Synthesis of isobutyl propionate using immobilized lipase in a solvent free system: Optimization and kinetic studies

Article abstract of DOI:10.1016/j.molcatb.2013.10.024

Isobutyl propionate is widely used in food and beverage industries as a rum flavor. This work presents the optimization and kinetic aspects of synthesis of isobutyl propionate by esterification of propionic acid with isobutyl alcohol using immobilized lipase Novozym 435 in a solvent free system (SFS). Process parameters such as reaction time, temperature, enzyme loading, speed of agitation, water concentration and acid to alcohol molar ratio were optimized to achieve maximum conversion. Higher conversion of 92.52% was obtained with the reaction conditions such as: temperature 40 C, enzyme loading 5% w/w, acid to alcohol molar ratio 1:3, time 10 h and stirring speed of 300 rpm. The bisubstrate kinetic models of the enzyme catalyzed reactions namely Ordered Bi-Bi, Random Bi-Bi and Ping-Pong Bi-Bi were applied to determine the initial rates and correlated with the experimental findings. Ping-Pong Bi-Bi model with substrate inhibition by both acid and alcohol gives the best fit with parameter values as V_max = 0.5 Mol/min/g catalyst, K_A = 0.631 M, K_B = 0.003 M, K_iA = 0.0042 M and K_iB = 0.1539 M for the concentration ranges of 2.25-10.21 M for propionic acid and 2.55-9.01 M for iso-butanol. The immobilized lipase could be reused for seven times with the % conversion of acid reaching to 83%; signifies that still it can be reused for several more times. SFS is the added benefit to produce such commercially valuable flavor ester.

Full text of DOI:10.1016/j.molcatb.2013.10.024

Journal of Molecular Catalysis B: Enzymatic 99 (2014) 143–149
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Synthesis of isobutyl propionate using immobilized lipase in a solvent
free system: Optimization and kinetic studies
∗
Vishakha V. Kuperkar, Vikesh G. Lade, Arushi Prakash, Virendra K. Rathod
Department of Chemical Engineering, Institute of Chemical Technology, Matunga (E), Mumbai 400019, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2013
Received in revised form 30 October 2013
Accepted 30 October 2013
Available online 8 November 2013
Isobutyl propionate is widely used in food and beverage industries as a rum flavor. This work presents the
optimization and kinetic aspects of synthesis of isobutyl propionate by esterification of propionic acid
with isobutyl alcohol using immobilized lipase Novozym 435 in a solvent free system (SFS). Process
®
parameters such as reaction time, temperature, enzyme loading, speed of agitation, water concentration
and acid to alcohol molar ratio were optimized to achieve maximum conversion. Higher conversion of
◦
9
2.52% was obtained with the reaction conditions such as: temperature 40 C, enzyme loading 5% w/w,
Keywords:
acid to alcohol molar ratio 1:3, time 10 h and stirring speed of 300 rpm. The bisubstrate kinetic mod-
els of the enzyme catalyzed reactions namely Ordered Bi–Bi, Random Bi–Bi and Ping-Pong Bi–Bi were
applied to determine the initial rates and correlated with the experimental findings. Ping-Pong Bi–Bi
model with substrate inhibition by both acid and alcohol gives the best fit with parameter values as
Vmax = 0.5 Mol/min/g catalyst, KA = 0.631 M, KB = 0.003 M, KiA = 0.0042 M and KiB = 0.1539 M for the concen-
tration ranges of 2.25–10.21 M for propionic acid and 2.55–9.01 M for iso-butanol. The immobilized lipase
could be reused for seven times with the % conversion of acid reaching to 83%; signifies that still it can
be reused for several more times. SFS is the added benefit to produce such commercially valuable flavor
ester.
Isobutyl propionate
Novozym® 435
Solvent free system (SFS)
Kinetic models
Ping-Pong Bi–Bi mechanism
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
unlike using chemical catalysts. Application of enzymes as a bio-
catalyst is the most frequently used technique of biosynthesis to
produce the flavors with high specificity, mild reaction conditions
and greater efficiency [5]. Lipases (triacylglycerol ester hydrolysis
EC 3.1.1.3) are enzymes that can catalyze esterification, transesteri-
fication, and hydrolysis reactions [6].
