Journal of Biomaterials and Nanobiotechnology, 2011, 2, 400-408
doi:10.4236/jbnb.2011.24049 Published Online October 2011 (http://www.SciRP.org/journal/jbnb)
Enzymatic Synthesis of Butyl Ferulate by
Silica-Immobilized Lipase in a Non-Aqueous
Medium
Chandresh Chandel, Ashok Kumar, Shamsher S. Kanwar
Department of Biotechnology, Himachal Pradesh University, Shimla, India.
Received March 18th, 2011; revised June 20th, 2011; accepted August 15th, 2011.
ABSTRACT
Butyl ferulate was synthesized using a silica-immobilized commercial lipase (Steapsin) in dimethylsulfoxide (DMSO).
Lipase-immobilized by surface adsorption onto silica pretreated with 1% glutaraldehyde showed 89% binding of pro-
tein. The esterification of butanol (100 mM) and ferulic acid (50 mM) by silica-bound biocatalyst was carried out at
45˚C for 6 h under shaking (120 rpm). The optimization of various reaction conditions like molar concentration of re-
actants, biocatalyst concentration, reaction time, temperature, addition of molecular sieves, salt ions, and repetitive
bio-catalysis in DMSO were studied, consecutively. The bound lipase (15 mg/ml) catalyzed the esterification of ferulic
acid and butanol with a yield of 64 mM under optimized reaction conditions. Among the salt ions Cu2+, Zn2+ and Al3+
ions moderately promoted the ester yield (66 mM) while Mg2+, NH4+, Fe2+ and Ca2+ were found to decrease the ester
yield. The by-product (H2O) produced in the reaction was scavenged by the molecular sieves (10 mg/ml) added to the
reaction mixture, which enhanced the formation of ester up to 74 mM. During the repetitive reactions, the bound lipase
produced 32 mM ester after 4th cycle of esterification. On scaling-up the reaction volume to 30 ml, 32.5 mM butyl feru-
late was synthesized under optimized conditions.
Keywords: Silica, Glutaraldehyde, Butyl Ferulate Synthesis, Molecular Sieves
1. Introduction
The lipases (EC 3.1.1.3.) are becoming increasingly at-
tractive in the pharmaceutical, cosmetic and oil industry
due to their stability, especially selectivity and successful
biotransformation. Currently, scientists are making ef-
forts to evolve newer derivatives of natural plant pro-
ducts that are more amenable as emoluments, constitu-
ents of cosmetics, toiletries and fragrances etc. Such pro-
ducts of plant origin are likely to be better acceptable for
human use with least side effects or cytotoxicity. Esters
of cinnamic acid, ellagic acid, ferulic acid etc. are orga-
nic compounds of biotechnological relevance that could
be suitable modified as flavor/fragrance compounds, pre-
cursors of pharmaceuticals and as additives in foods, cos-
metics and sun-screens. Ferulic acid (4-hydroxy-3-me-
thoxy cinnamate) is an effective antioxidative agent
among various hydroxycinnamic acids as it potentially
prevents the autoxidation of linoleic acid in the ethanol-
buffer system [1]. Moreover, esters of ferulic acid are
easy to apply on skin and show an increased antioxidant
activity instead of use of ferulic acid that has an acidic
nature and as such cannot be spread on the skin. The
activity of alkyl ferulate is somewhat influenced by the
chain length of alcohol moiety [2]. Comparing the effects
of ferulic acid and its esters, hexyl, octyl and 2-ethyl-1-
hexyl ferulates were significantly more active than feru-
lic acid itself, and higher or lower carbon chain homo-
logues than hexyl and octyl ferulates were less active as
anti-oxidant mo lecules [1,3]. Ethyl ferulate that is widely
distributed in plants possesses a UV absorbance maxi-
mum at ~320 nm [4]. Ferulic acid is believed to suppress
melanin generation by antagonizing tyrosine because its
chemical structure is similar to tyrosine; and it effec-
tively absorbs harmful long wave ultraviolet radiations
emerging from the Sun. Ferulic acid and its esters are
well known “oxidation inhibitors”.
