Synthesis of alkyl coumarate esters by celite-bound lipase of Bacillus licheniformis SCD11501

Article abstract of DOI:10.1016/j.molcatb.2013.12.017

Coumaric acid and its derivatives are known to inhibit UV-induced melanogenesis, skin erythema, angiogenesis, platelet accumulation and osteoclastogenesis besides regulation of bone formation, anti-oxidant and anti-microbial activities. The present study is a novel piece of work in which the syntheses of a series of alkyl coumarates (methyl coumarate, ethyl coumarate, propyl coumarate and butyl coumarate) have been achieved using celite-immobilized lipase of Bacillus licheniformis strain SCD11501 in a water-free medium at 55 C under shaking in a chemical reactor. The maximum yield(s) of coumaric acid based esters i.e. methyl coumarate (69.0%), ethyl coumarate (63.1%), n-propyl coumarate (59.8%) and n-butyl coumarate (55.1%) using celite-immobilized lipase could be achieved after optimizing various reaction parameters such as incubation time, incubation temperature, relative molar concentration of reactants, biocatalyst concentration and amount of molecular sieves added to the reaction system. Molecular sieves had an important effect on the ester synthesis resulting in an enhanced yield. Maximum yield was recorded for methyl coumarate (69.0%) possibly because methyl group causes less stearic hindrance to the catalytic site of the lipase due to which it becomes more accessible for immobilized lipase to undergo esterification. The characterization of synthesized esters was done through FTIR spectroscopy and ¹H NMR spectra.

Full text of DOI:10.1016/j.molcatb.2013.12.017

Journal of Molecular Catalysis B: Enzymatic 101 (2014) 80–86
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Synthesis of alkyl coumarate esters by celite-bound lipase
of Bacillus licheniformis SCD11501
Shivika Sharmaa, Priyanka Dograb, Ghanshyam S. Chauhanb, Shamsher S. Kanwara,∗
a Department of Biotechnology, Himachal Pradesh University Summer Hill, Shimla 171 005, India
b Department of Chemistry, Himachal Pradesh University Summer Hill, Shimla 171 005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Coumaric acid and its derivatives are known to inhibit UV-induced melanogenesis, skin erythema, angio-
genesis, platelet accumulation and osteoclastogenesis besides regulation of bone formation, anti-oxidant
and anti-microbial activities. The present study is a novel piece of work in which the syntheses of a series of
alkyl coumarates (methyl coumarate, ethyl coumarate, propyl coumarate and butyl coumarate) have been
achieved using celite-immobilized lipase of Bacillus licheniformis strain SCD11501 in a water-free medium
at 55 ◦C under shaking in a chemical reactor. The maximum yield(s) of coumaric acid based esters i.e.
methyl coumarate (69.0%), ethyl coumarate (63.1%), n-propyl coumarate (59.8%) and n-butyl coumarate
(55.1%) using celite-immobilized lipase could be achieved after optimizing various reaction parameters
such as incubation time, incubation temperature, relative molar concentration of reactants, biocatalyst
concentration and amount of molecular sieves added to the reaction system. Molecular sieves had an
important effect on the ester synthesis resulting in an enhanced yield. Maximum yield was recorded for
methyl coumarate (69.0%) possibly because methyl group causes less stearic hindrance to the catalytic
site of the lipase due to which it becomes more accessible for immobilized lipase to undergo esterification.
The characterization of synthesized esters was done through FTIR spectroscopy and 1H NMR spectra.
© 2014 Elsevier B.V. All rights reserved.
Received 27 September 2013
Received in revised form
13 December 2013
Accepted 24 December 2013
Available online 13 January 2014
Keywords:
Coumaric acid
Bacillus licheniformis strain SCD11501
Lipase
Esterification
Immobilization
1. Introduction
4-Coumaric acid (4-CA) and its derivatives possess wide spec-
trum of anti-microbial and anti-oxidant activities [6]. It is also
Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) constitute a
group of enzymes defined as carboxylesterases that catalyse the
hydrolysis of long-chain acylglycerols at the lipid–water interface
[1]. Lipase is an important class of hydrolytic enzyme with innu-
merable applications and industrial potential. Lipases are produced
by animals, plants and microorganisms [2]. Immobilization of
enzymes is one of the useful techniques to improve the application
of enzymes at the industrial level [3]. For successful immobiliza-
tion, the matrix must has the mesh size large enough to allow
molecules of the substrate and of the reaction product to diffuse
in and out and to keep the enzyme entrapped in the matrix, too [4].