Lipases are important class of enzymes because of their
properties like regiospecificity, stereospecificity and substrate
specificity [7] and their milder reaction conditions that reduce the
energy requirements [8]. Recently, lipases immobilized on vari-
ous supports like macroporous acrylic resin [9] polyacryclic resin,
polyurethane foams [5], Amberlite and Celite [10] and cotton cloth
[11] have come up for the industrial production of various specialty
esters, aroma compounds and active agents. These immobilized
techniques have provided more chances to use biocatalysts for var-
ious reactions in wider range of operating conditions in terms of pH,
temperature and pressure [12,13]. Novozym® 435 is commercial
immobilized lipase preparation supplied by Novozymes, immobi-
lized on macroporous polyacrylic resin beads. It has applications
ranging from biodiesel production to fine chemistry [14].
The esterification is a reaction wherein alcohol reacts with
acid in presence of catalyst to produce ester with elimination
of water. Short-chain esters are the class of compounds that are
widely distributed in nature and are major components of cos-
metics, food flavor, fragrance, pharmaceutical industries due to
its natural aroma. Currently commercial market for food flavor is
increasing rapidly. Esters obtained by chemical synthesis suffer
from drawbacks like high temperature and pressure, harsh reac-
tion conditions involving strong acid catalyst, hazardous chemicals,
longer reaction time and low conversion [1]. Other flaws associated
are tedious separation processes, toxicity, and unwanted harmful
byproducts [2]. Natural flavors extracted from fruits or plants are
too expensive and also incapable of fulfilling growing commercial
demands [3]. Therefore it is industrially and economically impor-
tant to synthesize flavors using cheaper and more broadly available
material to meet the consumer demand exploring the alternative
methods of the production [1,4]. Use of biocatalyst for the pro-
duction of flavor is green technology based approach and natural,
The variant shift has occurred in the lipase catalyzed reactions
based on the operating media viz. aqueous → biphasic → non-
aqueous media (organic). Since, major emphasis was given to
organic solvents to produce short-chain fatty acids ester in middle
∗ Corresponding author. Tel.: +91 22 33612020; fax: +91 22 33611020.
E-mail address: vk.rathod@ictmumbai.edu.in (V.K. Rathod).
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.10.024

1
44
V.V. Kuperkar et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 143–149
decades; lot of literature exists with combinations of substrates
with experimental and/or statistical determination of optimum
reaction conditions for maximum yield in shortest duration
directly without any solvent. If equimolar (5.98 Mol) mixture of
both reactants [A] and [B] solution has to be made, the reaction
mixture should contain [A] 7.5 cm3 and [B] 9.3 cm . Accordingly
all reaction mixtures were made based on predefined molar ratio.
Liquid samples free from catalyst particles were withdrawn period-
ically and further analyzed to determine the extent of reaction. The
procedure was repeated based on criterion for the optimization of
reaction parameter (reaction time, temperature, enzyme loading,
speed of agitation, concentration of water and molar ratio).
3
[
3,14–18]. However the use of toxic organic solvents is being pro-
gressively restricted for many applications due to industrial and
social implications. Recently the major shift has occurred in the
production of the esters where in the reactions are preferred in the
solvent free system; as it facilitates the downstream processing
thus reduction in cost and environmental hazards [2]. There are
few research studies concerning solvent free system for lipase cat-
alyzed production of flavor ester and it is found that the initial rates
are found to increase as compared to organic solvent [18,19].
The kinetic studies can provide better insight of the reaction
mechanisms of enzyme catalyzed reactions. The kinetics of the spe-
cific reaction can follow the specific kinetic model based on its
reaction mechanism. The kinetics information (operating condi-
tions and rate parameters) of the esterification reaction is useful for
the designing and scale-up of the reactor. However, kinetic stud-
ies of lipase-catalyzed esterification in organic solvents or SFS are
remarkably rare; most of these are based on the Michaelis–Menten
assumptions [5]. By virtue of the importance of kinetic mod-
els, the proper assessment of the dynamics of lipase-catalyzed
esterification reactions has been done using several models for
different combinations of substrates and enzymes over the years.
