Chapter
5
Synthesis of alkyl benzoate esters in nonaqueous media
Chapter 5- Synthesis of alkyl benzoate esters
5.1.
Introduction
In recent years, industrial biotechnology, also called white biotechnology, for the
production of chemicals has gained a great importance due to its wide application
from the pharmaceutical industry to bulk chemicals (Woodley et al., 2013). The
exploitation of enzymes as catalysts in chemical synthesis has been much in evidence.
Lipases and esterases are most widely used biocatalysts amongst them. Lipases have
been reported to catalyse many reactions in organic solvents which include
esterification, interesterification, transesterification, hydrolysis, amidation, thioesterification, trans-thioesterification and epoxidation (Shi et al., 2011; Vosmann et
al., 2008; Sovova et al., 2008; Kraai et al., 2008; Yadav and Shinde, 2012a). These
reactions were reported to kinetically proceed via Ping Pong bi–bi mechanism, ternary
complex ordered bi–bi mechanism or ternary complex random bi–bi mechanism. It
involve in some cases, an inhibition by either substrate or product or both (Kraai et
al., 2008 Perez et al., 2007; Romero et al., 2007; Segel, 1975; Vazquez Lima et al.,
1996; Yadav and Lathi, 2004). For instance, 4,8-dimethylnon-7-en-1-ol substrate
inhibition was observed in Novozym 435 catalysed cinnamate ester synthesiswith nheptane as solvent (Yadav and Shinde, 2012b) while in esterification of lauric acid
with geraniol in isooctane, lauric acid inhibition was proposed (Vazquez Lima et al.,
1996).
Alkyl benzoate esters are mainly used in perfume and flavour industry, in
cosmetics, pharmaceuticals, as dye carriers, in paint industry, in adhesives, as
plasticizer in surface coating and as the solvents of cellulose acetate, nitrocellulose,
and insect repellents (Krause et al., 2009; Thum, 2004; Asai et al., 1994). They are
synthesized by chemical (Asai et al., 1994) and enzymatic routes (Gryglewicz et al.,
2000; Krause et al., 2009; Shintre et al., 2002; Vosmann et al., 2008). Most
commonly used Fischer esterification process for synthesis of alkyl benzoate employs
sulphuric acid and para-toluene sulphuric acid (PTSA). These catalysts generate
tremendous acid waste which is hazardous to nature. Therefore, an environmentally
friendly and inexpensive process that could avoid homogeneous liquid acids is the
most desirable for organic transformations (Krishna et al., 2001; Yadav and Bokade,
1996; Yadav and Kirthivasan, 1995, 1997). Several general routes of ester preparation
have been listed amongst which heterogeneous solid acids as catalysts could be used
59
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
using high temperature (Yadav and Mehta, 1993). In contrast with solid acids,
biocatalysts allow synthesis of esters to be performed at moderate temperatures
(Yadav and Shinde, 2012a, 2012b; Yadav and Devendran, 2012). However,
biocatalytic methods reported in previous literature for alkyl benzoate synthesis
possess low reaction rates and require large reaction time to obtain good conversions.
Microwave is established as an efficient heating source for variety of chemical
reactions, where high yields and reaction selectivity can be achieved at reduced time
of reaction (Loupy, 2002; Liao and Chung, 2013; Hoseinzadeh Hesas et al., 2013;
Jeon et al., 2013). Hence it was thought to employ microwave irradiation to increase
the reaction rates and conversions. Microwave irradiation results in an instantaneous
localized superheating which is achieved due to dipole rotation or ionic conduction
(Hayes, 2002; Kappe et al., 2013; Yadav and Borkar, 2006; Liao and Chung, 2011;
Yadav and Shinde, 2012b). Kinetic modelling for enzymatic transesterification of
methyl benzoate with different alcohols under microwave irradiation has not been
reported so far. Kinetics and mechanism for the lipase catalyzed transesterification of
methyl benzoate with different alcohols under microwave irradiation was investigated
to propose a suitable model. The effect of various kinetic parameters such as solvents,
speed of agitation, biocatalyst concentration, temperature and substrate concentration
were studied systematically on conversion and rate of reaction.
