Green synthesis of isoamyl acetate via silica immobilized novel thermophilic lipase from Bacillus aerius

Article abstract of DOI:10.1134/S1068162016010118

Isoamyl acetate, a pear or banana flavor, is widely used in food, beverage, cosmetic, and pharmaceutical industries. In the present work, lipase from Bacillus aerius was immobilized on silica gel matrix in the presence of a cross-linking agent, glutaraldehyde, and its efficiency in synthesizing isoamyl acetate using esterification reaction was studied. The esterification of acetic acid and isoamyl alcohol by silica-bound lipase was studied as a function of time and temperatures. The incubation time of 10 h, temperature of 55°C, substrate molar ratio 1: 1, and the amount of lipase as 1% were found to be optimal for the esterification reaction. The bound lipase catalyzed the esterification of acetic acid by isoamyl alcohol with the yield of about 68% under the optimized reaction conditions. The product was identified as isoamyl acetate using gas-liquid chromatography, nuclear magnetic resonance, and Fourier transform IR spectroscopy analysis by the presence of an ester group at the wavenumber of 1720.5 cm^-1.

Full text of DOI:10.1134/S1068162016010118

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2016, Vol. 42, No. 1, pp. 69–73. © Pleiades Publishing, Ltd., 2016.
Green Synthesis of Isoamyl Acetate via Silica Immobilized Novel
Thermophilic Lipase from Bacillus aerius1
2
Sunil Kumar Narwala, Nitin Kumar Sauna, Priyanka Dograb, and Reena Guptaa,
aDepartment of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, 171005 India
bDepartment of Chemistry, Himachal Pradesh University, Summer Hill, Shimla, 171005 India
Received January 15, 2015; in final form, September 1, 2015
Abstract—Isoamyl acetate, a pear or banana flavor, is widely used in food, beverage, cosmetic, and pharmaꢀ
ceutical industries. In the present work, lipase from Bacillus aerius was immobilized on silica gel matrix in the
presence of a crossꢀlinking agent, glutaraldehyde, and its efficiency in synthesizing isoamyl acetate using
esterification reaction was studied. The esterification of acetic acid and isoamyl alcohol by silicaꢀbound lipase
was studied as a function of time and temperatures. The incubation time of 10 h, temperature of 55°C, subꢀ
strate molar ratio 1 : 1, and the amount of lipase as 1% were found to be optimal for the esterification reaction.
The bound lipase catalyzed the esterification of acetic acid by isoamyl alcohol with the yield of about 68%
under the optimized reaction conditions. The product was identified as isoamyl acetate using gasꢀliquid chroꢀ
matography, nuclear magnetic resonance, and Fourier transform IR spectroscopy analysis by the presence of
an ester group at the wavenumber of 1720.5 cm–1.
Keywords: lipase, immobilization, esterification, isoamyl acetate
DOI: 10.1134/S1068162016010118
INTRODUCTION
anionic exchange resin beads [4] and Rhizopus oryzae
NRRL 3562 lipase immobilized on silica gel matrix [5].
Lipase (triacylglycerol ester hydrolase, EC 3.1.1.3)
is an efficient enzyme with a broad variety of applicaꢀ
tions in the food industry, fine chemistry, and pharmaꢀ
ceutical industry. It catalyzes the hydrolysis of triaꢀ
cylglycerol to glycerol and fatty acids [1]. Lipases are
successfully used for the synthesis of esters. Flavor
esters are synthesized by the reaction between short
chain acids and alcohols.
Isoamyl acetate (3ꢀmethylꢀ1ꢀbutyl ethanoate) is
often called the essence of pear or banana and found
naturally in these fruits. It has a highly characteristic
smell and is one of the most frequently used fragrance
material in food, beverage, cosmetic, pharmaceutical
industries, and household products. Due to its strong
odor even at low concentrations and low toxicity,
isoamyl acetate is well known as a chemical used for
testing gas masks for tightness [6]. Isoamyl acetate is
traditionally produced by extraction of banana flavor
from natural sources, which results in difficulties with
its supply. Though the fermentation was proposed as
the alternative way, but, in the practice, this method
turned out to be too scarce and expensive for commerꢀ
cial exploitation [7].
