAB, Triton X-100 and Tween 80 based organogels using buffer of pH 7.2 containing lipase at 37 ◦C and 150 rpm. The reactions reached completion
on 9th day.
Surfactant
Solvent used for organogel preparation as well as for reaction medium
Isooctane
n-Heptane
Gelatin (%)
n-Hexane
Gelatin (%)
Gelatin (%)
Esterification (%)
Esterification (%)
Esterification (%)
AOT
CTAB
Triton X-100
Tween 80
14
16
18
16
78.13 0.8
19.21 0.9
11.01 0.7
10.84 0.7
14
20
16
16
97.82 0.9
74.74 1.1
12.46 0.8
27.13 0.9
14
20
20
16
75.38 1.6
32.20 0.8
4.27 0.7
12.50 0.7

T. Raghavendra et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 31–38
35
Fig. 2. Effect of Wo on esterification using AOT based organogels at 37 ◦C and
150 rpm.
Fig. 3. Effect of pH on esterification using AOT based organogels at 37 ◦C and
150 rpm.
temperature for optimum activity. It also conveyed the effective
protection to lipase offered by immobilization process against ther-
mal denaturation.
3.7. Effect of substrate concentration on esterification
As substrate concentration is a very significant parameter in
any enzyme catalyzed reaction, it is important to determine their
effects on the enzyme and reaction kinetics. It is also very essential
to set up reactions in such a way that result in maximum pro-
Fig. 1. (a) Synthesis of ethyl valerate using AOT/isooctane organogels in isooctane,
n-heptane, n-hexane and cyclohexane at 37 ◦C and 150 rpm for 9 days. The solid lines
represent lipase entrapped in MBGs and the dotted lines represent free lipase. (b)
Synthesis of ethyl valerate using AOT/n-heptane organogels in isooctane, n-hexane,
n-heptane and cyclohexane at 37 ◦C and 150 rpm for 9 days. The solid lines represent
lipase entrapped in MBGs and the dotted lines represent free lipase. (c) Synthesis
of ethyl valerate using AOT/n-hexane organogels in isooctane, n-hexane, n-heptane
and cyclohexane at 37 ◦C and 150 rpm for 8 days. The solid lines represent lipase
entrapped in MBGs and the dotted lines represent free lipase.
and Madamwar [54] and Schlatmann et al. [39]. Optimum temper-
ature was found to be 40 ◦C, showing 99.87% product formation
on 6th day whereas the reactions proceeded till days 8–9 at other
temperatures. Also, free lipase used as control showed extremely
meager ester production at higher temperatures when compared to
the immobilized lipase (Fig. 4). However, the productivities of free
as well as immobilized lipase were similar at 25 and 30 ◦C which
showed that the immobilized lipase preparation needed higher
Fig. 4. Effect of different temperatures on ethyl valerate synthesis by free and AOT
based organogels 150 rpm.