Butyl ferulate exhibits free radical scavenging activity
against DPPH radicals [1]. Also the same compound was
reported to play a chemopreventive role in cancer by in-
ducing tumor cells apoptosis. A significant cytochromec

Enzymatic Synthesis of Butyl Ferulate by Silica-Immobilized Lipase in a Non-Aqueous Medium401
release has been found when treating TM-3 cells with
butyl ferulate [5]. In the light of the afor esaid an attempt
was made to synthesize butyl ferulate using a silica-
bound commercial lipase “Steapsin”. Most common cin-
namic acids and short-chain esters are water soluble,
limiting their usefulness as waterproof sunscreens but
ferulic acid is insoluble in water and its derivatives there-
fore have been designed with long-chain hydrocarbons
(e.g., octyl-p-methoxy cinnamate), which renders them
water-insoluble and suitable for waterproof sunscreens
[6]. The -OCH3 group of octyl-p-methoxy cinnamate acts
as an electron-releasing group to improve the electron
excitation process [7]. When triolin ferulate was com-
bined with ferulyl-substituted oleins, it elevated the UV
absorptivity of a cinnamate ester with the water- insolu-
ble properties of a lipid [8]. The lipase-catalyzed trans-
esterification of ethyl ferulate with ethanol could poten-
tially lead to new sunscreen products, synthesized from
natural products, while providing a value-added use for
vegetable oils [9-11].
Ferulic acid that has little solubility in most alkanes
and aqueous media could be solublized in DMSO or di-
ethyl phthalate to achieve ester synthesis or trans-esteri-
fication [12,13]. The immobilized enzymes are preferred
for biocatalysts because of obvious easy recovery, in-
creased stability at raised temperature and or pH as well
as repetitive use in reaction system(s). The current work
focuses on the synthesis of butyl ferulate from butyl al-
cohol and ferulic acid by employing immobilized-lipase
under optimized reaction conditions such as catalyst loa-
ding, mole ratio, temperature, pH, effect of salt ions, mo-
lecular sieves and repetitiv e use of silica-bound lipase.
2. Material and Methods
Ferulic acid and butanol were procured from Merck
Schuchardt, Germany. DMSO from Sigma Aldrich, USA;
and molecular sieves 3 Å × 1.5 mm were from E. Merck
(India) Ltd., Mumbai. Commercial lipase: Steapsin was
obtained from Sisco Research Laboratory, Mumbai, In-
dia; and silica 100 - 200 mesh was from s-d fin e Chemi-
cals, Mumbai, India. All chemicals were of analytical
grade and were used as received.
2.1. Determination of Lipase Activity
The activity of free and immobilized Steapsin was mea-
sured by lipase assay [14] with minor modifications. The
reaction mixture contained 80 µl of p-nitrophenol palmi-
tate (p-NPP) stock solution (20 mM p-NPP prepared in
isopropyl alcohol), 80 µl of the test sample (lipase) and
Tris buffer (0.05 M, pH 8.5) to make final volume to 3 ml .
The reaction mixture was incubated at 45˚C for 10 min
in a water-bath. The reaction was stopped by keeping the
reaction mixture at –20˚C for 10 minutes. An appropriate
control with a heat-inactivated enzyme (5 min in a boil-
ing-water bath) was included with each assay. The ab-
sorbance of p-nitrophenol released in the reaction mix-
ture was measured at A410. Each of the assays was per-
formed in triplicate, and mean values ± standard devia-
tions were presented. One unit (1 IU) of lipase activity
was defined as the micromoles of p-nitrophenol released/
minute by the hydrolysis of p-NPP by 1 ml of soluble
enzyme or 1 g of silica-bound enzyme (weight of matrix
included) at 45˚C under assay conditions. All the addi-
tives including buffer were pre-incubated at 45˚C for
short period (3 min) before the enzyme was added to
start the reaction.