Most lipases are said to follow the Ping-Pong Bi–Bi mechanism
2
2
.3. Analytical method
.3.1. Identification of reaction product
Identification of synthesized isobutyl propionate in liquid sam-
ples was carried out by GC (CHEMITO 8610) equipped with flame
ionization detector using 3 m × 0.32 mm I.D. stainless steel column
packed with 10% OV-17 stationary phase. Nitrogen was used as car-
rier gas at pressure 0.8 bar. The temperature program was as follow:
◦
◦
◦
6
1
1
0 C for 1 min; 5 C/min up to 100 C; then steady temperature for
min. The injector and detector temperatures were both kept at
50 C. Injection volume of 2 ␮l was used. After primary identifi-
◦
cation on GC, titrimetric analysis (method explained below) was
used for routine measurements based on the comparison of both
analytical methods which gave about ± 3% deviation.
[
11,20–22] although Ordered reaction mechanism [23] and Ran-
dom mechanism [24] have also been reported in the literature.
Isobutyl propionate is an organic ester having an ethereal, rum-
like, fruity odor and therefore it is used as rum flavor to beverages,
candies, and baked goods [25]. Thus, this ester flavor has a high
commercial demand and it is less reported in literature. There-
fore, the objective of the present research work is synthesis of
isobutyl propionate in SFS using immobilized lipase as biocatalyst.
The optimization of process parameters was carried out based on
the investigations relating to the influence of reaction temperature,
enzyme load, speed of agitation, water concentration and substrate
ratio. Three kinetic mechanisms namely Ordered Bi–Bi mechanism,
Random Bi–Bi mechanism and Ping-Pong Bi–Bi mechanism were
tested for the validation of the experimental data.
2.3.2. Titrimetric analysis
The isobutyl propionate obtained was expressed in terms of
percent (%) conversion i.e. percent of propionic acid converted
with respect to the total acid in the reaction mixture by titrating
reaction mixture with 0.1N NaOH using phenolphthalein indicator
and methanol as a quenching agent.
2.3.3. Determination of initial rates of reaction
Initial rates of esterification were determined at various reac-
tion conditions depending on the molar ratio. The molar ratio of
acid to alcohol was varied from 4:1 to 1:4 in integral successions.
◦
The temperature was maintained at 40 C with 5% (w/w) enzyme,
Novozym® 435, loading. Reactions were carried out for 1 h. Aliquots
of the reaction mixture were taken every 15 min and analyzed by
titrimetric analysis as discussed above. Conversion data for <10%
conversion was used to determine initial reaction rates by plotting
conversion-time profiles.
2
. Materials and methods
2.1. Materials
Novozym® 435 (lipase B from Candida antarctica; immobilized
2
.4. Kinetics and mechanisms of the esterification reaction
on macroporous polyacrylic resin beads, bead size 0.3–0.9 mm,
3
bulk density 0.430 g/cm ) was generously gifted by Zytex Biotech
Two substrates i.e. propionic acid [A] and isobutyl alcohol [B]
Pvt. Ltd., Mumbai (India). Isobutyl alcohol [B] and Propionic acid
are bound to the immobilized lipase Novozym® 435 [E] in either
a specific or random order to form an [AEB] complex, which then
reacts to form the products viz. isobutyl propionate [P] and water
[
A] used were A.R. grade (with 99% purity) and were supplied by
HiMedia Laboratories Private Limited, Mumbai and Thomas Baker
Chemicals) Pvt. Ltd., Mumbai, respectively.
(
[
Q]. The reaction scheme for the synthesis of isobutyl propionate in
a solvent free system (SFS) can be shown as follows:
Reaction Scheme 1. Synthesis of isobutyl propionate by ester-
ification of propionic acid [A] with isobutyl alcohol [B] using
immobilized lipase Novozym® 435 in SFS.
2
.2. Experimental method
The experimental set up consisted of 4.5 cm i.d. three necked
Based on the experimental data the initial rates of esterification
were determined. These initial reaction velocities were then used
for identification of maximum velocity and, Michaelis–Menten,
inhibition and dissociation constants using three different bisub-
strate kinetic models of the enzyme catalyzed reactions viz.
