Microwave assisted lipase catalyzed synthesis of isoamyl myristate in solvent-free system

Article abstract of DOI:10.1016/j.molcatb.2012.06.011

Isoamyl myristate finds many applications in food, cosmetic and pharmaceutical industries as a flavor and fragrance compound. The current work focuses on solvent-free lipase catalyzed microwave assisted synthesis of isoamyl myristate from isoamyl alcohol and myristic acid. The activities of a number of commercially available lipases such as Novozym 435, Lipozyme RM IM and Lipozyme TL IM were evaluated, amongst which Novozym 435 was found to be the most active. Effect of solvent, agitation speed, type of enzyme, enzyme concentration, reactant concentrations and temperature was systematically studied on the esterification of isoamyl alcohol with myristic acid under microwave irradiation. Equimolar quantities of the reactants with 0.38% (w/v) Novozym 435 catalyst loading and agitation speed of 400 rpm leads to 96% conversion at 60 °C in 1 h. Based on the initial rates and concentration profiles, the reaction was found to obey the ping-pong bi-bi mechanism with inhibition by isoamyl alcohol and the kinetic parameters were evaluated by using non-linear regression analysis.

Full text of DOI:10.1016/j.molcatb.2012.06.011

Journal of Molecular Catalysis B: Enzymatic 83 (2012) 16–22
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Microwave assisted lipase catalyzed synthesis of isoamyl myristate in
solvent-free system
Ganapati D. Yadav∗, Pooja A. Thorat
Department of Chemical Engineering, Institute of Chemical Technology, Nathalal Parkh Marg, Matunga, Mumbai 400019, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Isoamyl myristate finds many applications in food, cosmetic and pharmaceutical industries as a flavor
and fragrance compound. The current work focuses on solvent-free lipase catalyzed microwave assisted
synthesis of isoamyl myristate from isoamyl alcohol and myristic acid. The activities of a number of com-
mercially available lipases such as Novozym 435, Lipozyme RM IM and Lipozyme TL IM were evaluated,
amongst which Novozym 435 was found to be the most active. Effect of solvent, agitation speed, type of
enzyme, enzyme concentration, reactant concentrations and temperature was systematically studied on
the esterification of isoamyl alcohol with myristic acid under microwave irradiation. Equimolar quan-
tities of the reactants with 0.38% (w/v) Novozym 435 catalyst loading and agitation speed of 400 rpm
leads to 96% conversion at 60 ◦C in 1 h. Based on the initial rates and concentration profiles, the reaction
was found to obey the ping-pong bi-bi mechanism with inhibition by isoamyl alcohol and the kinetic
parameters were evaluated by using non-linear regression analysis.
Received 14 January 2012
Received in revised form 13 June 2012
Accepted 18 June 2012
Available online 28 June 2012
Keywords:
Enzyme catalysis
Solvent-free synthesis
Novozym 435, Ping-pong bi-bi mechanism
Substrate inhibition
Kinetics
Isoamyl myristate
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Microwave assisted organic synthesis (MAOS) using both chem-
ical and biocatalysts has gained importance in Green Chemistry
Solvents are used rampantly in process industries mainly as
vehicles for transporting solids, dissolution of immiscible reagents,
creation of homogeneous phase to overcome mass transfer effects
and enhancing rates of reactions. Solvents also help to dissipate
heat of reaction, and are used as diluents to get better selectivities.
However, they are a major source of volatile organic compounds
(VOCs) and pollution. Solvent-free system offers many advan-
tages. The absence of solvents facilitates downstream processing
since fewer components are present in the reaction mass and
the elimination of solvents from the production step makes the
process cost effective and environmentally friendly [1–3]. Solvent-
free energy efficient enzymatic synthesis of wax ester was scaled
up by Petersson et al. [4] for industrial purpose. Lipases and
supported lipase catalyzed reactions in non-aqueous media have
been studied by different groups including ours for industrially
significant esterifications [5–10], transesterifications [11,12], oxi-
dations/epoxidations [13–15], optical resolution of pharmaceutical
intermediates [16,17], hydrolysis [18], and other transformations
[19,20].