2.2. Determination of Protein Content
Protein concentration in the free or matrix-bound lipase
was determined as described previously [15] using bo-
vine serum albumin as a standard.
2.3. Immobilization of Enzyme onto Silica
The matrix was washed three times with Tris buffer 0.05
M pH 8.5 to remove soluble impurities. The silica (3.5 g),
pre-equilibrated in an excess volume of Tris buffer
(0.05M, pH 8.5), was incubated with commercial lipase
(Steapsin 3.60 IU/ml and 18.2 mg/ml protein) at 8˚C
overnight. The volume of the supernatant, amount of
unbound protein, and the lipase activity were estimated.
The bound-lipase activity was assayed in silica-bound
lipase 20 mg/reaction volume of 2 ml). The bound pro-
tein in the matrix was determined by subtraction of the
unbound protein in the sup ernatant from the total protein
used for immob ilization. The silica was pretreated with a
glutaraldehyde (12 ml; 1%, v/v in 0.05 M Tris pH 8.5)
before immobilization of the lipase by adsorption
[16,17].
2.4. Esterification of Butanol and Ferulic Acid
Butyl ferulate synthesis was performed by using 100 mM
ethanol, 50 mM ferulic acid and silica-bound lipase
(~2.95 IU/mg) taken in 2.0 ml of DMSO in Tefloncap-
ped glass-vial (5 ml capacity). The reaction was per-
formed at 45˚C for 6 h along w ith buffer-treated silica as
a control under shaking (120 rpm). Each of the esterifi-
cation reactions was performed in triplicate, unless other-
wise stated, and the mean values and standard deviation
(SD) were determined. Samples of the reaction cocktail
(10 l) were withdrawn at intervals and analyzed by gas
liquid chromatography (GLC) for presence of butyl fe-
rulate.
2.5. Analysis of Butyl Ferulate by GLC
The butyl ferulate produced in the reaction cocktail was

Enzymatic Synthesis of Butyl Ferulate by Silica-Immobilized Lipase in a Non-Aqueous Medium 402
O
O
O
OH
CH
CH3 OH
H
3O
O
O
O
CH
CH
H
3
3
Ferulic acidn-Butanol Butyl ferulate
Immobilized lipase
45oC, DMSO
Figure 1. Lipase catalyzed esterification of ferulic acid and n-butanol to produce butyl fer ulate.
2.6.4. Effect of Temperature on Ester Synthesis
analyzed by GLC equipped with a flame ionization de-
tector and a packed type column (10% SE-30 Chrom
WHP, 2 meter length, mesh size 80 - 100, internal dia me-
ter 1/8 inches, maximum temperature limit 300˚C; Netel
Chromatograph, Thane, India). N2 was used as a carrier
gas (30 cm3/min). The injector was set at 260˚C; detector
at 270˚C and the column/ oven temperature was kept at
250˚C. The sample size for the GLC analysis was 2 l.
The effect of reaction temperature (25, 35, 45, 55, and
65˚C) on the synthesis of butyl ferulate was studied in
Teflon-capped glass-vials (5 ml). The reaction mixture
containing butanol, ferulic acid (100 mM:50 mM) in
DMSO and silica-immobilized lipase (30 mg/reaction
volume of 2 ml) were incubated at each of the selected
temperatures for 6 h under shaking. The amount of ester
synthesized was determined by GLC.
2.6. Optimization of Parameters for Synthesis of
Butyl Ferulate 2.6.5. Effect of Salt Ions on the Esterification of
Ferulic Acid and Butanol.