Ordered Bi–Bi, Random Bi–Bi and Ping-Pong Bi–Bi. These three
generalized two-substrate two-product i.e. Bi–Bi reactions models
were selected as they take into account that the product forma-
tion occurs only after the formation of an enzyme–two substrate
complex [26].
baffled glass reactor of 50 ml capacity; provided with six-bladed
turbine impeller. The entire assembly was immersed in a thermo-
static water bath, which was maintained at the desired temperature
◦
with an accuracy of ± 2 C. Electric motor with speed controller was
provided for agitation. The experiment was performed as: 0.1 mol
of each reactant was added to the reactor and mixture was agitated
at 200 rpm for 5 min and then 5% w/w enzyme was added to ini-
tiate the reaction. The molar concentration of [A] and [B] for SFS
can be expressed in volume as shown in Figs. 1–4. This is because
pure [A] having 13.36 M and pure [B] having 10.83 M were used

V.V. Kuperkar et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 143–149
145
H3C
CH
O
H3C
H3C
OH
CH CH2
O
CH3
Novozyme435
C
CH3
H3C
CH2
C
O
+
H O
2
HO
CH2
+
Isobutanol
Propionic acid
Isobutyl propionate
Water
Scheme 1.
2
.4.1. Ordered Bi–Bi mechanism
An Ordered Bi–Bi mechanism is very common with bisubstrate
According to Ping-Pong Bi–Bi with inhibition by both the sub-
strates mechanism:
enzymes. This is a ternary-complex mechanism with a strict order
of attachment of substrate to enzyme [9,27]. In this mechanism,
an enzyme reacts with two substrates in an ordered way affording
two products, which are also released in an ordered fashion. If the
substrate [B] has affinity attaches to the free enzyme and not the
enzyme–substrate complex [E.A], it forms [E.B] (a dead-end com-
plex) by competitively attaching to the active site. This is known
as competitive inhibition by substrates. The reaction mechanism is
given below
Vmax[A][B]
v0 =
(8)
[
A][B] + KB[A] + (1 + [A]/K ) + K [B](1 + [B]/K )
iA
A
iB
where v0 is the initial rate of reaction; Vmax is the maximum
velocity; [A] and [B] are the initial concentrations of the two sub-
strates; KA is Michaelis–Menten constant for substrate [A]; KB is
Michaelis–Menten constant for substrate [B], KiA is the inhibition
constant for [A] and KiB is the inhibition constant for [B].
K
KB
K
KP
K
Q
3. Results and discussion
iA
PQ
E + A⇔E.A + B⇔E.A.B ⇔ E.P.Q⇔E.Q + P⇔E + P + Q
(1)
3
.1. Optimization of reaction variables
K
i
E + B⇔E.B
(2)
The optimization of the reaction condition such as reaction time,
For an Ordered Bi–Bi system, where the two substrates lead to
two products, the initial forward velocity in the absence of products
is given by the equation:
temperature, enzyme loading, alcohol to acid molar ratio, speed of
agitation was undertaken. Moreover the reusability of the enzyme
was tested for evaluation of economic feasibility of the process.
Each of the reaction parameter has been discussed in detail in the
following sections.
Vmax[A][B]
v0 = [
(3)
A][B] + KB[A] + K [B](1 + [B]/K ) + KiAKB(1 + [B]/Ki)
A
iA
3.1.1. Effect of temperature
2.4.2. Random Bi–Bi mechanism
For preliminary analysis, the effect of reaction time on progress
When substrates attach to the enzyme in random order and
of reaction is carried out as it is an important parameter helps to
determine optimum time to achieve maximum yield of product.
Therefore, the effect of reaction time on lipase catalyzed esterifi-
cation for isobutyl propionate was carried out in the experimental
glass reactor which was placed in a thermostatic water bath with
give products, a random bi–bi mechanism is followed. The attach-
ment of one substrate does alter the affinity of the other substrate
towards the enzyme. A rapid-equilibrium random ordered reaction
mechanism is outlined below:
◦
temperature of 40 C, stirring speed at 200 rpm, enzyme loading 5%
K
KB
kcat
iA
E + A⇔E.A + B⇔E.A.B ⇔ E + P + Q
(4)
(5)
w/w and reactant molar ratio 1:1. It was observed that the percent-
age conversion increased with time and % conversion of 82% was
obtained in initial 8 h. However, a marginal change in the conver-
sion was observed after 8 h. This was taken the basis and further
experiments were carried out till 10 h.