[21–24]. Microwave irradiation leads to an instantaneous local-
ized superheating due to dipole rotation or ionic conduction and
the energy transfer occurs within 10−9 s with each cycle of electro-
magnetic energy and is faster than the rate at which the molecules
can relax (∼10−5 s) which results in non-equilibrium conditions
and high instantaneous temperatures. An increase in temperature
causes greater movement of molecules leading to a greater number
of energetic collisions and hence enhancement in reaction rate and
product yield [23,24].
We have reported a number of microwave assisted lipase-
catalyzed reactions such as esterification [7,25], transesterification
[7,26], epoxidation [14,27], hydrolysis [28] and N-acetylation [29]
and found that the reaction rates had increased by 2.5–4.5 times
under microwave irradiation. The mechanistic and kinetic aspects
have also been discussed in detail in these publications amongst
others.
Organic esters are employed as solvents, fragrance, flavors, and
precursors in a variety of industries. Particularly, aliphatic esters are
greatly used in flavor industry, mainly as fixatives and modifiers,
whereas aromatic esters in fragrance compositions [30]. Isoamyl
myristate is an important ester which is used as flavor and fragrance
agent in a variety of food products. It has also been tested in vitro
for antibacterial activity which is found to be comparable with the
drug Ciprofloxacin [31].
∗
Corresponding author. Tel.: +91 22 3361 1001/1111/2222;
fax: +91 22 3361 1002/1020.
The synthesis of isoamyl esters has been reported in the litera-
ture. However, majority of them have been directed more towards
E-mail addresses: gdyadav@yahoo.com, gd.yadav@ictmumbai.edu.in
(G.D. Yadav).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.06.011

G.D. Yadav, P.A. Thorat / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 16–22
17
low molecular weight fatty acid esters, such as acetate, propionate
2.3.3. Enzymatic reaction
and butyrate. Solvent-free esterification reactions for the synthe-
sis of isoamyl valerate, isoamyl isovalerate and amyl isobutyrate
have been attempted earlier [32,33]. Synthesis of isoamyl myris-
tate has only been reported by a reaction of myristic acid with
isoamyl alcohol in the presence of sulfuric acid under reflux [31].
Thus, in the current work the esterification of isoamyl alcohol with
myristic acid under microwave irradiation was investigated and the
effects of various parameters such as solvent, agitation speed, type
of enzyme and its concentration, reactant concentration and tem-
perature were systematically studied to deduce mechanism and
kinetics.
The range of parameters was determined on the basis of
the results obtained in preliminary experiments, considering the
experimental set-up limits, and the working conditions limit for
each enzyme. Thus, it was possible to fix the upper temperature
level at 70 ◦C, in order to avoid the loss of enzymatic activity caused
by temperature effect; there was no deactivation of the enzyme
at 60 ◦C [9]. It has been reported that the external mass trans-
fer resistance was absent at or beyond 400–500 rpm in earlier
studies on enzyme catalysis [14–16] and optimum speed of agi-
tation for microwave reactors has been in the range of 300–500
for lab reactors [25–29]; so a speed of 400 rpm was chosen dur-
ing all experiments. Earlier studies of esterification reaction under
microwave reaction have used equimolar quantities of substrates
to avoid inhibition by one or both substrates or product(s).
A typical reaction mixture consisted of equimolar quantities of
isoamyl alcohol and myristic acid (0.068 mol each) to make the
total liquid volume of 25 mL. To make volume of 25 mL in solvent
optimization studies, 0.0409 mol of both substrates and 10 mL of
solvent were added. Then 0.38% (w/v) of immobilized enzyme was
added to initiate the reaction. The reaction mixture was agitated at
a speed of 400 rpm at 60 ◦C under microwave irradiation of 50 W.
Samples were withdrawn periodically from the reaction mixture
and analyzed by GC. The parameters were optimized by changing
one parameter at a time while maintaining others constant. For all
the parameter optimization studies, the total volume of the reac-
tion mass was kept 25 mL to maintain uniform volume for all the
experiments. This would give the same enzyme loading (mg/cm3)
and mixing conditions (same impeller height) in the microwave
reactor.