5.2.
Experimental section
5.2.1. Enzyme and chemicals
The Lipozyme RM IM, Lipozyme TL IM and Novozym 435 were obtained as free
gift samples from Novo Nordisk, Denmark. Lipase AYS Amano was a gift sample
from Amano Enzyme Inc. Japan. Lipozyme RM IM is Rhizomucor miehei lipase
immobilized on anionic exchange resin (activity of 30 U g-1, based on tristearin assay)
whereas Lipozyme TL IM is Thermomyces lanuginosus immobilized on silica.
Novozym 435 is Candida antarctica lipase B (CALB) immobilized on a macroporous
polyacrylic resin beads (bead size 0.3–0.6 mm, bulk density 0.430 g cm-3, water
content 3%, activity of 7,000PLU g-1). Lipase AYS Amano is Candida rugosa lipase
in form of lyophilized powder (activity 30,000 U g-1).
60
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
Chemicals used in the study were AR grade, purchased from reputed firm and used
as such with no further purification: Methyl benzoate, n-butanol, n-pentanol, nhexanol, n-octanol, benzyl alcohol, isoamyl alcohol, 2-ethyl-1-hexanol, n-heptane, nhexane, toluene, tetrahydrofuran and 1,4-dioxan (S.D. Fine Chemicals Pvt. Ltd.,
Mumbai, India).
5.2.2. Analytical method
The analysis was performed on a Chemito Gas Chromatograph (Model Ceres 800
plus) equipped with flame ionisation detector. BPX-1 capillary column (Make: SGE,
USA, 100% Dimethyl Polysiloxane; 25m× 0.22mm; 0.5µm film thickness) was used
for the analysis. The temperature of the column was 70°C for 2 min and then it was
increased to 90 °C at a ramp rate of 10 °C min-1 and held constant for 1 min. Further it
was increased to 230 °C at a ramp rate of 10 °C min-1 and held constant for 1 min.
Nitrogen was used as a carrier gas at a flow rate of 1 mL min-1. Both injection and
detection temperatures were set to 250 °C. n-Decane (2 % v/v) was used as an internal
standard to quantify the collected data for conversions and rates of reactions. Product
was confirmed by GC-MS (Make: PerkinElmer, USA, Model: Clarus 500).
5.2.3. Experimental set-up and procedure
5.2.3.1. Conventional heating
The experimental set up used for conventional heating studies was the same as
described in section 3.2.2 of Chapter 3. In a typical experiment, the reaction mixture
contained 20 mmol of methyl benzoate and 10 mmol of n-hexanol, diluted to 15 mL
with n-heptane as a solvent. It was agitated at 60 °C for 15 min at a speed of 300 rpm,
and a known quantity of enzyme was then added to initiate the reaction. Samples were
withdrawn periodically, filtered to remove fine particles, if any, and analyzed by GC.
5.2.3.2. Microwave heating
The experimental set up used for microwave heating studies was the same as
described in section 4.2.3.2 of Chapter 4. The CEM Discover microwave reactor
could be used to carry out reactions up to microwave power of 300 W. The
61
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
experiments were carried at constant temperature. A constant microwave irradiation
was provided (30–40 W). Experimental conditions were maintained same as in
conventional heating as described in section 5.2.3.1, unless otherwise stated.
5.2.4. Enzyme kinetics
Different kinetic parameters were studied to elucidate the kinetics of lipase
catalyzed transesterification of methyl benzoate with n-hexanol. Concentrations of
substrates were systematically varied over a wide range to study its effect on rate of
reaction using 300 mg Novozym 435. Various concentrations of n-hexanol (B) from 3
to 20 mmol were studied at different fixed quantity of methyl benzoate (A) (10-30
mmol). In other set of experiments, different concentrations of methyl benzoate (A)
from 10 to 30 mmol were studied at a fixed quantity of n-hexanol (3-20 mmol). The
quantified data obtained was used to calculate initial rates of reaction.
O
O
O
Methyl benzoate
+
Novozym 435
HO
O
+
OH
Microwaves
n- Hexyl benzoate
n- Hexanol
Methanol
Scheme 5.1. Microwave irradiated Novozym 435 catalysed synthesis of n-hexyl
benzoate
5.3.