K
KA
kcat
iB
E + B⇔E.B + A⇔E.A.B ⇔ E + P + Q
The final equation for the initial rate is given by Eq. (6)
The enzyme activity and stability are the most important fac-
tors to be considered while dealing with the enzymatic reactions.
However these factors are the function of the reaction tempera-
ture. Generally, an increase in the reaction temperature increases
the reaction rate but the stability of the enzyme may decline and
even enzyme may be denatured. Therefore, the effect of reaction
temperature on the formation of isobutyl propionate was studied
Vmax[A][B]
v0 = [
(6)
A][B] + KB[A] + K [B]KiAKB
A
2
.4.3. Ping-Pong Bi–Bi mechanism
The Ping-Pong Bi–Bi model describes a specialized Bi–Bi mech-
anism in which the binding of substrates and release of products
is ordered. In Ping-Pong mechanism, at any point of time only one
substrate [A] is bound to the enzyme and acyl–enzyme complex
◦
at different temperatures ranging from 30 to 60 C, keeping other
parameters constant i.e. stirring speed at 200 rpm, enzyme loading
5
% (w/w), substrate molar ratio 1:1. Fig. 1B depicts that the initial
[
E.A] is formed. Then the product [P] is detached and formed as it
reaction rate increases from 0.0028 to 0.0594 Mol/min/g enzyme
is fragment of the original substrate [A]. The rest of the substrate
is covalently attached to the enzyme E, which is designated as [F].
The second substrate [B] binds and reacts with the enzyme to form
a covalent adduct with the covalent fragment of [A] still attached to
the enzyme to form product [Q] which is released and the enzyme
is restored to its initial form [E]. A ping-pong mechanism with inhi-
bition by both the substrates is given below
◦
as temperature increased from 30 to 50 C. However, % conversion
increased from 66 to 82% with an increase in temperature from 30
◦
◦
to 40 C but at 50 C final conversion was reduced to 75% as shown
in Fig. 1A. This is attributed to thermal denaturation of enzyme at
higher temperature and hence decline in conversion with time [6].
An increase in % conversion as well as initial rate with increase in
temperature is due to fact that the temperature may reduce mix-
ture viscosity, enhance mutual solubility, improve diffusion process
of substrates, and enhance the interactions between catalytic
K
K
K
K
K
K
Q
A
EA
P
B
FB
E + A⇔E.A⇔F.P⇔F.B⇔F.B⇔E.Q⇔E + Q
(7)

1
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V.V. Kuperkar et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 143–149
Fig. 1. Effect of temperature for the esterification of propionic acid with isobutyl alcohol using immobilized lipase Novozyme 435 in SFS. (A) % coversion vs. time (B) initial
rate vs. temperature.
particles and substrates. However, high temperature may disrupt
the active conformation of enzyme which leads to loss of activity
reduced to 75 and 72% for 7.5%, 10% w/w loading respectively. This
might be due to high amount lipase making active sites of enzyme
unexposed to the substrates [7] or agglomeration of immobilized
enzyme in solvent free system [28]. However, the initial rate was
found to increase from 0.0042 to 0.0241 Mol/min/g enzyme when %
enzyme loading was increased from 2.5 to 7.5 but decreased there-
after (see Fig. 2B). At 5% w/w enzyme loading rapid formation of
enzyme–substrate complex explains the higher conversion. Sim-
ilarly, an excess enzyme also hampers the mixing by increasing
viscosity leading to the less effective transfer of the substrates to
the active sites of the excess enzyme molecules in turn reducing
the overall mass transfer. Considering the above findings, 5% w/w
enzyme loading was set to be optimum.
®
and selectivity at higher temperature [7]. Novozym 435 can work
◦
even at 100 C as it is a heat-tolerant enzyme but, at this high tem-
perature denature enzyme faster with time, so it is recommended
to operate at lower temperatures. Moreover, working at lower tem-
perature minimizes thermal energy for economical optimization
◦
[
8]. Therefore 40 C was selected as an optimum temperature to
carry out further studies.