2. Materials and methods
2.1. Enzymes
Novozym 435 (component
B of the lipase from Candida
antarctica immobilized on macroporous polyacrylate resin),
lipozyme RM IM (Rhizomucor miehei lipase) immobilized on anionic
resin and lipozyme TL IM (Thermomyces lanuginosus lipase) were
all procured as gift samples from NovoNordisk, Denmark. Olive
oil assay and titrimetry methods were used to analyze immobi-
lized enzyme [12]. With this assay method activity was found to be
6900 Units/g for the fresh enzyme which was similar to activity in
the validation certificate provided by the manufacturer.
2.2. Chemicals
All chemicals were analytical grade reagents procured from
firms of repute and used as such without further purification.
Myristic acid, isoamyl alcohol, toluene, 1,4-dioxane, n-hexane, n-
heptane, acetonitrile and other analytical reagents were obtained
from S.D. Fine Chemicals Pvt. Ltd., Mumbai, India. GC–MS was also
done to confirm their purity.
2.4. Analysis
The concentrations of the reactants and products were deter-
mined on Chemito Gas chromatograph (model 8610) equipped
with a flame ionization detector. A 2 m × 3.2 mm stainless steel col-
umn packed with SE-30 was used for analysis. After an initial hold
of 30 s at 55 ◦C, the column temperature was increased to 235 ◦C
at a ramp rate of 25 ◦C min−1. Nitrogen was used as the carrier gas
at a flow rate of 1 mL min−1. Both injection and detection tempera-
tures were set to 290 ◦C. Synthetic mixtures were prepared of pure
samples, and calibration was done to quantify the collected data
for conversions and rates of reactions. Purity of chemicals used and
formation of isoamyl myristate was confirmed by GC–MS analysis
(Clarus 500 GC/MS, PerkinElmer, USA).
2.3. Experimental setup
2.3.1. Conventional reactor
The studies were carried out in a 3 cm i.d. mechanically agi-
tated glass reactor of 50 mL capacity, equipped with four baffles
and six-bladed turbine impeller. The entire reaction assembly was
immersed in a thermostatic water bath. The reaction temperature
was maintained with an accuracy of 1 ◦C.
3. Results and discussion
2.3.2. Microwave reactor
The effects of various parameters on conversion and rates of
reaction were studied systematically. The reaction is shown by
Scheme 1.
The microwave experiments were conducted in commercially
available “Discover” system of CEM Corporation, USA (Model CEM-
SP 1245), with proper temperature and pressure feedback systems
for complete control of the reaction conditions. It is a mono-mode
microwave system. The frequency of microwave generated in the
magnetron is 2455 MHz. It automatically regulates the energy con-
sumption to maintain constant temperature at the set temperature
with accuracy of 1 ◦C. Typically 50 W power input was given
and it was continuous with automatic control. The reactor was
a 120 mL capacity, 4.5 cm i.d. cylindrical glass vessel with provi-
sion for mechanical stirring. A standard six-blade-pitched turbine
impeller of 1.5 cm diameter was used for agitation. The actual reac-
tor volume exposed to the microwave irradiation was 45 mL with
5.5 cm height. The temperature in the reactor was digitally con-
trolled with an accuracy of 1 ◦C. The quantities of reactants and
enzyme for the microwave reaction procedure were identical to
those used for conventional heating.
3.1. Screening of different lipases
Immobilized enzymes have various advantages like easy recov-
ery, reusability, and stability and thus can be designed in
continuous operations on large scale. Three commercially avail-
able lipases, Novozym 435, Lipozyme TL IM, and Lipozyme RM IM,
were evaluated to choose the most efficient enzyme for synthesis
of isoamyl myristate. Novozym 435 is a versatile enzyme and has
been found to be very effective even in solvent-free systems [34].
In the case of Novozym 435, the conversion was 97% compared to
47% with Lipozyme RM IM and 8.5% with Lipozyme TL IM in the
first 60 min (data not shown).