Results and Discussion
5.3.1. Conventional heating vs. microwave irradiation
Enzymatic transesterification of methyl benzoate and n-hexanol (Reaction scheme
5.1) under conventional heating versus microwave irradiation conditions was
compared. A 6.5-fold increase in initial rate was observed under microwave
irradiation when compared with that under conventional heating which resulted in
reduced reaction time to achieve same conversion. This suggests that microwave
capturing nature of the reactants (methyl benzoate and n-hexanol) was contributing to
the elevated reaction rate. Recently, similar effect for enzymatic transformation under
microwave irradiation for various reactions was reported (Yadav and Shinde, 2012b,
62
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
Yu et al., 2010). Enzyme under microwave irradiation may possibly behave to some
extent in a different way and turn out to be more effective. This is due to
conformational modification in enzyme which is capable of assisting the substrate to
come close to active site of enzyme more easily under microwave irradiation. The
control experiments were conducted without enzyme as well as only under microwave
irradiation in the absence of enzyme. In either case, no conversion was observed. This
clearly indicates the synergistic effect between microwave irradiation and enzyme
catalysis.
5.3.2. Effect of various biocatalysts
Enzymatic transesterification of methyl benzoate with n-hexanol was selected as the
model reaction to study effect of different immobilized lipases on conversion and rate
of reaction. It can be seen from Fig. 5.1 that the conversion varied noticeably with the
type of lipase. The Lipozyme RMIM and Novozym 435 showed conversion of 79 %
and 13 %, respectively whereas very less conversion was obtained with Lipozyme TL
100
90
Conversion (%)
80
70
60
50
40
30
20
10
0
0
1
2
3
4
Time (h)
5
6
7
Figure 5.1: Effect of various biocatalysts [Reaction Condition: methyl benzoate, 20
mmol; n-hexanol, 10 mmol; solvent, toluene up to 15 ml; temperature, 50°C; speed of
agitation, 300 rpm; catalyst, 0.02 g cm-3;
(
Novozym 435,
Lipozyme RM IM,
Lipozyme TL IM,
× Lipase AYS Amano)]
63
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
IM and Lipase AYS Amano. The objective to study these different enzymes was to
discover if any significant activation could be attained owing to microwave
irradiation, regardless of their well-known applications. Novozym 435 being most
efficient biocatalyst amongst studied enzymes was used in further experiments.
5.3.3. Effect of various solvents
Different solvents such as 1,4-dioxan, tetrahydrofuran, toluene, n-hexane and nheptane were used to study its effect on conversion and rate of reaction (Fig. 5.2).
Nature of solvent has great impact on activity of enzyme which requires essential
water activity for maintaining the native, catalytically active enzyme conformation in
the organic solvent. For non-aqueous enzymatic transformations, hydrophobic
solvents are more favoured against hydrophilic solvents. Because hydrophilic solvents
cause striping of the necessary water layer around the enzyme, resulting in reduced
enzyme activity (Wehtje and Adlercreutz, 1997; Yadav and Devendran, 2012).
Maximum conversion of 97 % was obtained using n-heptane as a solvent and hence
was used for further studies.