The effects of various parameters such as reaction time,
relative molar concentration of reactants, reaction tempe-
rature, pH, and effect of detergents, chelating agen ts, salt
ions and repetitive use of immobilized lipase on yield of
butyl ferulate were separately evaluated. All esterifica-
tion reactions were performed using 20 mg of silica-
bound lipase/reaction volume (2 ml) at 45˚C under sha-
king condition (120 rpm) unless stated otherwise.
The effect of salt ions on the synthesis of butyl ferulate
was evaluated by pre-incubating the immobilized en-
zyme separately with each of the salt ions NH4+, Mg2+,
Ca2+, Mn2+, Zn2+ and Al+3 (1 mM in Tris buffer) for 30
min at 45˚C. The silica-bound lipase was spun down by
centrifugation (10,000 rpm, 5 min) and the buffer con-
taining salt ion(s) was completely decanted by inversion.
The sedimented bound-enzyme was washed in DMSO
and spun down again to perform the esterification as
above. The synthesis of butyl ferulate was recorded, the-
reafter.
2.6.1. Effect of Molar C oncentration of React an ts
The concentration of feru lic acid and bu tanol w ere varied
one at a time (25, 50, 75 and 100 mM). The reaction was
carried out for 18 h at 45˚C under shaking. The subse-
quent esterification reactions were carried out at opti-
mized molar concentrations of ferulic acid and butanol.
2.6.6. Effect of Molecular Sieves on the Synthesis of
Butyl Ferulate
The reaction mixture containing 50 mM ferulic acid and
100 mM butanol when incubated at 45˚C with varying
concentration of molecular sieves (10 - 200 mg) and to
observe the effect of varying concentration of molecular
sieves on the amount of ester synthesized was recorded
in comparison to the control without molecular sieves.
2.6.2. Effect of Reaction Time on Synthesis of Butyl
Ferulate
The reaction mixture containing silica-bound lipase, and
100 mM of butanol and 50 mM ferulic acid in DMSO
was incubated at 45˚C in a water-bath-incubator shaker
for 18 h. At intervals of 2 h, the solvent phase was sam-
pled and analyzed by GLC for the presence of butyl fe-
rulate. The optimized reaction time was considered in the
subsequent experiments.
2.6.7. Reusability of Silica Immobilized Lipase in
Continuous Cycles of Esterification.
The immobilized lipase was used for the synthesis of
butyl ferulate in DMSO, repetitively up to 4th cycle of 6
h each at 45˚C under shaking. After first cycle of esteri-
fication, the biocatalyst was recovered (by centrifuging
and decanting the reaction mixture), and this biocatalyst
was used to catalyze fresh esterification reaction. The
reaction system contained 100 mM of butanol and 50
mM of ferulic acid treated with molecular sieves (10
mg/ml reaction volume) and the reaction was carried out
under optimized conditions.
2.6.3. Effect of the Biocatalyst Concentration
The synthesis of butyl ferulate was performed by placing
varying amounts of the immobilized lipase (5 - 30 mg/ml
in triplicates) in 2 ml of a reaction mixture containing
100 mM each of ferulic acid and butanol in DMSO at
45˚C for 6 h under shaking. GLC analysis was done for
the quantification of the butyl ferulate produced in the
reaction mixture.

Enzymatic Synthesis of Butyl Ferulate by Silica-Immobilized Lipase in a Non-Aqueous Medium403
2.6.8. Effect of Volumetric Scale up of Reaction
System on the Synthesis of Butyl Ferulate
The volume of reaction mixture containing 50 mM:100
mM ferulic acid and butanol, respectively was scaled up
from initial 2 ml to 5, 10, 15 and 30 ml with a corres-
ponding increase in the amount of biocatalyst and the
molecular sieves in the reaction system.