Dimensional analysis of Candida antarctica lipase B (Novozym
435) based on the crystal structure clearly shows that amino acid

18
G.D. Yadav, P.A. Thorat / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 16–22
OH
Lipase
+
H2O
OH
O
MW 60ºC
+
O
O
Isoamyl alcohol
Myrisꢀc acid
Isoamyl myristate
Water
Scheme 1. Synthesis of isoamyl myristate in solvent-free system.
residue A281 is a part of an ␣-helix (␣-10) located at the top
of the substrate-binding pocket in a highly hydrophobic environ-
ment [35]. This micro-environment around the active site pocket
of Novozym 435 favors proper interaction of substrate in non-
aqueous media. The flapping lid of Mucor miehei (Lipozyme RM IM)
and Thermomyces lanuginosus (Lipozyme TL IM) projects into the
binding pocket of the enzyme [36] thereby creating steric hindrance
in binding of myristic acid at the active site. Since Novozym 435 is
known for its high activity even in solvent-free media and does not
contain a flapping lid, there is less steric hindrance as compared to
other lipases and it shows greater activity.
It has been shown that the activity of Novozym 435 was the best
amongst all the commercial lipases in a wide range of 30–60 ◦C
for conventional heating [7–10] and also under the influence of
microwave irradiation [13]. Lipozyme TL IM is mainly intended for
interesterification of bulk fats and production of frying fats [37].
Novozym 435 is a thermostable lipase and mainly useful for the
synthesis of esters and amides [38]. The purpose of using these
enzymes was to find out if significant activation could be achieved
through microwave irradiation, despite their known use. Since
Novozym 435 was found to be the best catalyst, it was used in all
further studies.
deduce that the abnormal solvent effect may be related to the
microwave characteristics of selective heating. It is then interesting
to investigate how the reactions with and without solvents perform
under microwave irradiation. In the present reaction when per-
formed under solvent-free conditions, the micro-environment is
surrounded by myristic acid which has a high log P value of 5.8 and
thus preserves the essential water coat leaving the biocatalyst in an
active state. Non-polar solvents (such as toluene, hexane, heptanes
and 1,4-dioxane) possess very low dielectric constants, loss tangent
(tan ı) values and dielectric loss values [41]. Non-polar solvents are
thus viewed as transparent for microwave radiation, as there is a
very weak solvent–microwave interaction leading to direct heating
of the substrates [23]. Similar is the fact in the absence of solvents in
solvent-free reactions. Microwave irradiation also exerts a stronger
selective heating of reactant isoamyl alcohol due to its low log P of
1.03 by affecting dipole molecular rotation and ion induction of
this polar reactant, thus the energy transfer is direct and fast to
the reactant in this solvent-free reaction [42]. The current results
showed that combined effects of both substrates in this solvent-
free reaction were comparable to results obtained in reactions with
non-polar solvents. Under the influence of microwave, the initial
rates in this solvent-free reaction were superior to that of reactions
in other organic solvents (Fig. 1).
3.2. Synergism of microwave with lipase catalyzed reactions
3.4. Effect of speed of agitation
There is always a trace of residual water remaining in the
immobilized enzyme beads, which is heated rapidly due to
material–wave interactions [39], leading to thermal effects (con-
nected to dipolar and charge space polarization) and purely
non-thermal (accelerating the molecular rotation and electron spin
oscillation of the polar parts of enzyme) effects and this leads to
improved enzyme activity [40]. The thermal gradients and flow of
heat are the reverse of those in materials heated by conventional
means and this is mainly responsible for the increased activity and
selectivity [41]. Thus, microwave irradiation acts synergistically
with lipase catalysis. The reaction at 60 ◦C with Novozym 435 using
conventional heating gave 56% conversion in 60 min while under
the influence of microwave (energy input: 50 W) led to a conversion
of 96% in 60 min. The initial rate was increased by 2.25 times under
the influence of microwave irradiation in the first 15 min of the
reaction. All further studies were carried out under microwave irra-
diation as considerable increase in total conversion was observed
using microwave irradiation.