100
90
Conversion (%)
80
70
60
50
40
30
20
10
0
0
1
2
3
4
Time (h)
5
6
7
Figure 5.2: Effect of various solvents [Reaction Condition: methyl benzoate, 20
mmol; n-hexanol, 10 mmol; solvent, up to 15 ml; temperature, 50°C; speed of
agitation, 300 rpm; Novozym 435, 0.02 g cm-3;
(
n-heptane,
n-hexane,
toluene, × 1,4-dioxan,
tetrahydrofuran)]
64
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
100
90
Conversion (%)
80
70
60
50
40
30
20
10
0
0
1
2
3
4
Time (h)
5
6
7
Figure 5.3: Effect of speed of agitation [Effect of various biocatalyst (Reaction
Condition: methyl benzoate, 20 mmol; n-hexanol, 10 mmol; solvent, n-heptane up to
15 ml; temperature, 50°C; Novozym 435, 0.02 g cm-3; speed of agitation,
(
100 rpm,
200 rpm,
300 rpm, × 400 rpm)]
5.3.4. Effect of speed of agitation
Optimal speed of agitation and enzyme loading of optimum particle size can be
used to minimize external mass transfer and internal diffusion limitations. Hence,
effect of speed of agitation in the range of 100–400 rpm was studied using Novozym
435 as biocatalyst and n-heptane as solvent (Fig. 5.3). The conversion profile and
initial rate data was obtained. An increase in conversion from 37 to 97 % was found
when speed of agitation was increased from 100 to 300 rpm. On the other hand, there
was no significant increase in rate and conversion at 300 and 400 rpm. At higher
speed of agitation, the biocatalyst particles were thrown out of the reaction medium
on the rector wall, resulted in reduced effective biocatalyst loading. Thus, further
studies were carried out at an optimum speed of 300 rpm.
5.3.5. Effect of different alcohols
Transesterification of methyl benzoate was studied with different alcohols such as
n-butanol, n-pentanol, n-hexanol, n-octanol, benzyl alcohol, 2-ethyl-1-hexanol and
65
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
100
90
Conversion (%)
80
70
60
50
40
30
20
10
0
0
1
2
3
4
Time (h)
5
6
7
Figure 5.4: Effect of different alcohols [Reaction Condition: methyl benzoate, 20
mmol; alcohol, 10 mmol; solvent, n-heptane up to 15 ml; temperature, 50°C; speed of
agitation, 300 rpm; Novozym 435, 0.02 g cm-3; (+ n-butanol,
hexanol,
n-octanol,
isoamyl alcohol, × benzyl alcohol,
n-pentanol,
n-
2-ethylhexyl alcohol)]
isoamyl alcohol under otherwise similar conditions. It was observed that the
conversion obtained with primary alcohols viz. n-butanol, n-pentanol, n-hexanol, noctanol and isoamyl alcohol was 72 %, 80 %, 97 % and 87 %, respectively (Fig. 5.4).
Transesterification with branched chain alcohol, 2-ethyl-1-hexanolshowed lower
conversions of 50 % whereas aromatic alcohol i.e. benzyl alcohol showed good
conversions of 79 %. The difference in conversion with chain length and nature of
alcohol can be related to effect of number of factors such as molecular size of alcohol,
solubility in reaction solvent and the affinity of lipase for specific individual alcohol
(Varma and Madras, 2010). Similar kind of effect causing variation in conversion
with change of chain length, type of alcohol have been reported in the literature with
respect to esterification and transesterification reactions catalyzed by lipase in the
organic solvents (Yadav and Lathi, 2004a; Shintre et al., 2002).
5.3.6. Effect of catalyst amount
The effect of amount of Novozym 435 varied from 0.007 to 0.027 g cm-3 for
transesterification of methyl benzoate was investigated under microwave irradiation
66
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
while molar ratios of substrates were maintained constant. It can be seen from Fig. 5.5
the conversion increased linearly with an increase in biocatalyst amount up to the
loading of 0.02 g cm-3. Above 0.02 g cm-3 biocatalyst loading, no significant increase
in the conversion was found which clearly shows that the biocatalyst loading was
much higher than the needed and the rate was limited by the external mass transfer.
Hence, biocatalyst loading of 0.02 g cm-3 under specified conditions was considered
be the most efficient and optimal.
5.3.7. Effect of n-hexanol concentration
The transesterification of methyl benzoate with n-hexanol was studied at different
moles of n-hexanol, keeping the moles of methyl benzoate (20 mmol) constant at
constant liquid volume. Maximum conversion and rate of reaction were achieved at
10 mmol of n-hexanol (Fig. 5.6). By increasing the moles of n-hexanol from 5 to 10
mmol, conversion and reaction rate was increased. The conversion was decreased
with further increase in n-hexanol concentration. This could be related to the
inhibitory effect of n-hexanol at high concentration on Novozym 435 enzyme.