Though esters are commonly produced using
chemical catalysts (acidic or basic by their nature)
there are certain limitations which restrict the usage of
these catalysts, such as the necessity to remove inorꢀ
ganic salts, high temperature, and side reactions
occurring along with the esterification [2]. Due to
enzyme specificity, the enzymatic catalysis ensures
formation of pure products. The cost of lipase producꢀ
tion is one of the main obstacles for industrial applicaꢀ
tion of lipases. Immobilization of lipases decreases the
cost of production owing to their reusability, that
makes them more potent and attractive for industrial
applications, together with better thermal stability,
activity in nonꢀaqueous media, improved handling,
recovery, and recycling the biocatalyst [3]. Previously,
isoamyl acetate was synthesized using Rhizomucor
miehei lipase immobilized on macroporous weak
The enzyme catalysis in industrial applications
requires the development of efficient processing methꢀ
ods and optimization of reaction conditions. In the
present work, the extracellular thermoalkaliphilic
purified lipase of Bacillus aerius has been immobilized
by adsorption onto activated silica matrix using glutꢀ
araldehyde as a crosslinking agent. Immobilized
enzyme was then used for synthesis of isoamyl acetate
by esterification of acetic acid with isoamyl alcohol.
The effects of incubation time, reaction temperature,
relative molar concentration of reactants, and enzyme
concentration on the rate of synthesis of isoamyl aceꢀ
tate were separately evaluated. The synthesized ester
1
The article is published in the original.
Corresponding author: eꢀmail: reenagupta_2001@yahoo.com.
2
69

70
SUNIL KUMAR NARWAL et al.
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
4
6
8
10
12
40
45
50
55
60
Incubation time, h
Temperature,
°
C
Fig. 2. Effect of temperature on the synthesis of isoamyl
acetate. Reaction conditions: reaction time 10 h, acetic
acid/isoamyl alcohol ratio of 1 : 1 (mol/mol), and 1%
lipase by acid weight.
Fig. 1. Timeꢀcourse of isoamyl acetate synthesis at 55°C.
Reaction conditions: acetic acid/isoamyl alcohol ratio of
1 : 1 (mol/mol); 1% lipase by acid weight.
was characterized by FTꢀIR, NMR, and GLC techꢀ esterification of acrylic acid with methanol into
niques.
methyl acrylate in a shorter period of 6 h at 55°C [12].
RESULTS AND DISCUSSION
Effect of Reaction Temperature
Immobilization of the Lipase onto Silica
Changes in the reaction temperature can affect the
activity and stability of the enzyme and thus the rate of
the reaction. The maximum ester synthesis (68.38%)
Lipase from Bacillus aerius gave maximum binding
efficiency of 75.64% with 1.44 U/mg of enzyme activꢀ
ity on silica matrix when crossꢀlinked with 4% of gluꢀ
taraldehyde solution (v/v). The immobilization kinetꢀ
ics revealed that the lipase was optimally immobilized
on silica matrix within 2 h of incubation. Previously,
the binding efficiency of 75% was observed when
lipase from Bacillus coagulans was immobilized on
nylonꢀ6 [8].
was observed at 55°C (Fig. 2) under the above reaction
conditions. A rise in temperature increases the activity,
however, at high temperatures enzyme becomes denaꢀ
tured, so that its activity decreases. Gogoi and Dutta
have found a reaction temperature of 65°C to be optiꢀ
mal for the synthesis of isoamyl acetate using immobiꢀ
lized lipase from Mucor miehei [6]. A reaction temperꢀ
ature of 65°C was considered optimal for synthesis of
4ꢀnitrophenyl acetate by immobilized Bacillus coaguꢀ
lans lipase [12]. In another study, the optimal temperꢀ
ature for the synthesis of ethyl ferulate using celiteꢀ
Effect of Incubation Time
The study of the time course of an enzymatic reacꢀ
tion is a good indicator of enzyme performance and
reaction progress. It can pinpoint the shortest or adeꢀ
quate time necessary to obtain a good yield and miniꢀ
mize the process cost. The yield of isoamyl acetate
increased steeply with the time during early stages of
the esterification reaction to attain the major part of
bound commercial lipase was found to be 45°C [13].