This is a solid–liquid system having myristic acid and isoamyl
alcohol in the liquid phase and the immobilized enzyme (Novozym
435) as a solid phase. However, as the reaction proceeds, the water
100
90
80
70
60
50
40
30
20
10
0
3.3. Effect of solvent
The influence of organic solvents on the enzymatic reactions
has been summarized [41]. The enzyme activity is higher in the
environment surrounding by non-polar (log P > 4) and mid-polar
solvents (2 < log P < 4), whereas the lowest activity is expressed in
polar solvents (log P < 2). Solvents having log P greater than 4 do
not distort the essential water coat around the particle, thereby
leaving the biocatalyst in an active state. The highest microwave
effect is expressed in the polar solvent instead of the non-polar
solvents [42], although polar solvent is anticipated as more ‘unfa-
vorable’ to enzymatic reactions under conventional heating. They
0
20
40
60
80
100
Time (min)
Fig. 1. Screening of different solvents. Reaction conditions – isoamyl alcohol:
0.0409 mol, myristic acid: 0.0409 mol; solvent up to 25 cm3 (0.068 mol of both reac-
tants for solvent-free reaction); catalyst: 0.38% (w/v); speed of agitation: 400 rpm;
temperature: 60 ◦C; microwave irradiation: 50 W. ( ) solvent-free; ( ) n-hexane;
(
) n-heptane; ( ) toluene; ( ) 1,4-dioxane; ( ) acetonitrile.

G.D. Yadav, P.A. Thorat / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 16–22
19
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
0
20
40
60
80
100
Time (min)
Time (min)
Fig. 3. Effect of temperature. Reaction conditions – isoamyl alcohol: 0.068 mol,
Fig. 2. Effect of catalyst loading. Reaction conditions – isoamyl alcohol: 0.068 mol,
myristic acid: 0.068 mol; solvent-free, catalyst: 0.38% (w/v); speed of agitation:
400 rpm; temperature: 30–60 ◦C; microwave irradiation: 50 W. ( ) 30 ◦C; (
40 ◦C; ( ) 50 ◦C; ( ) 60 ◦C; ( ) 70 ◦C.
)
myristic acid: 0.068 mol; solvent-free, catalyst: 0.19–0.58% (w/v); speed of agitation:
400 rpm; temperature: 60 ◦C; microwave irradiation: 50 W. ( ) 0.19% (w/v); (
0.29% (w/v); ( ) 0.38% (w/v); ( ) 0.58% (w/v).
)
3.6. Effect of temperature
generated in situ forms immiscible liquid phase and the original
two-phase system is converted into a three-phase system. The
pores in the enzyme are filled with aqueous phase, and a thin film
of aqueous phase which surrounds the matrix should be retained
for the enzyme to be active. The effect of external mass transfer
resistance to the transfer of the reactants toward the outer surface
of the solid particle was studied in the range of speed of agitation
of 200–800 rpm. The initial rate of reaction increased with speed
of agitation from 200 to 400 rpm and remained constant at higher
speeds (data not shown). Indeed, the conversion after 30 min for
400 and 500 rpm remained practically constant at 95%. This indi-
cated that there was no resistance to mass transfer of the reactants
to the external surface of the catalyst particle [19]. At speeds beyond
600 rpm, there was a marginal decrease in conversion either due
to attrition of enzyme beads or beads sticking to the wall of the
reactor [19]. Thus, there was no resistance to external mass trans-
fer. Therefore, further experiments were performed at a speed of
400 rpm.
The reaction temperature is a crucial parameter in biocataly-
sis [44]; therefore, we selected four different temperatures in the
range of 30–70 ◦C for the synthesis of isoamyl myristate. It was
found that the overall conversion as well as rate of reaction was
higher under microwave irradiation vis-à-vis conventional heating
at the same temperature. Further, the rate enhancement is due to a
combined effect of the microwave absorption properties of reaction
mixture, due to their polar and ionic characteristics, as suggested
by the “dipolar polarization mechanism” [45]. The enzyme is in the
immobilized form, in the proximity of the substrates in solvent-free
system and seems to behave slightly differently due to the non-
thermal effects of microwave energy. It was clearly observed that
initial rates and conversion increased with increase in the reaction
temperature. However, a decrease in the conversion was observed
with a further rise in the temperature from 60 ◦C (Fig. 3). This could
be attributed to the deactivation of the lipase at high tempera-
tures. Therefore, temperature of 60 ◦C seemed to be the optimum
for the reaction system. Fig. 4 shows the Arrhenius plot. Activation
energies calculated respectively as 4.72 and 4.95 kcal/mol for con-
ventional and microwave heating were similar to those reported for
most lipase catalyzed reactions [44]. Desnulle [46] has reported a
value of 5.3 kcal/mol in an aqueous emulsion system. The lines are
almost parallel in the Arrhenius plot (Fig. 4). This would suggest
that there is no change in mechanism due to the mode of heating
but the rate constant is augmented in microwave heating because
of a greater collision frequency of reaction molecules.