Immobilized enzymes can provide an easily separable and reusable
system (together with enhanced product recovery) which is often
also more resistant to deactivation by heat and/or denaturing sol-
vents as compared to the parent free enzyme [5].
a common dietary polyphenolic antioxidant that induces pro-
oxidant effects in human [7]. Coumaric acid (CA) can be obtained
from basic hydrolysis of coumarin through a reaction process con-
sisting of opening the lactone ring and cis–trans isomerization.
Coumarin derivatives also have diverse biological properties such
as enzyme inhibition, hypotoxicity, anti-carcinogenic and anti-
coagulant activity [8]. CA is known to suppress UV-induced
skin erythema and melanogenesis, inhibit angiogenesis, pre-
vent platelet aggregation, inhibit osteoclastogenesis and stimulate
bone formation. Through its free radical scavenging ability, 4-
CA prevents oxidative damage to bio-molecules and renders
organoprotection against drug-induced toxicities [9]. 4-CA: Coen-
zyme A ligase (4CL) is involved in monolignol biosynthesis for
lignification in plant cell walls. It ligates coenzyme A (CoA)
with hydroxycinnamic acids, such as 4-coumaric and caffeic
acids, into hydroxycinnamoyl-CoA thioesters [10]. CA, a common
dietary polyphenolic antioxidant, can dose-dependently induce
pro-oxidant effects in human endothelial cells [7]. 4-CA formulated
in a cream permeated skin tissue ex vivo and its topical application
mitigated UVB-induced inflammatory erythema and skin pigmen-
tation in vivo in human skin [11]. In animals and human the alkyl
∗
Corresponding author. Tel.: +91 177 2831948;
fax: +91 177 2831948/+91 177 2832153.
E-mail address: kanwarss2000@yahoo.com (S.S. Kanwar).
1381-1177/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.12.017

S. Sharma et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 80–86
81
coumarates are synthesized through the phenylpropanoid path-
2.3.1. Unit of lipase activity
way. The beneficial effects of alkyl coumarates have been attributed
to their antioxidant activity particular against oxidative attacks by
their radical-scavanging activity.
The unit (U) of enzyme activity was defined as ␮mole(s) of
p-nitrophenol released from p-NPP per min by one ml of free
enzyme or one gram of celite-immobilized enzyme (weight of
matrix included) under standard assay conditions.
Alkyl coumarate esters inhibit the oxidation of low-density
lipoproteins more efficiently and exhibited a higher antiradical
activity than coumaric acid, a natural antioxidant. In addition,
alkyl coumarates find extensive applications in chemical, phar-
maceutical and cosmetics industries [12]. Alkyl coumarates have
also been reported to inhibit several key molecular targets like
histone-deacetylase, mitogen-activated protein kinases, cancer
and immune disorders. Because of many imporatnet biological
properties of alkyl coumarates, the present work was undertaken to
optimize the reaction process parameters to efficiently synthesize
different C-chain length alkyl esters of coumaric acid in a water-
free medium employing celite-bound lipase of Bacillus licheniformis
SCD11501.
2.4. Esterification reaction
Coumaric acid esters were synthesized by using 1 M alco-
hol (methanol, ethanol, n-propanol or n-butanol), 1 M coumaric
acid and celite-bound purified lipase (63.9 U/g, hydrolysis reac-
tion activity) taken in Teflon coated glass-vials (20 ml capacity).
The reaction was performed at 55 ◦C for 10 h in a chemical reac-
tor under stirring. The syntheses (Fig. 1) of the methyl coumarate,
ethyl coumarate, n-propyl coumarate and n-butyl coumarate was
optimized by studying the effect of various physico-chemical
parameters such as incubation time, reaction temperature, rela-
tive molar concentration of reactants, biocatalyst concentration
and concentration of molecular sieves added to the reaction system
using celite-bound lipase. The formed esters were separated in dif-
ferent test tubes on the basis of their solubility in hot-water using
a separating funnel. The coumaric acid was soluble in hot water
while the formed esters (methyl coumarate, ethyl coumarate, n-
propyl coumarate and n-butyl coumarate formed in different test
tubes) were insoluble in hot water and were separated out using
a separating funnel. The amount of ester synthesized in each case
was determined and represented as % yield and characterized by
FTIR and NMR techniques.