3. Results
3.1. Immobilization of the Commercial Lipase
A commercial lipase “Steapsin” was optimally immo-
bilized on to silica that retained 95.3% of total protein
used for immobilization. The activity of free lipase was
3.6 IU/ml. After immobilization onto silica, the bound-
lipase exhibited 2.95 IU activity and retained approxi-
mately 98% of its orig in al hydro lytic activit y. To ach iev e
the biocatalysis it is essential to determine the optimum
values of temperature, pH, time of reaction, and molar
concentration of reactants for higher yield of products
[18-20].
3.2. Optimization of Various Reaction
Parameters for Esterification Reaction
3.2.1. Effec t of Mol ar Concentration of Re act ants
The maximum ester synthesis (64 mM) was observed
when concentration of ferulic acid and butanol was kept
50 mM: 100 mM (Figure 2). The reactant concentration
above or below this ratio resulted in a decline in the syn-
thesis of butyl ferulate. In contrast a little esterification
could be achieved by immobilized lipase when concen-
tration of ferulic acid was kept constant (100 mM) and
butanol concentration was varied from 25 to 100 mM.
Thus further esterification reactions were carried out at
optimized molar concentrations of ferulic acid and bu-
tanol (50 mM:100 mM, respectively).
3.2.2. Effect of Incubation Time on Synthesis of Butyl
Ferulate
Kinetics of immobilized-lipase catalyzed synthesis of
butyl ferulate was studied for 18 h at 45˚C in DMSO
under continuous shak ing. The butyl ferulate was formed
with a maximum conversion rate of approximately 64
mM within a short period of 6 h (Figure 3). Thus in the
subsequent experiments reaction time of 6 h at 45˚C for
immobilized-lipase was considered optimum for the syn-
thesis of butyl ferulate.
3.2.3. Effect of the Biocatalyst Concentration
The synthesis of butyl ferulate was performed by placing
varying amounts of the immobilized lipase (5-30 mg/ml
in triplicates) in 2 ml of a reaction mixture containing
100 mM butanol and 50 mM ferulic acid in DMSO at
45˚C for 6 h under shaking (Figure 4). GLC analysis
Figure 2. Effect of molar concentrations of reactants on the
synthesis of butyl ferulate. The reaction mixture containing
varying ratio of ethanol and ferulic acid in DMSO was in-
cubated at 45˚C under shaking for 6 h.
Figure 3. Effect of reaction time on the synthesis of butyl
ferulate. The reaction mixture containing 100 mM of bu-
tanol and 50 mM ferulic acid and, silica-bound biocatalyst
in DMSO was incubated at 45˚C under shaking.
Figure 4. Effect of biocatalyst concentration on synthesis of
butyl ferulate. The reaction mixture containing 100 mM
butanol and 50 mM ferulic acid and, silica-bound biocata-
lyst in DMSO was incubated at 45˚C under shaking for 6 h.

Enzymatic Synthesis of Butyl Ferulate by Silica-Immobilized Lipase in a Non-Aqueous Medium 404
shows approx imately 65 mM yield of butyl feru late with
15 mg/ml immobilized enzyme.
3.2.4. Effect of Temperature on the Synthesis of Butyl
Ferulate
The silica-immobilized lipase performed the esterifica-
tion reaction much efficiently at 45˚C with the maxi-
mum yield 62 mM (Figure 5). As increase above 30˚C
showed a corresponding increase in the rate of esteri-
fication and maximum yield was observed at 45˚C but
any increase in temperature beyond 45˚C resulted in a
decline in the ester synthesis.
3.2.5. Effect of Salt ions on the Synthesis of Butyl
Ferulate
Among the salt ions; Cu2+, Zn2+ and Al3+ moderately pro-
moted the ester formation up to 66 mM in comparison to
control 62 mM while Mg2+, NH4+, Fe2+ and Ca2+ were
found to decrease the yield of the butyl ferulate (Figure
6).