3
.1.2. Effect of enzyme loading
Novozym 435 is a kind of lipases immobilized on macroporous
®
acrylic resin wherein enzyme molecules could be attached on the
surface of the support and/or in the inner part of the support. When
the reaction is carried out in a SFS; excessive enzyme particles in
the reaction mixture may lead to decrease in mass transfer effi-
ciency due to internal diffusion problem. The optimum enzyme
loading has to be found out for any enzymatic reaction as enzymes
are main cost component attached to the process. Therefore, keep-
ing high esterification yield and economic feasibility of synthesis
of isobutyl propionate using immobilized lipase Novozym® 435
in viewpoint, the effect of enzyme loading was investigated. The
range of the enzyme loading was chosen as 2.5–10% (w/w) keeping
the other reaction parameters constant. The curves of % conver-
sion with time and initial rate with % enzyme loading are shown
in Fig. 2. From Fig. 2A, it is observed that the % conversion was
increased up to 82% for 5% w/w enzyme loading, thereafter it was
3.1.3. Effect of speed of agitation
In case of immobilized enzyme, the reactants have to diffuse
from the bulk liquid to the external surface of the particle and from
there into the interior pores of the catalyst where the actual reac-
tion takes place and products are being formed. These, products
need to diffuse out from the enzyme particles to the bulk liquid [29].
External mass transfer limitations can be minimized by carrying out
the reaction at an optimum speed of agitation and low enzyme load-
ing [30]. Therefore, experiments were carried out at varying speed
of agitation from 100 to 400 rpm, so as to study its effect on solvent
free esterification reaction. Fig. 3A illustrates that, as speed of agita-
tion increased from 100 to 300 rpm, the % conversion was found to
increase from 88 to 92.5% respectively. However, the % conversion
Fig. 2. Effect of enzyme loading for the esterification of propionic acid with isobutyl alcohol using immobilized lipase Novozyme 435 in SFS. (A) % coversion vs. time (B)
initial rate vs. % enzyme loading.

V.V. Kuperkar et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 143–149
147
Fig. 3. Effect of speed of agitation for the esterification of propionic acid with isobutyl alcohol using immobilized lipase Novozyme 435 in SFS. (A) % coversion vs. time (B)
initial rate vs. speed of agitation (rpm).
decreased at 400 rpm to 90.04%. The similar observations can be
made from the initial rate vs. speed of agitation as shown in Fig. 3B.
An increase in % conversion and initial rate for increase in the speed
of agitation from 100 to 300 rpm can be attributed to the fact that
mass transfer resistance has decreased due to the increase in the
turbulence. However, the decrease in the % conversion and initial
rate at 400 rpm can be reasoned by the shearing effect on enzyme
at higher stirring speed. Although highest 92.25% conversion was
obtained at 300 rpm in comparison with 200 rpm; enzyme tends to
leave its support after 5–6 h thereby losing its recyclability prop-
erty. Therefore the mild stirring speed of 200 rpm was selected as
optimum for the esterification reaction in SFS keeping in view not
to hamper the catalytic efficiency (reusability) of the immobilized
lipase.
respectively. Moreover, the initial rate of the esterification reaction
is found to be highest (0.063 Mol/min/g enzyme) for 0 M water con-
centration and it tends to decrease from 0.043 to 0.012 Mol/min/g
enzyme when water concentration is increased from 0.1 to 0.3 M.
A decrease in % conversion and initial rate for increase in concen-
tration of water can be attributed to the fact that the addition
of water shifts the equilibrium towards the backward reaction
(hydrolysis) rather than towards forward reaction (esterification)
[32].
3.1.5. Effect of molar ratio
The effect of molar ratios of substrates is an important param-
eter in enzymatic esterification reactions in a SFS. For preliminary
analysis, acid to alcohol molar ratios were varied in the range of
1
:1–1:4 for concentration of 0.1 M for each substrate. One way of
3
.1.4. Effect of water concentration
It is well known fact that a large amount of water favors
pushing the equilibrium of the reaction in a forward direction is
to increase the nucleophile concentration [33] and also negative
effect of acid on lipase enzyme was observed in literatures. How-
ever, high alcohol concentration may slow down the reaction rates.