36
T. Raghavendra et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 31–38
Table 3a
Effect of valeric acid concentration on ester production using AOT based organogel
using buffer of pH 7.2 containing lipase at 37 ◦C and 150 rpm. Ethanol concentration
was kept constant at 0.1 M. The reactions were carried out for 8 days.
Valeric acid concentration (M)
Esterification (%)
Residual substrate (%)
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
41.57 1.2
52.90 1.1
63.21 0.8
90.23 0.7
98.89 0.9
96.67 0.7
85.73 1.2
86.66 0.8
0.11 0.7
0.37 0.9
0.58 0.8
2.66 0.7
5.50 1.2
17.41 1.1
18.44 0.9
21.27 0.8
duction with minimal wastage/usage of substrates. To determine
the optimum ratio of the substrates, the concentrations of ethanol
and valeric acid were varied one at a time keeping the other con-
stant and carrying out esterification reaction. In case of variation
of valeric acid concentration (ethanol 0.1 M), 98.89% esterification
was observed with 0.06 M valeric acid on 8th day and both the
substrates were nearly completely utilized (Table 3a). In reactions
with less than 0.06 M valeric acid, esterification increased till a
maximum of 90% with complete utilization of the acid but some
residual presence of the alcohol and in those with more than 0.06 M
valeric acid, the vice-versa was observed i.e. considerable quan-
tities of unutilized valeric acid was present. When the same was
repeated keeping valeric acid at a constant concentration of 0.06 M
(obtained from the previous study) and varying ethanol concentra-
tion from 0.04 to 2.0 M, highest yield was obtained at 0.08–0.12 M
concentrations (Table 3b). The reactions proceeded very quickly
at low alcohol content (0.05–0.08 M) and reached a maximum
in 5–6 days while higher concentrations of alcohol exhibited an
inhibitory effect on reaction slowing it down drastically showing
minimum esterification of 45% at 0.16 M ethanol concentration
(Table 3b). Similar phenomena have been observed by Gogoi et
al. [50] and Somashekar and Divakar [52]. Thus, the molar ratio
of valeric acid to alcohol was found to be 1:1.6 correspond-
ing to maximum esterification with complete utilization of both
substrates.
Fig. 5. Reusability of AOT/isooctane, AOT/n-hexane and AOT/n-heptane organogels.
The up arrows indicate intermittent treatment of the organogels with 1 M
AOT/isooctane dry reverse micellar solution for removal of excess water. The reac-
tions were carried out at 37 ◦C and 150 rpm for 9 cycles.
of excess water formed as a byproduct of esterification reaction. As
water may result in hydrolysis reaction behaving as a substrate,
it is important to remove this water. Thus, 1 M dry AOT/isooctane
reverse micellar treatment was given to the MBGs for 24 h. Signifi-
cant increase or maintenance in esterification (no further decrease)
was observed in all three MBGs demonstrating partial recovery
of the lost activity (Fig. 5). The AOT/isooctane, AOT/n-hexane and
AOT/n-heptane organogels exhibited 44%, 75% and 76% residual
esterification respectively in 9th run. These results indicate bet-
ter immobilization efficiency and reaction systems depicted by
the high reaction rates even after 8–9 runs as compared to others
[40,47,55].
3.9. Thermostability studies
It is well known that proteins, including enzymes are sensi-
tive biological molecules which tend to get denatured under harsh
physical and chemical environments (except for a few belonging
to extremophiles). High temperatures may result in alteration of
spatial structure of many proteins rendering denaturation and con-
sequently loss of their activity. On the other hand, high temperature
may also induce minor structural changes resulting in superactiv-
ity of the enzyme [56]. In this study, a similar phenomenon was
observed at 50 and 60 ◦C wherein incubation of the organogels for
prolonged periods at these temperatures led to increase in ester
synthesis providing evidence of some structural change resulting
in high activities (Fig. 6). This may also be attributed to slow adap-
tations to high temperature as incubation for 1–2 h led to loss in
activity while in longer incubation this loss was restored, perhaps
by adaptation to the temperature. Also, all the organogels were
physically stable at all three temperatures. More than 80% esterifi-
cation was observed even after 10 h incubation at 50 and 60 ◦C but
incubation at 70 ◦C for more than 6 h resulted in drastic reduction
in ester production (Fig. 6). However, in case of free lipase, it was
observed that though the enzyme activity was low compared to its
immobilized counterpart, but the performance in terms of esteri-
fication was more or less constant at all three temperatures even
after 10 h of incubation (Fig. 6). These observations point towards
an inherent stability of the enzyme against high temperatures
which was further enhanced by immobilization. Nevertheless, the
product yields were lower than unincubated free enzyme (Figs.
1a–c and 6). However, many reports have shown total or high loss
of activities of enzymes (free or immobilized) at such high tem-
peratures and shorter incubation periods [47,57,58]. Hence, the
results obtained in this study may be a combined consequence of an
3.8. Reusability of the immobilized enzyme preparation
The MBGs were subjected to reusability examination for
determining the efficiency of immobilization. The enzyme prepa-
ration was given solvent washes after every cycle and reused in
fresh media supplemented with substrates. Three organogels viz.
AOT/isooctane, AOT/n-hexane and AOT/n-heptane were reused 9
times with intermittent dry AOT/isooctane reverse micellar treat-
ment after declination in ester production. It was observed that
the MBGs catalyzed the reaction appreciably for first three cycles
after which their activity started declining slowly (Fig. 5). A sharp
decline in ester production was observed in 4th and 8th run with
respect to all the three organogels. This may be due to accumulation
Table 3b
Effect of ethanol concentration on ester production using AOT based organogel using
buffer of pH 7.2 containing lipase at 37 ◦C and 150 rpm. Valeric acid concentration
was kept constant at 0.06 M. The reactions were carried out for 8 days.
Ethanol concentration (M)
Esterification (%)
Residual ethanol (%)
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.2
81.45 1.2
88.67 0.9
97.67 0.7
98.68 0.9
98.25 1.1
73.61 1.1
45.22 0.7
59.82 0.9
0.60 1.1
1.23 1.2
4.54 0.9
14.65 0.9
19.38 1.2
21.33 1.2
25.76 0.9
29.76 0.7

T. Raghavendra et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 31–38
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4. Conclusion
Production of ethyl valerate by condensation of ethyl alco-
hol and valeric acid was performed using CRL immobilized
in MBGs under nearly non-aqueous conditions. AOT proved to
be the ideal surfactant for organogels among other surfactants
yielding as high as 98–99% product under optimum conditions.
Gelatin was the gelling agent of choice due to the stronger,
stable organogels and higher esterification rates. The combi-
nation of the two organic solvents viz. isooctane as reverse
micellar constituent and cyclohexane as reaction medium exhib-
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deleterious effects on MBGs and the reaction. The optimum tem-
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enzyme showed high activities at acidic to neutral pH (5–7),
pH 7 being the optimum while a steep decline was seen in
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inhibition. The organogels could be reused for 9 cycles with
excellent activity retention. Also, they were highly stable at
50, 60 and 70 ◦C for 1–10 h of incubation prior to reaction.
This confirms the staturation of microemulsion based organogels
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esters.
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Authors acknowledge University Grants Commission, New
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