3.2.6. Effect of Molecular Sieves on the Synthesis of
Butyl Ferulate
With increasing the concentration of molecular sieve
from 10 mg/ml to 200 mg/ml the yield of butyl ferulate
elevated marginally to 74 mM in comparison to control
(67 mM) when 10 mg/ml of molecular sieves were used
in the reaction mixture (Figure 7). A further increase of
the concentration of the molecular sieve from 20 to 200
mg/ml resulted in a gradual decrease in synthesis of es-
ter.
3.2.7. Reusability of Silica-Immobilized Enzyme
It was observed that the silica-immobilized lipase cata-
lyzed the esterification appreciably for first three cycles
after which its activity started to decline (Figure 8). Ap-
proximately, 33% decrease in the ester formation was
observed after 4th cycle of esterification.
3.2.8. Volumetric Scale up of Reaction System for the
Synthesis of Butyl Ferulate
The volume of reaction mixture containing 50 mM: 100
mM ferulic acid and butanol, respectively was when
scaled up from initial 2 ml to 5, 10, 15 and 30 ml with a
corresponding increase in the amount of biocatalyst and
molecular sieves, a decrease in the amount of ester pro-
duced in the reaction cocktail was noticed with a cor-
responding increase in volume of the reaction system and
a lower yield (32.6 mM) was recorded at 30 ml reaction
volume (Figure 9).
4. Discussion
The exploitation of commercial lipases for preparing
value-added specialty products from lipids by esterifi-
cation/trans-esterification depends on understanding and
Figure 5. Effect of reaction temperature on the synthesis of
butyl ferulate. The reaction mixture containing 100 mM
butanol and 50 mM ferulic acid, and silica-bound biocata-
lyst in DMSO was incubated at stated temperature under
shaking for 6 h.
Figure 6. Effect of salt-ions on the synthesis of butyl feru-
late. The silica-bound lipase pre-exposed to the selected salt
ions (1 mM) for 1 h at 45oC was used to achieve esterifica-
tion of butanol and ferulic acid (100 mM: 50 mM) in DMSO
at 45oC under shaking in 6 h.
Figure 7. Effect of molecular sieves on the synthesis of butyl
ferulate. The reaction mixture containing 100 mM butanol
and 50 mM ferulic acid, and silica-bound biocatalyst in
DMSO was incubated at 45oC under shaking for 6 h.
C
opyright © 2011 SciRes. JBNB

Enzymatic Synthesis of Butyl Ferulate by Silica-Immobilized Lipase in a Non-Aqueous Medium405
Figure 8. Repetitive synthesis of butyl ferulate by silica-
bound lipase. The silica-bound lipase was used to achieve
esterification of butanol and ferulic acid (100 mM:50 mM)
in DMSO at 45
oC in repetitive cycles of 6 h each under
shaking.
Figure 9. The volume of reaction mixture containing 50
mM:100 mM ferulic acid and butanol, respectively was
scaled up from initial 2 ml to 5, 10, 15 and 30 ml with corre-
sponding increase in the amount of biocatalyst and molecu-
lar sieves.
controlling reaction selectivity towards different sub-
strates [21]. Various features of reaction selectivity of
lipases are modulated by exogenous factors such as type
of organic solvent, choice of co-substrates/reactants, wa-
ter activity, pH, temperature and nature of support matrix
[22-25]. The lipase after immobilization onto silica was
exposed to glutaraldehyde that acts as a crosslinking
agent and is effective against dilution induced disso-
ciation of enzyme [26]. Glutaraldehyde stabilizes the
bound protein (enzyme) onto the silica and maintains the
structural integrity as well as its biocatalytic activity. Th e
immobilized enzyme showed maximum hydrolytic ac-
tivity at pH 8.5 and temperature 45˚C in comparison to
the free enzyme that gave maximum activity at pH 8 and
temperature 35˚C. Immobilization, therefore, endowed
an effective protection to silica-bound lipase against
thermal denaturation and thus possibly facilitates the
dispersal of enzyme on a solid surface to provide a far
greater interfacial area and accessibility of substrate to
the enzyme relative to the use of enzyme powders [27].