Therefore, it is necessary to optimize the actual excess nucleophile
concentration to be employed in a given reaction. The isobutyl pro-
pionate conversion was increased from 82.5 to 90% when molar
ratio was increased from 1:1 to 1:3 however only marginal change
in the % conversion can be seen at molar ratio 1:4 (Fig. 5). This
invariant % conversion at molar ratio of 1:4 indicates the inhibitory
effect of alcohol on enzyme which was also shown in case of isoamyl
acetate [19], isoamyl myristate [34], geranyl propionate [35]. How-
ever, only this evidence does not prove that the inhibitory effect of
alcohol on enzyme. This has to be extended to the variation in the
acid concentration to analyses the enzymatic kinetic rate data so
the hydrolysis and inhibits the esterification reaction. However,
small amount of water is an important factor to be considered
while dealing with lipase catalyzed reactions both for optimal cat-
alytic activity of the enzyme (thermodynamic equilibrium of the
reaction) as well as the maintenance of three dimensional struc-
tural integrity (reducing probability of contact of inhibitor with
enzyme) [3,31]. The effect of initial water on enzymatic activ-
ity was examined by adding water ranging from 0.1 M (1.8 cm )
to 0.3 M (5.4 cm ) to the reaction mixture moreover it was com-
pared with SFS (i.e. 0 M water concentration). It is clear from
Fig. 4A that the highest % conversion of 90.4% is obtained at SFS
whereas as we increase the initial concentration of water from 0.1
to 0.3 M the % conversion substantially decreases from 77.3 to 60.5%
3
3
Fig. 4. Effect of addition of water in the esterification of propionic acid with isobutyl alcohol using immobilized lipase Novozyme 435. (A) % coversion vs. time (B) initial rate
vs. concentration of water.

1
48
V.V. Kuperkar et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 143–149
Fig. 7. Comparison of calculated initial rates by the all mechanism with the exper-
imental values.
Fig. 5. Effect of acid to alcohol molar ratio for the esterification of propionic acid
with isobutyl alcohol using immobilized lipase Novozyme 435 in SFS.
of water loss from the micro-environment of enzyme particles or
blocking the active sites by one of the product [34].
as to arrive at the actual inhibition effect and mechanism under-
lying the reaction scheme. This was extended further to study the
actual inhibitory effect of both the substrate by kinetic model fitting
shown in the Section 3.2.
3.2. Kinetic model based on initial rate measurements
The effect of substrate concentration on the rate of the esteri-
fication of propionic acid with isobutyl alcohol using immobilized
lipase Novozym® 435 in a SFS was investigated using three different
mechanisms. The initial rates of esterification were calculated using
data from the reaction times giving less than 10% conversion of the
limiting reagent and plotted against acid concentration in Fig. 7.
As propionic acid concentration increased from 2.25 M (B:A = 4:1)
to 8.27 M (B:A = 1:2); the initial velocity reaches to highest value
3
.1.6. Enzyme reusability
Novozym 435 is very efficient enzyme and used for esterifica-
®
tion effectively on large extent and hence its reusability is one of the
important factor for its use on industrial scale to decide economic
viability of the process and to restrict the cost factor. At the end
of the reaction, the immobilized lipase was filtered, washed with
acetone and kept in desiccator for 24 h and then reused for further
batches. With optimized parameters; it was observed that there
were marginal changes in percentage conversion up to seven cycles
of reuse and further conversion was reduced by difference of 6%
only (see Fig. 6). The reusability of immobilized enzyme varies with
the reaction systems being used based on the substrates applied in
the reaction scheme. This could be a result of the decrease in the
amount of effective biocatalyst adsorbed to the solid support or the
denaturation of the enzyme by propionic acid after several reaction
cycles. Also, decrease in the activity of the enzyme could be because
(0.11 M/min/g catalyst) from 0.023 Mol/min/g enzyme and again
start decreasing. However the isobutyl alcohol concentration was
also variable since the experimentations were done in a solvent free
system. The similar trend was observed for the same system when
solvent system was used [36].