3.5. Effect of catalysts loading
The effect of catalyst loading was studied in the range of
0.19–0.58% (w/v) Novozym 435, maintaining all other parameters
the same. Both reaction rate and conversion increased with increas-
ing catalyst loading. This indicated that the reaction was kinetically
controlled. Increase of catalyst loading from 0.19 to 0.38% (w/v)
increased the conversion by around 10%. However, further increase
of catalyst loading to 0.58% (w/v) increased the conversion only
marginally (Fig. 2). This would suggest that all available active
sites of the enzyme are occupied by substrate molecules. Any
further increase in enzyme loading will have no effect since no sub-
strate molecules are available and these sites would be vacant [43].
However, beyond 0.38% (w/v) of catalyst loading, there was no sub-
stantial increase in final conversion. It appeared that mass transfer
limitations were set in at higher loadings beyond 0.38% (w/v). Obvi-
ously, the lower the amount of enzyme, the more cost effective the
process; thus, 0.38% (w/v) of Novozym 435 was used as optimum
catalyst loading in further studies.
3.7. Effect of mole ratio of isoamyl alcohol to myristic acid
The mole ratio of isoamyl alcohol to myristic acid was varied as
1:1, 2:1 and 3:1. Different concentrations of isoamyl alcohol were
varied in the range of 2.7, 4.2, 5.1 mol/L, for the known concentra-
tion myristic acid of 2.7, 2.1, 1.7 mol/L calculated with respect to a
constant total volume of 25 mL of the solvent-free system to achieve
the above mentioned mole ratios. Fig. 5 shows that the maxi-
mum conversion (96%) and rate of reaction were obtained with
a 1:1 mole ratio. The enzyme is immobilized within macroporous

20
G.D. Yadav, P.A. Thorat / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 16–22
-9
-9.2
100
90
80
-9.4
70
-9.6
Microwave
60
50
40
30
20
10
0
irradiaꢀon
-9.8
-10
Convenꢀonal
heaꢀng
-10.2
-10.4
-10.6
0.00295
0.00305
0.00315
1/T (K-1)
0.00325
0.00335
0
20
40
60
80
100
Time (min)
Fig. 4. Arrhenius plot for the synthesis of isoamyl myristate.
Fig. 6. Reusability of the catalyst. Reaction conditions – isoamyl alcohol: 0.068 mol,
myristic acid: 0.068 mol; solvent-free, catalyst: 0.38% (w/v); speed of agitation:
polyacrylic resin, which is hydrophilic; it may trap water within the
support as well as it may form a film around the resin [37]. External
mass transfer limitation due to water was eliminated by increas-
ing the speeds of agitation [47]. The decrease in the conversion and
rate of reaction with increasing concentration of isoamyl alcohol
could be ascribed to the inhibitory effect of isoamyl alcohol on the
Novozym 435. Isoamyl alcohol contains a hydrophobic hydrocar-
bon tail and a polar head of the alcohol functional group. Since the
enzyme is hydrophobic, there may be hydrophobic–hydrophobic
interaction between the enzyme and the hydrocarbon chain of
isoamyl alcohol, and thus the dissociation of isoamyl alcohol
from the enzyme is prolonged. Because of a close contact with
the neighboring hydrophobic residues, the enzyme–isoamyl alco-
hol complex would have to be partially dehydrated, which may
destabilize the native conformation of the enzyme; this leads to
“molecular lubrication” [27]. Therefore, it may be concluded that
400 rpm; temperature: 60 ◦C; microwave irradiation: 50 W. ( ) fresh; (
) 1st
reuse; ( ) 2nd reuse; ( ) 3rd reuse.
isoamyl alcohol at higher concentration interacts with the enzyme
to form dead-end inhibitory complex.