2. Materials and methods
2.1. Materials
Celite 545 (S.D. Fine-Chem Ltd., Hyderabad, India); p-
nitrophenyl acetate (p-NPA), p-nitrophenyl benzoate (p-NPB),
p-nitrophenyl caprylate (p-NPC), p-nitrophenyl formate (p-NPF),
p-nitrophenyl laurate (p-NPL), p-nitrophenyl palmitate (p-NPP)
and glutaraldehyde (Lancaster Synthesis, England); molecular
˚
sieves (3 A × 1.5 mm), methanol, ethanol, n-propanol and n-butanol
(MERCK, Mumbai, India); Tris buffer and coumaric acid (Hime-
dia Laboratory, Ltd., Mumbai, India) were procured from various
commercial suppliers. All chemicals were of analytical grade and
were used as received. A chemical reactor with stirring and heat-
ing (MiniBlockTM, China) was used to perform ester synthesis using
celite-immobilized lipase of B. licheniformis SCD11501.
2.5. Optimization of reaction conditions for the synthesis of
methyl coumarate, ethyl coumarate, n-propyl coumarate and
n-butyl coumarate
Esterification was carried out by reacting coumaric acid with
methanol, ethanol, n-propanol or n-butanol separately in different
ratios in the presence of celite-bound lipase at different tempera-
tures for different time periods in a chemical reactor.
2.2. Microorganism
An aerobic, rod-shaped (cocco-bacillary), thermophilic
endospore forming bacterium of genus Bacillus was originally
isolated from hot springs of Tatapani, District: Mandi (Himachal
Pradesh), India. This bacterium was identified B. licheniformis
strain SCD11501 (GenBank Accession Number JN998712.1) by
Xcelris Labs Ltd., Ahmedabad-380054, India on the basis of 16S
RNA sequence analysis.
2.5.1. Effect of incubation time on synthesis of methyl coumarate,
ethyl coumarate, n-propyl coumarate and n-butyl coumarate
The reaction mixture (2 g) comprised of celite-immobilized
lipase (10 mg); coumaric acid (1 M): alcohol such as methanol,
ethanol, n-propanol or n-butanol (1 M) taken in different glass vials
in a water-free system. The glass vials were incubated at 55 ◦C
in a chemical reactor for 4, 6, 8, 10, 12 and 14 h under shaking.
The amount of ester synthesized in each case was determined and
represented as % yield in each case.
2.3. Purification and immobilization of lipase of Bacillus
licheniformis strain SCD11501
2.5.2. Effect of reaction temperature on the synthesis of methyl
coumarate, ethyl coumarate, n-propyl coumarate and n-butyl
coumarate
An extracellular solvent- and thermo-tolerant lipase produced
by B. licheniformis strain SCD11501 was purified (37.0 U/mg pro-
tein) to homogeneity by successive techniques of salting out
using ammonium sulphate, dialysis and DEAE anion-exchange
chromatography to 10.5-fold with an overall yield of 8.4% exper-
imental data not provided here). To avoid mixing of biocatalyst
with the product, the lipase was immobilized by physical adsorp-
tion (∼95% binding of protein/lipase) onto Celite-545 matrix and
immobilization of protein onto the matrix was stabilized by glu-
taraldehyde treatment/cross-linking. The celite-bound lipase was
completely dehydrated under vacuum in a freeze drier for 2 h and
this celite-bound lipase (63.9 U/g celite matrix) was used there-
after to perform bio-catalytic reactions in a chemical reactor under
stirring. The lipase activity of the celite-bound lipase-treated with
glutaraldehyde was comparatively higher than that of celite-bound
biocatalyst without glutaraldehyde treatment [13].