100
90
Conversion (%)
80
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
Time (h)
Figure 5.5: Effect of catalyst loading [Reaction Condition: methyl benzoate, 20
mmol; n-hexanol, 10 mmol; solvent, n-heptane up to 15 ml; temperature, 50°C; speed
of agitation, 300 rpm;
Novozym 435, (
0.007 g cm-3,
0.013 g cm-3,
0.02 g cm-3, × 0.027 g cm-3)]
67
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
100
90
Conversion (%)
80
70
60
50
40
30
20
10
0
0
1
2
3
4
Time (h)
5
6
7
Figure 5.6: Effect of n-hexanol concentration [Reaction Condition: methyl benzoate,
20 mmol; n-hexanol 5-30 mmol; solvent, n-heptane up to 15 ml; temperature, 50°C;
speed of agitation, 300 rpm; Novozym 435, 0.02 g cm-3;
5 mmol;
10 mmol,
20 mmol, × 30 mmol)]
5.3.8. Effect of temperature
The effect of different temperature on conversion and initial rate under conventional
heating and microwave irradiation was investigated. As discussed in section 5.3.1,
under microwave irradiation overall conversion and the rate of reaction for
transesterification of methyl benzoate was higher (Fig. 5.8) than that under
conventional heating (Fig. 5.7). With an increase in temperature in range of 40–70 °C
under microwave irradiation, an initial rate and conversion was found to be increased
from 3.01 × 10-3 to 19.16× 10-3 mol L-1 min-1 g-1 of enzyme and from 37 % to 97 %,
respectively. This is attributed to the momentum provided by microwave energy to
overcome energy barrier and thus the reaction is completed more quickly than
conventional heating. Further, under microwave irradiation, high instantaneous
heating of the substance(s) above the normal bulk temperature results in greater
number of more energetic collisions is the primary factor for the observed rate
enhancements. The activation energy values were obtained by the Arrhenius plot (Fig.
5.9) as 13.79 and 14.14 kcal mol-1 under microwave and conventional heating
respectively.
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© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
100
90
Conversion (%)
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10 12 14
Time (h)
16
18
20
22
24
Figure 5.7: Effect of temperature (conventional heating) [Reaction Condition: methyl
benzoate, 20 mmol; n-hexanol, 10 mmol; solvent, n-heptane up to 15 ml; speed of
agitation, 300 rpm; Novozym 435, 0.02 g cm-3,
(
40 °C,
50 °C,
60 °C, × 70 °C)]
100
90
Conversion (%)
80
70
60
50
40
30
20
10
0
0
1
2
3
4
Time (h)
5
6
7
Figure 5.8: Effect of temperature (microwave heating) [Reaction Condition: methyl
benzoate, 20 mmol; n-hexanol, 10 mmol; solvent, n-heptane up to 15 ml; speed of
agitation, 300 rpm; Novozym 435, 0.02 g cm-3,
(
40 °C,
50 °C,
60 °C, × 70 °C)]
69
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
-4
-5
ln r
-6
-7
-8
-9
-10
0.00285
0.00295
0.00305
1/T (K-1)
Figure 5.9: Arrhenius plot (
0.00315
microwave,
0.00325
conventional)
100
Conversion (%)
Activity retained (%)
90
80
70
60
50
40
30
20
10
0
Fresh
1st Reuse
2nd Reuse
Reusability
Activity retained (%)
3rd Reuse
Conversion (%)
Figure 5.10: Effect of reusability
5.3.9. Effect of reusability
The reusability of Novozym 435 under optimized process parameter conditions was
studied to examine the stability and recyclability of the enzyme. The enzyme after
first use was filtered, washed with n-hexane after each use, dried at room temperature
and reused for further studies. Similar steps were followed for each further reusability
70
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
studies. Marginal decrease in conversion from 97 % to 82 % after three reuses was
observed for transesterification of methyl benzoate with n-hexanol which may
possibly be because of the loss of enzyme during handling (Fig. 5.10). Thus, the
Novozym 435 was quite stable for reuse.