3.8. Reusability of the catalyst
Novozym 435 reusability was studied out to ascertain its sta-
bility during the reaction. After each run, the enzyme was filtered,
washed with n-hexane and the solvent was evaporated prior to
next run. The dried enzyme was added with the makeup quantity
to make the loading 0.38% (w/v) for the further reusability reaction.
The activity was reduced after each reuse (Fig. 6).
During reusability study, the enzyme activity was measured
after each recycle by olive oil assay [12]. Novozym 435 is Can-
dida antarctica lipase immobilized on macroporous polyacrylate
resin beads. With this assay method, the activity was found to be
6900 Units/g for the fresh enzyme. After third reuse the activity was
reduced to 5400 Units/g, which is equivalent to 21.3% reduction.
The probable reason for decrease in activity is a combined effect of
different processes simultaneously at play including (i) denaturing
of enzyme by the product, (ii) stripping of water from the micro-
environment of enzyme particles, (iii) retention of some product
blocking the active sites, (iv) onset of solid–liquid mass transfer
resistance, and (v) onset of intra-particle diffusion resistance. The
detail explanation is as follows.
This is a three phase reaction with organic phase containing
reactants (continuous phase) and the product ester, polymer par-
ticle containing the enzyme (disperse phase), and water generated
in situ as co-product (dispersed phase). The reactants diffuse inside
the solid (polymer) matrix containing the enzyme and there is a
counter diffusion of the product ester and the co-product water.
Water is known to increase protein flexibility by forming multiple
hydrogen bonds with the enzyme molecule in organic compounds
[47] which has been cited in a number of papers on non-aqueous
media reaction [7,27,48]. A critical amount of water is necessary
for enzymes to be active in non-aqueous media as well as the
outer water layer surrounding the support should not be stripped
away by the solvent. Water is originally present in the pores of the
support. Novozym 435 contains this critical amount of water. Addi-
tional water generated in the system is likely to affect the enzyme
100
90
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
Time (min)
Fig. 5. Effect of isoamyl alcohol concentration. Reaction conditions – isoamyl
alcohol: 2.7, 4.2, 5.1 mol/L for myristic acid concentration: 2.7, 2.1, 1.7 mol/L respec-
tively; solvent-free, catalyst: 0.38% (w/v); speed of agitation: 400 rpm; temperature:
60 ◦C; microwave irradiation: 50 W. ( ) 1:1; ( ) 2:1; ( ) 3:1.

G.D. Yadav, P.A. Thorat / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 16–22
21
activity. Concentrations of isoamyl alcohol as well as myristic acid
8
7
6
5
4
3
2
1
0
are high in the solvent-free media in the current study (2.73 mol
each), as compared to other enzymatic reactions in non-aqueous
media (concentrations in mmol). Water generated in situ diffuses
out of the pore matrix and through the water film surrounding the
support (polymer) matrix, and then into the bulk organic phase,
which would be saturated with water, and then it forms a separate
phase. Similarly the product also diffuses out of the matrix in the
same way but is dissolved in the organic phase which is a continu-
ous phase. In this case, intra-particle water and product diffusion is
a parallel process and their transfer from external surface through
bulk organic phase is a series process. Besides, as the organic ester
concentration increases, it is likely to strip off the water from the
support. A number of non-polar solvents like hexane or toluene are
found to be better than polar solvents for enzymatic reactions in
non-aqueous media [9,49]. Generation of water beyond the critical
amount in solvent-free system may not affect the ongoing reaction
rate in a well agitated system. The denaturing of enzyme sites by
high concentration of product is also a possibility. The removal of
product during hexane washing would remove all products from
active sites.
0
0.2
0.4 0.6
0.8
1
1.2
1.4 1.6
1.8
1/myristic acid (1/M)
Once the enzyme is removed from the reaction mixture and is
stationary, there is an organic layer surrounding the resin with
less water layer. During subsequent runs, the enzyme is dena-
tured due to inhibitory effect of isoamyl alcohol which is used in
high concentrations as well as the water layer is also stripped off
offering more resistance. Organic substrates with poor solubility in
aqueous medium diffuse with difficulty through the intra-particle
water layer to the active centers of the enzyme. Once the agita-
tion is stopped and removed from microwave field for filtration
and washed, it may not remove the intra-particle product totally
thereby reducing the number of active sites.