The effect(s) of reaction temperature (40, 45, 50, 55 and 60 ◦C)
on the synthesis of methyl coumarate, ethyl coumarate, n-propyl
coumarate and n-butyl coumarate were studied for 8 h under shak-
ing. The reaction mixture (2 g) comprised of celite-immobilized
lipase (10 mg); coumaric acid (1 M): alcohol such as methanol,
ethanol, n-propanol or n-butanol (1 M) taken in different glass vials
in a water-free system. The amount of esters synthesized was deter-
mined and represented as % yield in each case.
2.5.3. Effect of relative molar concentration of reactants on the
synthesis of methyl coumarate, ethyl coumarate, n-propyl
coumarate and n-butyl coumarate
The concentration of one of the reactants (coumaic acid)
was kept constant at 1 M and the concentration of the other

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S. Sharma et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 80–86
Methanol/ 55oC
HO
O
OCH
Methyl coumarate
HO
Ethanol/55oC
HO
O
O
OH
OC2H5
Ethyl coumarate
Coumaric acid
Propanol/55oC
Butanol/55oC
Propyl coumarate
But l couma ate
Fig. 1. Scheme of the synthesis of methyl coumarate, ethyl coumarate, n-propyl coumarate and n-butyl coumarate.
reactant (methanol, ethanol, n-propanol or n-butanol) was var-
ied (0.25 to 4 M) in the in a water-free system. The reaction
mixture (2 g) comprised of celite-immobilized lipase (10 mg);
coumaric acid (1 M): alcohol taken in different glass vials
and esterification was carried out at 55 ◦C for optimized time
(8 h) under continuous shaking. The amount of esters syn-
thesized was determined and represented as % yield in each
case.
reaction product(s). FTIR spectrum was recorded on Perkin Elmer
spectrophotometer in transmittance mode in KBr and NMR spec-
troscopy (INOVA 400 MHz spectrophotometer) using TMS as
internal standard in CHCl3.
3. Results and discussion
3.1. Optimization of reaction conditions for the synthesis of ethyl
coumarate, methyl coumarate, n-propyl coumarate and n-butyl
coumarate
2.5.4. Effect of amount of biocatalyst on synthesis of methyl
coumarate, ethyl coumarate, n-propyl coumarate and n-butyl
coumarate
The syntheses of methyl coumarate, ethyl coumarate, n-propyl
coumarate and n-butyl coumarate were studied by taking different
amount of immobilized lipase (0.5–3% of acid weight) in reac-
tion mixture (2 g) containing 1 M of coumaic acid: 1 M methanol,
ethanol, n-propanol or n-butanol in a water-free system at 55 ◦C
for 8 h under shaking. The amount of esters synthesized was deter-
mined and represented as % yield in each case.
Esterification was carried out by reacting coumaric acid and
methanol, ethanol, n-propanol or n-butanol in different molar
ratios in the presence of celite-bound lipase at different temper-
atures for different time periods in a chemical reactor.
3.2. Effect of incubation time on synthesis of methyl coumarate,
ethyl coumarate, n-propyl coumarate and n-butyl coumarate
The effect of reaction time on synthesis of esters of coumaric
acid using celite-immobilized lipase was studied at a temperature
of 55 ◦C under continuous shaking (Fig. 2) upto 14 h. Maximum
yield of methyl coumarate, ethyl coumarate, n-propyl coumarate
and n-butyl coumarate (64.3%, 60.6%, 54.4% and 49.1%, respectively)
was recorded at 8 h. The synthesis of coumaric acid ester(s) was
time dependant and methyl coumarate (64.3%) was produced max-
imally after 8 h of reaction. Thereafter is seems there was a loss of
activity of lipase that might be on account of extended exposure of
celite-bound biocatalyst because of heat-denaturation leading to
decreased esterification. In a previous study, reaction time of 6 h at
45 ◦C for immobilized-lipase was considered optimum for the syn-
thesis of butyl ferulate [14]. Earlier, a maximum molar conversion
of methyl butyrate and octyl acetate has been obtained at reaction
times of 14 and 12 h. After the specified time intervals (12 h for
octyl acetate and 14 h for methyl butyrate) the molar conversion
was relatively constant, which might be due to the attainment of
reactions at the equilibrium [15].