The optimum values of temperature, pH, time of reaction,
and molar concentration of reactants must be determined
to carry out the esterification reaction expecting higher
yield [20].
In the present study, the op timal yield of butyl feru late
(64 mM) was obtained at the molar concentration in fa-
vor of alcohol (100 mM butanol:50 mM ferulic acid) and
optimum temperature for ester formation by silica-
bound lipase was found to be 45˚C (62 mM yield). At
lower temperature (25˚C), a decreased conversion (43
mM) was observed. This suggested that at higher tem-
perature, the conversion rate is controlled by reaction
temperature. In contrast, at lower temperature, the re-
action rate is limited by mass transport phenomena. In-
crease or decrease in temperature of reaction mixture
might interfere with the porosity, hydrophobic character
and diffusion of the reactants and/or products at the cat-
alytic site of the enzyme [28]. In a recent study, the syn-
thesis of ethyl ferulate was carried out at equimolar con-
centration of ethanol and ferulic acid by celite-bound
lipase [29]. Butyl ferulate synthesis increased rapidly
during the early stage of reaction (0 - 6 h). Moreover,
concentration of biocatalyst, molecular sieves and the
selected salt ion showed cu mu lative effect to enhance the
yield of butyl ferulate in the reactio n system. The immo-
bilized lipase retained more than 50% of its original ac-
tivity after 3rd repetitive cycle of esterification. In the
present study, the immobilized lipase efficiently cata-
lyzed the esterification of butanol and ferulic acid into
butyl ferulate in a short period of 6 h at 45˚C under op-
timized conditions. The bound lipase had a greater sta-
bility/activity at enhanced temperature than the free li-
pase. Temperature had an important effect on the phy-
sical state of substrate dispersion also. Higher tem- pera-
ture and liquefaction tend to make the substrate more
diffusible and hence easily acceptable to the enzyme [30].
But ferulic acid is unstab le at high temperatures where it
undergoes oxidation [31]. Moreover, in the present study,
we have used silica-immobilized lipase to catalyze the
esterification of ferulic acid and butanol and interaction
among the various parameters was evaluated in such a
way to get the maximum yield of ester. In a previous
study, lipase immobilized onto on a poly (MAc-co-
DMA-cl-MBAm) hydrogel showed approximately 94%
binding capacity for lipase. It gave a higher yield for
both hydrolysis and esterification for the synthesis of
isopropyl myristate as compared to other polymers [32].
The esterification of butanol and ferulic acid by immo-

Enzymatic Synthesis of Butyl Ferulate by Silica-Immobilized Lipase in a Non-Aqueous Medium 406
bilized lipase was found to be maximum when molar
concentration of the hydrophobic reactant; akohol was
increased from 1:1 to 2:1 (butanol: ferulic acid) in the
reaction mixture. On the other hand, an increase in molar
concentration in favor of ferulic acid brought about a
decrease in the amount of butyl ferulate synthesized. It
appeared that such a decrease in the ester formation
might be because of change brought about by excess
concentration of ferulic acid that possibly alter the
charge distribution at the catalytic site that comprised a
triad of serine, aspartic (or glutamic) acid and histidine;
serine being a highly conserved residue in immobilized
lipase as reported previously in lipase sourced from Ba-
cillus spp. BTS-1 [33].