Effect of Relative Molar Concentration of the Reactants
In esterification reactions, the rate of reaction can
the final conversion in 10 h (Fig. 1). At 10 h, approxiꢀ be pushed forward either by using excess molar quanꢀ
mately 68.55% of ester was produced. Thus, in the tities of nucleophile (alcohol) or by removing product
subsequent experiments, a reaction time of 10 h was from the reaction mixture. The equimolar ratio of aceꢀ
considered the optimum for the ester synthesis at tic acid and isoamyl alcohol in a solventꢀfree system
55°C for the immobilizedꢀlipase. Longer reaction was optimal to yield isoamyl acetate (68.38%) in the
time leads to the reduction of ester formation because reaction (Fig. 3). Kumar et al. have found the reacꢀ
of the esterification reaction reversal [9]. Kumar et al. tants molar ratio of 1 : 3 (ethanol : propionic acid) to
found optimum incubation time of 12 h for the synꢀ be optimumal for the synthesis of ethyl propionate in
thesis of ethyl propionate using lipase from Bacillus hexane [10]. The optimal synthesis of butyl acetate by
coagulans BTSꢀ3 [10]. A reaction time of 3 h for lipase from Bacillus coagulans immobilized on Nylonꢀ6
immobilized lipase from Bacillus coagulans BTSꢀ3 was was achieved when the acid and alcohol were used in
considered optimum for synthesis of 4ꢀnitrophenyl equimolar ratio (at 100 mM) in the reaction mixture
acetate [11]. In a previous study, immobilization of [8]. Earlier Kumari et al. have reported that the molar
Pseudomonas aeruginosa lipase onto a synthetic polyꢀ ratio of 3 : 1 was optimal for the synthesis of isoamyl
(
AAcꢀcoꢀHPMAꢀclꢀEGDMA) hydrogel catalyzed the acetate using lipase from Rhizopus oryzae [5].
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 1
2016

GREEN SYNTHESIS OF ISOAMYL ACETATE
71
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
0.5
1.0
1.5
2.0
1 : 1
1 : 2
1 : 3
1 : 4
Amount of catalyst, %
Molar ratio, mol/mol
Fig. 4. Effect of lipase concentration on the synthesis of
isoamyl acetate.
Fig. 3. The effect of acetic acid/isoamyl alcohol molar
ratio on the esterification reaction. Reaction conditions:
temperature 55
10 h.
°
C
, 1% lipase by acid weight, reaction time
Reaction conditions: reaction time 10 h, temperature
55°C, acetic acid/isoamyl alcohol ratio of 1 : 1 (mol/mol).
Effect of the Biocatalyst Amount
ing). The 1H NMR spectrum of the ester had peaks at
2.21 ppm due to methyl group protons confirming the
presence of a –C=O group adjacent to these protons.
Signals at 4.6, 1.52, and 1.81 ppm were due to protons
of isoamyl group in the vicinity of the ester group. A
doublet at 0.91 ppm was due to two protons in the
same environment.
The amount of enzyme plays a crucial role in any
biocatalytic process, especially for large scale producꢀ
tion. The influence of this parameter on the reaction
was therefore assessed to determine the minimal
amount of the lipase required for achieving good yield
of the product. We observed that with an increase of
enzyme concentration the initial rate of esterification
was increasing almost linearly at low lipase concentraꢀ
tions. Then the rate started to decrease slowly and after
reaching the critical value it remained unchanged. The
maximum yield of isoamyl acetate (68.38%) was
obtained with 1% of immobilized lipase (Fig. 4). As
reported for the synthesis of isoamyl acetate, 16% of
immobilized lipase from Rhizopus oryzae was used to
get maximal yield of the ester [5]. Recently it has been
found that 5.78% of immobilized lipase from Candida
antarctica was optimal for the synthesis of isoamyl
acetate [7].
EXPERIMENTAL
General
Silica gel matrix (pore size 60–150 mesh), glutaralꢀ
dehyde,
nyl benzoate
ꢀnitrophenyl palmitate (
p
ꢀnitrophenyl acetate
ꢀNPB), ꢀnitrophenylformate
ꢀNPP), and acetic acid
(
p
ꢀNPA),
p
ꢀnitropheꢀ
(p
p
(pꢀNPF),
p
p
were purchased from Lancaster Synthesis, England;
Tris buffer was from HIMEDIA Laboratory Ltd.,
Mumbai, India; and isoamyl alcohol was from
MERCK, Mumbai, India. All chemicals were of anaꢀ
lytical grade and were used as received.
Reusability
Preparation of Lipase from Bacillus aerius
The residual activity of silicaꢀbound lipase was calꢀ
culated after every cycle to estimate the inactivation of
the immobilized lipase. Silicaꢀbound lipase retained
almost 50% activity after five cycles (table). In a previꢀ
ous study, the lipase from Bacillus coagulans immobiꢀ
lized on a molecular sieve retained 58.5% of its original
activity after five cycles of esterification [11]. The comꢀ
mercial lipase immobilized on silica gel matrix retained
almost 52% activity after five cycles of reuse [14].
The lipase producing bacteria were isolated from
the soil and water samples of a hotꢀspring named Tatꢀ
tapani, Kullu, and sweet shops around Shimla. The
thermophilic Bacillus aerius (identified at IMTECH,
Reusability of immobilized lipase in ester synthesis
Cycle No.