2.5.5. Effect of molecular sieves on the synthesis of methyl
coumarate, ethyl coumarate, n-propyl coumarate and n-butyl
coumarate
Molecular sieves (1% to 5% of reaction volume) were added to
the reaction system to study their effect on the synthesis of alkyl
coumarate using immobilized lipase (20 mg) in a reaction mixture
(2 g) containing 1 M of coumaric acid: 1 M methanol, ethanol, n-
propanol or n-butanol in a water-free system at 55 ◦C for 8 h under
shaking. The synthesized ester in each case was determined and %
yield was recorded.
2.5.6. Characterization of methyl coumarate, ethyl coumarate,
n-propyl coumarate and n-butyl coumarate
Synthesized esters i.e. methyl coumarate, ethyl coumarate,
n-propyl coumarate and n-butyl coumarate were characterized
by FTIR (Fourier transform infrared spectroscopy) and NMR
(Nuclear magnetic resonance) to get evidence of the formation of

S. Sharma et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 80–86
83
Fig. 4. Effect of relative molar concentration of reactants on the synthesis of methyl
coumarate, ethyl coumarate, n-propyl coumarate and n-butyl coumarate.
conformational structure which is stabilized when immobilized on
hydrophobic supports such as silica and celite. Hence, the immo-
bilized thermostable lipases provide a higher conversion rate;
minimal risk of microbial contamination; higher solubility of the
substrates and lower viscosity of the reaction medium. Thermosta-
bility is dependent on the structure of the enzyme, the environment
(solvent), pH, temperature, the presence of additives (organic
solvents, ions) and type of immobilization. In the present study,
the B. licheniformis SCD11501 that was used as a source of lipase
was found to possess a GC content of 55% on the basis of
its 16S rRNA nucleotide sequence thereby indicating the ther-
mophilic nature of the organism used as a source of lipase.
A maximum increase in the yield of esters of coumaric acid
was recorded at a temperature of 55 ◦C thereafter the yield
declined.
Fig. 2. Effect of incubation time on synthesis of methyl coumarate, ethyl coumarate,
n-propyl coumarate and n-butyl coumarate.
3.3. Effect of reaction temperature on the synthesis of methyl
coumarate, ethyl coumarate, n-propyl coumarate and n-butyl
coumarate
An increase in temperature of reaction mixture might interfere
with the porosity, hydrophobic character, diffusion of the reactants
and/or products at the catalytic site of enzyme as well as catalytic
activity of the enzyme. The reaction temperature above or below
55 ◦C decreased the production of esters (Fig. 3) of coumaric acid in
the solvent-free reaction system. This might be on account of dena-
turation of the lipase as well as alteration in the 3-D structure of
celite-bound lipase. The recorded yields of synthesized esters such
as methyl coumarate, ethyl coumarate, n-propyl coumarate and n-
butyl coumarate were 65.3%, 61.5%, 57.8% and 52.6%, respectively
at 55 ◦C in 8 h under continuous shaking. Any marked increase or
decrease in temperature of reaction mixture might interfere with
the biological activity of the enzyme added to the reaction sys-
tem [16]. Little information is available about the use of lipase in
the synthesis of alkyl esters of coumaric acid in literature. Previ-
ously, optimum temperature for butyl ferulate (62 mM) synthesis
in DMSO using silica-bound lipase was found to be 45 ◦C [14].
Generally, thermostable enzymes have a more rigid and packed
3.4. Effect of relative molar concentration of reactants on the
synthesis of methyl coumarate, ethyl coumarate, n-propyl
coumarate and n-butyl coumarate
The esterification was performed by varying the molar con-
centration (0.25, 0.5, 0.75, 1, 2, 3 or 4 M) of selected alcohol
(methanol, ethanol, n-propanol or n-butanol) while the concen-
tration of coumaric acid was kept constant (1 M) in a water-free
system. The molar ratio of 1:1 for alcohol and coumaric was found
to be optimum for the synthesis of methyl coumarate (66.0%)
Fig. 3. Effect of reaction temperature on the synthesis of methyl coumarate, ethyl
coumarate, n-propyl coumarate and n-butyl coumarate.
Fig. 5. Effect of amount of biocatalyst on synthesis of methyl coumarate, ethyl
coumarate, n-propyl coumarate and n-butyl coumarate.