Esterification of butanol and ferulic acid by silica-
bound lipase in the presence of molecular sieves, and
certain salt ions (Cu2+, Zn2+ and Al3+) enhanced the ester
synthesis. Water that is often produced as a by-product
of the esterification reactions performed by biocatalysts
in organic media has several adverse effects on the reac-
tion and enzyme activity/performance. Esterification is
generally a water-limited reaction because the equilib-
rium catalyzed by hydrolytic enzymes is in favor of hy-
drolysis [34]. Moreover, water inhibits the catalytic reac-
tion besides when an immobilized en zyme with a suppo rt
which has hydrophilic nature is used, water causes ag-
gregation of support particles [35] resulting in a de-
creases in the rate of enzyme activity, as seen in the pre-
sent study. Esterification of butyl alcohol and ferulic acid
by silica-immobilized lipase of in the absence of a water
scavenger/molecular sieves exhibited approximately 66
mM esterification. Addition of molecular sieves rise up
the esterification rate up to 74 mM. The addition of mo-
lecular sieves usually improve the equilibrium conver-
sion [36,37] yet in many cases negative effects such as
the formation of di-ester and degradation of unstable
substrates have also been reported [38,39]. The effect of
various salt ions was also studied on the esterification
potential of immobilized lipase. Al3+ showing a rise in
the yield of butyl ferulate, whereas Mg2+, NH 4+, Fe2+ and
Ca2+ ions have mild inhibitory effect. In a previous study
on Burkholderia multivorans V2 the exposure to metal
ions such as Ca2+, Mg2+ and Mn2+ stimulated the lipase
activity while Cu2+, Fe2+ and Zn2+ antagonized the bio-
catalytic potential of lipase [19 ]. The inhibitory nature of
transition metals has been thought to be due to interac-
tion of ions with charged side chain groups of surface
amino acids, thus influencing the conformation and sta-
bility of the enzyme [40]. However, in present study
Cu2+, Zn2+ and Al3+ ions were found to have stimulatory
effect, which is in contradictio n to the abo ve observ ation ,
reported earlier [19]. Upon immobilization on to silica,
the lipase became quite stable and efficient in retaining
its activity up to four cycles of repetitive esterifications.
Further, when an attempt was made to scale up the re-
action volume to 30 ml to produce but yl ferulate, strangely
any increase in the reaction volume beyond 2 ml resulted
in a corresponding decline in the concentration of the
ester produced (32.6 mM) in the reaction mixture. It ap-
peared that a simple volumetric increase to achieve the
bulk production of the synthesized ester is not possible
and some other parameters might affect the rate of esteri-
fication in organic solvent system. Thus it might be pos-
sible that an increase in the surface area/the area of con-
tact of biocatalyst surface and or enhanced shaking
should be explored to see its effect on the increasing
yield of the ester in the volumetrically scaled up batch
reaction system. The main advantage of enzyme immo-
bilization is the reduced cost because lipase can be re-
peatedly used to achieve esterification over a couple of
fresh cycles of ester synthesis. The silica-bound lipase
when repeatedly used to perform esterification under
optimized conditions in DMSO resulted in 32 mM butyl
ferulate after 4th cycle of esterification.
5. Conclusions
In this work, we demonstrated the synthesis butyl fe-
rulate, a medically important ester that is used as an anti-
oxidant/UV blocking agent, antibacterial/anti-fungal agents,
cosmetics ingredient and an inducer of apoptosis in
mammalian tumor cells. The synthesis of butyl ester was
achieved successfully using silica-bound lipase in DM-
SO in a reaction time as small as 6 h under optimized
conditions of mole ratio, reaction temperature (45˚C)
under shaking in the presence of molecular sieves. The
silica-bound lipase could be repetitively used to perform
esterifications reactions up to 4th cycle. The scale up of
optimized reaction system possibly required further op-
timization of parameters like surface area to volume ra-
tion of the reaction system as well as agitation of the rea-
ction cocktail to achieve consistently higher yield of bu-
tyl ferulate.
6. Acknowledgements
This work has been funded under a Major Research Pro-
ject F. No. 34-269/2008 (SR) dated Dec. 30, 2008 awarded
to one of the authors [SSK] by University Grants Com-
mission, New Delhi, India; and the annual grant sanc-
tioned by Department of Biotechnology, Ministry of
Science & Technology, Government of India to De-
partment of Biotechnology, Himachal Pradesh Univer-
sity, Shimla, India.
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