Fig. 7. Lineweaver–Burk double inversion plot for different concentrations of
isoamyl alcohol at 60 ◦C. The initial rate of reaction is (M s−1 g-enz−1) for a fixed
catalyst loading of 0.38% (w/v). Concentrations of isoamyl alcohol: ( ) 0.8 mol/L;
(
) 1.1 mol/L; ( ) 1.4 mol/L; ( ) 2 mol/L.
of one product before the second substrate can associate to the
enzyme–substrate complex. The shape of the curve indicates sub-
strate inhibition at high concentrations of isoamyl alcohol.
A mechanism in which a product is released between addi-
tions of two substrates is common in group transfer or “substituted
enzyme” reactions. The mechanism is called “ping-pong bi-bi”
mechanism as each substrate addition is followed by a product
release. From these observations, a ping-pong bi-bi mechanism
with alcohol inhibition is postulated. These assumptions are used to
design a reaction mechanism that is depicted in Cleland’s notation
(Scheme 2).
By analogy to the classical mechanism of esterification by
lipases, it is assumed that myristic acid (A) binds first to the free
enzyme (E) and forms a noncovalent enzyme-acid complex (EA),
which releases the first product, water (P) and F modified enzyme.
The second substrate, isoamyl alcohol (B), reacts with F to give
the complex FB and gives the product isoamyl myristate (Q) and
free enzyme (E). Along with this, B also forms the dead-end com-
plex [EiB] by binding to free enzyme [E]. The rate equation for the
ping-pong bi-bi mechanism is as follows [50]:
3.9. Kinetic model
To analyze the reaction mechanism in detail as in other solvent-
free systems, reactions were carried out under earlier optimized
parameters, and 0.38% (w/v) Novozym 435 was used in each of the
experiment. Hence the effects of concentration of both the reac-
tants on the rate of reaction were studied systematically over a
wide range. In one set of experiments, the mole ratio of myristic
acid to isoamyl alcohol was increased from 1:1 to 2.5:1. In other
set of experiments the mole ratio of isoamyl alcohol to myristic
acid was increased from 1:1 to 2.5:1. That is – different concen-
trations of myristic acid were varied in the range of 0.8–2 mol/L,
for known concentration of isoamyl alcohol (0.8–2 mol/L). Samples
were taken from the reaction mixture periodically and analyzed by
GC.
The Lineweaver–Burk plots of reciprocal rate versus recipro-
cal concentration of myristic acid are illustrated in Fig. 7. The
Lineweaver–Burk plots demonstrate that the intercept decreases
with increasing concentration of isoamyl alcohol, although the
reverse is the case when the concentration of isoamyl alcohol is
low, which implies that isoamyl alcohol acts as an inhibitor. Based
on the kinetics study including effect of mol ratio and enzyme
reusability, it was confirmed that the isoamyl alcohol caused deacti-
vation of the enzyme. For the initial rate analysis, it is assumed that
isoamyl alcohol acts as a dead-end inhibitor and the inhibition step
is irreversible. The Lineweaver–Burk plot also demonstrates that
the isoamyl alcohol inhibition is stronger at low concentrations of
myristic acid.
v
m[A][B]
ꢀ =
(1)
KmB[A] + KmA[B](1 + [B]/Ki) + [A][B]
where v is the initial rate of reaction, vm is maximum rate of
reaction, [A] is initial concentration of myristic acid, [B] is initial
concentration of isoamyl alcohol, KmA is the Michaelis constant for
myristic acid, KmB is the Michaelis constant for isoamyl alcohol and
Ki is the inhibition constant due to isoamyl alcohol.
A B
P
B
Q
(1)
E
EA
FP
F
FB
EQ
E
A sequential mechanism can be ruled out as there is no com-
mon intersection for the family of lines in Fig. 7. The slope of the
lines is not influenced by the concentration of the fixed substrate
at low concentrations of isoamyl alcohol and myristic acid [50].
This is indicative of a mechanism that requires the dissociation
ki
EiB
E + B
Scheme 2. Cleland’s notation for a ping-pong bi-bi mechanism with single substrate
inhibition.

22
G.D. Yadav, P.A. Thorat / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 16–22
Table 1
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Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2012.06.011.
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