84
S. Sharma et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 80–86
˚
Fig. 6. Effect of molecular sieves (3 A × 1.5 mm) on synthesis of methyl coumarate, ethyl coumarate, n-propyl coumarate and n-butyl coumarate.
and ethyl coumarate (61.9%) while maximum yield for n-propyl
coumarate (58.2%) and n-butyl coumarate (53.6%) was observed
at 1:2 molar concentrations of the reactants at 55 ◦C in 8 h under
continuous shaking (Fig. 4). Previously, it has been reported that
optimal synthesis of butyl acetate by lipase from Bacillus coagulans,
immobilized on nylon-6 was achieved when ester and alcohol were
used in equimolar ratio (100 mM) in the reaction mixture [17]. In
a recent study, the synthesis of ethyl ferulate was carried out in
DMSO at equimolar concentration of ethanol and ferulic acid by
celite-bound lipase [18]. Previously, maximum yield of biodiesel
was attained after 12 h at the stoichiometric molar ratio of 3:1 of
methanol and butyric acid using a bacterial lipase [19].
Fig. 7. FTIR results (a) methyl coumarate, (b) ethyl coumarate, (c) n-propyl coumarate and (d) n-butyl coumarate.

S. Sharma et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 80–86
85
3.5. Effect of amount of biocatalyst on synthesis of methyl
coumarate, ethyl coumarate, n-propyl coumarate and n-butyl
coumarate
Enzyme concentration is known to influence the trans-
esterification behavior. To establish the optimal amount of
celite-immobilized lipase that could be used for efficient
esterification reaction, different quantities of celite-bound-lipase
(0.5–3%; w/v) were used and ester synthesis was monitored by
FTIR and NMR. The maximum yield of methyl coumarate (68.2%)
and ethyl coumarate (63.3%) was detected at 1% concentration of
biocatalyst (Fig. 5). However, a concentration of 1.5% was optimal
for maximum yield of ester in case of propyl coumarate (58.8%) and
butyl coumarate (54.0%). In a recent study, 10 mg/mL (i.e. 1 g/mL)
of celite-bound lipase was found to give an optimum yield of ethyl
ferulate in DMSO [18].
3.6. Effect of molecular sieves on synthesis of methyl coumarate,
ethyl coumarate, n-propyl coumarate and n-butyl coumarate
When the reaction mixture was treated with varying amounts
(1% to 5% of reaction volume of 2 g) of molecular sieves to remove
traces of water molecules produced as by-product of the esterifi-
cation reaction at 8 h at 55 ◦C, a relatively higher amount of methyl
coumarate (69.0%), ethyl coumarate (63.1%), n-propyl coumarate
(59.8%) and n-butyl coumarate (55.1%) was recorded when the
molecular sieves were used at a concentration of 2% of reaction
mixture (Fig. 6). Any further addition of molecular sieves to the
reaction mixture prompted a decreased in the amount of ester
synthesized. Molecular sieves, a class of synthetic water scavengers
often improve the ester yield by withdrawing water molecules,
produced as by-product in the esterification reaction thus pro-
moting the forward reaction. Esterification is generally a water
limited reaction [20] and excess of water activity inhibits the for-
ward catalytic reaction in a water restricted/organic medium. The
formation of water also causes aggregation of support particles
resulting in a decrease in the rate of enzyme activity. Esterifica-
tion of isopropyl alcohol and ferulic acid by silica-immobilized
lipase in the absence of a water scavenger/molecular sieves
exhibited approximately 84% esterification [21]. The addition of
molecular sieves usually improved the equilibrium conversion
[18], yet in many cases negative effects such as the forma-
tion of diester and degradation of unstable substrates have also
been reported [22]. However, in the present study, addition of
molecular sieves to the reaction system modulated the ester
yields.