Several mechanisms have been proposed to explain lipase
catalyzed reactions. However, in our study we have used three dif-
ferent mechanisms i.e. Ordered Bi–Bi, Random Bi–Bi and Ping-Pong
Bi–Bi for evaluation of the initial rates based on their mechanism.
For greater accuracy, non-linear regression with minimization of
sum of square of errors (SSE) was applied using Microsoft Excel
2
010 and model parameters were evaluated. The comparison of
initial velocity calculated by the rate equation given by mechanism
Ordered Bi–Bi, Random Bi–Bi and Ping-Pong Bi–Bi as a function
of acid concentration is shown in Table 1 and Fig. 7. It is evident
from Table 1 and Fig. 7 that the kinetic parameter values evaluated
by Ordered Bi–Bi and Random Bi–Bi are non-significant. Moreover
the sum of squares of errors is higher as compared to Ping-Pong
Bi–Bi. Although, several mechanisms have been proposed in theory,
lipases are generally said to follow the Ping-Pong Bi–Bi Mechanism
[
21]. Our results indicate the same too.
The esterification of propionic acid with isobutyl alcohol using
immobilized lipase Novozym® 435 in SFS includes the reaction
steps in which the lipase may react with iso-butanol to yield
a dead-end enzyme iso-butanol complex or it may react with
propionic acid to yield the effective lipase propionic acid com-
plex. Then the lipase propionic acid complex is transferred to an
enzyme–acyl intermediate and water is released. This is followed
by the interaction of the enzyme–acyl complex with iso-butanol
to form another binary complex, which then yields the ester and
Fig. 6. Reusability of the immobilized lipase Novozyme 435 in SFS for the esterifi-
cation of propionic acid with isobutyl alcohol.

V.V. Kuperkar et al. / Journal of Molecular Catalysis B: Enzymatic 99 (2014) 143–149
149
Table 1
Comparison of calculated evaluated kinetic parameters for all mechanisms.
Model/Parameters
Ordered Bi–Bi
Random Bi–Bi
Ping-Pong Bi–Bi
Ping-Pong Bi–Bi (Varma and Madras [36])
Vmax (M/min/g catalyst)
0.73
19.65
0.50
486.66
–
50.31
4.3E−03
67.78
3159.52
26.28
194.78
–
0.500
0.631
0.003
0.0042
0.1539
–
0.127
0.278
0.0014
0.00012
0.00061
–
KA (M)
KB (M)
KiA (M)
KiB (M)
Ki (M)
SSE
–
4.4E−03
9.6E−05
–
free lipase. If the enzyme–acyl complex reacts with acid it forms a
dead-end complex leading to acid inhibition. In line with the reac-
tion mechanism discussed above, the best fit model parameters are
obtained by non-linear regression for Ping-Pong Bi–Bi mechanism
with acid and alcohol inhibition were Vmax = 0.5 M/min/g enzyme,
KA = 0.631 M, KB = 0.003 M, KiA = 0.0042 M and KiB = 0.1539 M. The
apparent Michaelis–Menten constants obtained from the analy-
sis KA and KB, give light to the fact that the enzyme has greater
affinity for iso-butanol than propionic acid (specificity (Ks) is
given by kcat/Km where Km is the Michaelis–Menten constants and
Vmax = kcat[Eo] where [Eo] is the total enzyme concentration). The
Michaelis–Menten constant for most of the enzyme catalyzed reac-
tions is in the range of 10 –10 M [37] which is consistent with
the observed kinetic constants found in this study. The values of the
specificity constant suggest that the enzyme specificity is higher
in the immobilized lipase system with respect to iso-butanol [38].
The inhibition constants have a direct relationship with inhibitory
effect of the substrate.
yields as compared to those incorporating organic solvents. Three
different bi–bi mechanisms are tested for the development of the
kinetics of esterification of iso-butanol and propionic acid in SFS.
The best fit model parameters obtained by non-linear regression
for Ping-Pong Bi–Bi mechanism with acid and alcohol inhibi-
tion are Vmax = 0.5 M/min/g catalyst, K = 0.631 M, KB = 0.003 M,
A
KiA = 0.0042 M and KiB = 0.1539 M.
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