Relative yield, %
1
2
3
4
5
6
100
Spectral Data
83.24
71.57
63.21
50.18
41.98
The structure of the target product isoamyl acetate
was confirmed by the data of FTꢀIR spectra and H
NMR spectrum. FTꢀIR spectrum showed the peaks at
1720.5 cm–1 (–C=O stretching), 1246.9 cm–1 (–C–
O–C– stretching), and 2959.9 cm–1 (–C–H stretchꢀ
1
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 1
2016

72
SUNIL KUMAR NARWAL et al.
Chandigarh) was grown in the medium of the followꢀ
ing composition (g/L): yeast extract, 2.0; peptone,
5.0; sodium chloride, 5.0; beef extract, 1.5; and
ammonium chloride, 1.0, supplemented with cottonꢀ
seed oil (10 mL/L) emulsified with 0.5% Gum arabic)
at pH 8.5. The seed culture (7.5% inoculum) was
transferred into 50 mL production medium (250ꢀmL
Erlenmeyer flask) and kept for 48 h under shaking
Esterification Reaction
Isoamyl alcohol (1 M) and acetic acid (1 M) were
taken in a screwꢀcapped glass vial. To this mixture, the
enzyme (Bacilus aerius lipase, ~1.44 U/mg) was added
as a catalyst and the reaction mixture was incubated
with constant shaking. The effect of reaction time (4,
6, 8, 10, and 12 h), temperature (40 to 60°C), acetic
acid to isoamyl alcohol molar ratio (1 : 1 to 1 : 4) and
immobilized lipase amount (1–4% of acid weight) on
ester synthesis were investigated in a solvent free reacꢀ
tion system. The reaction mixture was washed with hot
water to remove the reactants using separating funnel.
The silica bound lipase of Bacillus aerius was used
repetitively upto 6th cycle of esterification. After the
first cycle of reaction, the immobilized lipase was
recovered (by centrifuging and decanting the reaction
mixture) and this biocatalyst was used to catalyze the
fresh reaction.
conditions at 110 rpm at 55
centrifuged at 10000 rpm for 10 min at
°
C
. The culture broth was
4°C. The lipase
activity was assayed both in the supernatant and pellet
for determining extracellular and intracellular enzyme
activity, respectively. The enzyme produced by therꢀ
mophilic Bacillus aerius was purified to 9ꢀfold with
7.2% recovery by ammonium sulfate precipitation and
chromatography on a DEAEꢀcellulose column (size
12
×
2 cm) activated with 0.1 N NaOH and 0.1 N
NaCl. Subsequently the column was equilibrated with
0.1 M TrisꢀHCl buffer (pH 9.5). The dialyzed sample
(3 mL) was loaded on the column and eluted with
0.1 M TrisꢀHCl (pH 9.5) containing NaCl gradient of
0.3, 0.5, 0.7, and 1 M. According to the data of SDSꢀ
PAGE, the enzyme was obtained in a monomeric form
with molecular weight of 33 kDa.
The ester was quantified by FTꢀIR and NMR specꢀ
troscopy and GLC. FTꢀIR spectra were recorded on a
Nicollet 5700 apparatus in KBr pellets. 1H NMR specꢀ
trum was registered on an Advance Bruker IIꢀ400 MHz
in deuterated chloroform (CDCl3) solution with TMS
as an internal standard (0 ppm) and chemical shifts
recorded in parts per million ( , ppm). GLC apparatus
δ
Chrom WHP was equipped with a flame ionization
detector and a column (10% SEꢀ30, 2 m, mesh size
80–100, internal diameter 1/8 inches, maximum temꢀ
Determination of Lipase Activity
The activity of free and silicaꢀbound lipase was
measured by a colorimetric method [15]. The reaction
perature limit 300
India); nitrogen was used as
30 cm3/min); the injector was warmed to 250
the detector was set at 280 . The quantification was
°
C
; Netel Chromatograph, Thane,
carrier gas
and
mixture (3 mL) contained 60
palmitate ( ꢀNPP) stock solution (20 mM
prepared in isopropyl alcohol) and 40 lipase and
Tris buffer (0.1 M, pH 9.5). The mixture was incubated
at 55 for 10 min on a water bath. The reaction was
stopped by cooling the reaction mixture at –20 for
μ
L
of
pꢀnitrophenol
a
p
p
ꢀNPP
)
(
°C
μL
°C
accomplished using isoamyl acetate as internal stanꢀ
dard. The ester yield was determined using GLC as
follows:
°C
°
C
10 min. The absorbance of pꢀnitrophenol released was
measured at 410 nm. The enzyme activity was defined
Weight of ester formed (g)
% yield = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ × 100.
as the micromoles of
minute on hydrolysis of
p
ꢀnitrophenol released per
Total weight of reaction mixture (g)
pꢀNPP by 1 mL of free
enzyme or by 1 mg of silicaꢀbound enzyme (weight of
matrix included) under standard assay conditions. The
protein was assayed by a standard method [16].