3.7. Characterization of methyl coumarate, ethyl coumarate,
n-propyl coumarate and n-butyl coumarate
Characterization of the coumaric acid esters (methyl coumarate,
ethyl coumarate, n-propyl coumarate and n-butyl coumarate) was
carried out by FTIR spectroscopy (Nicollet 5700) and 1H NMR spec-
tra (INOVA 400 MHz spectrophotometer) in deuterated chloroform
(CDCl3) solution with internal standard TMS (0 ppm) and chemical
shifts were recorded in parts per million (␦/ppm). FTIR spectrum of
methyl coumarate clearly showed a sharp peak at 1730 cm−1 which
was due to –C=O group of ester and when compared with the spec-
trum of coumaric acid this peak of ester was not present in it which
clearly confirmed that the lipase-catalyzed esterification reaction
had occurred (Fig. 7 a). The other peak(s) at value 2829.5 cm−1
clearly confirm C–H stretching which was due to methyl group pre-
sented in methyl coumarate. Again in next three spectrum of ethyl,
propyl and butyl coumarate the characteristic peaks of ester with
value 1730.2, 1739.9, 1736.6 cm−1 respectively confirmed the ester
synthesis as these were the peaks due to –C=O stretching of a ester
Fig. 8. NMR spectrum of (a) methyl coumarate, (b) ethyl coumarate, (c) n-propyl
coumarate and (d) n-butyl coumarate.

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S. Sharma et al. / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 80–86
Table 1
Characteristic peaks of various esters formed from coumaric acid.
S. No.
FTIR characteristics peaks
Functional groups
1H NMR peaks value in
Functional groups
Compound
Values in (cm−1
1693,3500
1730,1215.5, 2829.5
1730.21,172.32,939.9
)
Values in ppm
1.
2.
3.
4.
5.
Coumaric acid
–C=O, –OH
–COOH, benzene ring
12.05, 7.5, 6.5
3.89, 6.95, 7.5
2.3, 3.92, 6.95, 7.5
2.2, 2.3, 3.5, 6.95, 7.5
1.5, 2.2, 2.3,3.5, 6.95, 7.1
Methyl coumarate
Ethyl coumarate
n-Propyl coumarate
n-Butyl coumarate
–C=O, –O–C–O, –C–H
–C=O, –O–C–O, –C–H
–C=O, –O–C–O, –C–H
–C=O, –O–C–O, –C–H
–C=O, –C–H, benzene ring
–C–O–C2H5, benzene ring
–C–O–C3H7, benzene ring
–C–O–C4H9, benzene ring
1739.91,192.52,832.5
1736.3,1172.5,2991.2
group (Fig. 7b–d). The peaks at value near 2700–2900 cm−1 were
due to C–H stretching of ethyl, propyl and butyl group presented
in the respective esters.
of synthesized esters was done through FTIR spectroscopy and 1
NMR spectra.
H
The 1H NMR spectrum of coumarate series (methyl coumarate,
ethyl coumarate, n- propyl coumarate and n-butyl coumarate)
were compiled (Fig. 8). Various peaks corresponding to methyl
coumarate, ethyl coumarate, n-propyl coumarate and n-butyl
coumarate, respectively were recorded (Fig. 8a–d; Table 1). Spec-
trum of methyl coumarate has peaks with (␦/ppm) 3.95 (singlet, 3H
of ester group attached with CH3) and 6.95 (doublet, 1 H of benzene
ring; Fig. 8a). As the chain length of the ester increased the corre-
sponding peaks due to the methylene and methyl protons of the
alkyl group also appeared with ␦/ppm values in the range 3.2–1.9
confirming the synthesis of different esters with various alcohols.
For ethyl coumarate the signals due to –C2H5 and benzene ring
attained value at 2.3 (–CH3), 3.92 (–CH2), and 6.95 ppm (Fig. 8b).
Similarly, for n-propyl and n-butyl coumarate the peaks due to the
absorption by different protons of –C3H7 and –C4H9 ␦ values at 2.3
(–CH3), 2.2 (–CH2), 3.92 (–CH2) and 6.95 ppm and 1.5 (–CH2), 2.2
(–CH2), 2.3 (–CH2), 3.92 (–CH2), 6.95, respectively, were observed
(Fig. 8c and d).
Acknowledgements
The financial support from Department of Biotechnology,
Ministry of Science and Technology, Government of India to Depart-
ment of Biotechnology, Himachal Pradesh University, Shimla
(India) is thankfully acknowledged. Moreover, the authors do not
have any conflict of interest amongst themselves or with their par-
ent institution.
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