ACKNOWLEDGMENTS
The financial support from Department of Bioꢀ
technology, Ministry of Science and Technology, Govꢀ
ernment of India, to Department of Biotechnology,
Himachal Pradesh University, Shimla (India) is
thankfully acknowledged.
Immobilization of Lipase onto Silica
Two grams of silica gel matrix (60–150 mesh) was
washed with 0.1 M Tris buffer (pH 7.0) and then cenꢀ
trifuged at 10000 rpm at 4°C for 10 min. The supernaꢀ
tant was discarded and the pellet was washed out 4–
5 times with Tris buffer. The matrix was then kept at
REFERENCES
1. Kharrat, N., Ali, Y.B., Marzouk, S., Gargouri, Y.T.,
and Chaabouni, M.K., Process Biochem., 2011, vol. 46,
pp. 1083–1089.
2. Anschau, A., Aragao, V.C., Porciuncula, B.D.A.,
Kalil, S.K., Burkert, C.A.V., and Burkert, J.F.M.,
J. Braz. Chem. Soc., 2011, vol. 22, pp. 2148–2156.
3. Narwal, S.K. and Gupta, R., Biotechnol. Lett., 2013,
vol. 35, pp. 479–490.
4. Divakar, S.H.S., Prapulla, S.G., and Karanth, N.G.,
4
°
C
overnight in Tris buffer. Then 1–5% (v/v) glutꢀ
araldehyde solution was added to the matrix and the
mixture was kept at 55 under shaking for different
°C
time periods (1–5 h). The matrix was washed 3–
4 times with Tris buffer (pH 7.0) to remove unbound
glutaraldehyde. Four milliliter of lipase from Bacillus
aerius (1.91 U/mL) was then incubated with the
matrix for 1 h under shaking condition. The supernaꢀ
tant was discarded.
J. Biotechnol., 2001, vol. 87, pp. 193–201.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 1
2016

GREEN SYNTHESIS OF ISOAMYL ACETATE
73
5. Kumari, A., Mahapatra, P., Garlapati, V.K., 11. Raghuvanshi, S. and Gupta, R., J. Indus. Microbiol.
Banergee, R., and Dasgupta, S., Food Technol. Biotechꢀ
nol., 2009, vol. 47, pp. 13–18.
Biotechnol., 2009, vol. 36, pp. 401–407.
12. Kanwar, S.S., Verma, M.L., Maheshwari, C., Chauꢀ
han, S., Chimni, S.S., and Chauhan, G.S., J. App.
Polym. Sci., 2007, vol. 104, pp. 4636–4644.
6. Gogoi, S. and Dutta N.N., Ind. J. Chem. Technol.
2009, vol. 16, pp. 209–215.
,
7. Azudin, N.Y., Don, M.M., and Shukor, S.R.A., Chem.
Eng. Trans., 2013, vol. 32, pp. 1057–1062.
8. Pahujani, S., Shukla, S.K., Bag, B.P., Kanwar, S.S.,
and Gupta, R., J. App. Polym. Sci., 2007, vol. 106,
pp. 2724–2729.
13. Kumar, A. and Kanwar, S.S., Bioresour. Technol., 2011,
vol. 102, pp. 2162–2167.
14. Narwal, S.K., Saun, N.K., and Gupta, R., J. Oleo Sci.
2014, vol. 63, pp. 599ꢀ605.
,
9. Eevera, T., Rajendran, K., and Saradha, S., Renewable
Energy, 2009, vol. 34, pp. 762–765.
10. Shukla, S.K., Pahujani, R., Ola, P., Kanwar, S.S., and
15. Winkler, W.K. and Stuckmann, M., J. Bacteriol., 1979,
vol. 138,pp. 663–670.
Gupta, R., Acta Microbiol. Immunol. Hungar., 2006, 16. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Ranꢀ
vol. 53, pp. 217–229.
dall, R.J., J. Biol. Chem., 1951, vol. 193, pp. 265–275.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 42
No. 1
2016