5.3.10. Kinetic model based on initial rate measurements
From the initial rate (V) measurements, it was observed that the rate increased with
increasing the concentration of the methyl benzoate (A). When the concentration of nhexanol (B) was increased keeping the concentration of methyl benzoate constant, the
rate of reaction increased and reached the maximum at critical concentration. Further
increase in n-hexanol concentration resulted in the reaction rate to fall and thus the
substrate inhibition was notable. Therefore, it may be concluded that n-hexanol at
higher concentrations reacts with the enzyme to form dead end inhibitory complex.
There was no evidence of inhibition by methyl benzoate (A) at any concentration
tested. The Lineweaver Burk plot 1/V versus 1/[B] for varied initial concentrations of
A shows that lines are not parallel, ruling out the possibility of a Ping Pong bi-bi
mechanism. In fact, the lines intersected at a point suggesting a ternary complex
mechanism (Fig. 5.11). The lipase-catalyzed reaction mechanism involves the
formation of an acyl enzyme complex with the acyl donor, which is methyl benzoate
(A). This explanation rules out a random mechanism and it can only be an ordered bibi mechanism. According to this, the lipase (E) will react with acyl donor (A) to form
a complex (EA). The second substrate (B) then reacts to form a ternary complex
(EAB). At higher concentrations of n-hexanol (B) a dead end complex (EiB) is formed.
These assumptions are used to design a reaction mechanism that is depicted in
Cleland’s notation, as shown below.
E + A
EA + B
EAB
+B
EB’
EPQ
E + P + Q
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© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
The rate equation is as follows:
V=
Vmax [ A][ B ]
(5.1)
 [ B] 
 [ B] 
K iA K mB 1 + B  + K mB [ A] + K mA [ B ] 1 + B  + [ A][ B ]
 Ki 
 Ki 
Where [A] is methyl benzoate concentration (mol L-1), [B] is n-hexanol
concentration (mol L-1), KmA is the Michaelis constant for methyl benzoate (mol L-1),
KmB is the Michaelis constant for n-hexanol (mol L-1), KiA is the dissociation constant
for enzyme– methyl benzoate complex and KiB is the inhibition constant due to nhexanol. V and Vmax are the initial rate and maximum rate (mol L-1 min-1),
respectively.
Kinetic constants were obtained by using Polymath 6.0 software as follows: Vmax
(mol L-1) 0.012, KmA (mol L-1) 1.014, KmB (mol L-1) 0.026, KiA 0.015 and KiB 0.151. A
parity plot of experimental versus simulated rate gave straight line passing through
origin with excellent correlation coefficient. This demonstrates that the proposed
model is valid (Fig. 5.12).
2000
1800
1/V (L.min/mol)
1600
1400
1200
1000
800
600
400
200
0
0
1
2
3
1/B (L/mol)
4
5
6
Figure 5.11: Lineweaver-Burk plot of 1/initial rate (L min mol-1) versus 1/[ nhexanol] (L mol-1) at different constant methyl benzoate concentration
(
10 mmol,
20 mmol,
30 mmol).
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© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.
Chapter 5- Synthesis of alkyl benzoate esters
0.006
rsimulated (mol/L.min)
0.005
0.004
R² = 0.9809
0.003
0.002
0.001
0
0.000
0.001
0.002
0.003
0.004
0.005
0.006
rexperimental (mol/L.min)
Figure 5.12: Parity plot
5.4.
Conclusions
Industrially important alkyl benzoate esters were synthesised using Novozym 435 as
biocatalyst
under
synergistic
effect
of
microwave
irradiation.
Enzymatic
transesterification of methyl benzoate was also studied with different alcohols such as
n-butanol, n-pentanol, n-hexanol, n-octanol, benzyl alcohol, isoamyl alcohol and 2ethyl-1-hexanol. Optimized kinetic parameters obtained were 300 rpm, speed of
agitation; n-hexane, solvent; 0.02 g cm-3, Novozym 435 loading and 60 °C,
temperature. A kinetic model was postulated based on initial rate data and conversion
profiles. The ternary complex ordered bi–bi mechanism with n-hexanol substrate
inhibition was assumed for enzymatic transesterification of methyl benzoate with nhexanol. The proposed mechanism appropriately fitted the data well for microwave
assisted Novozym 435 biotransformation reaction. The enzyme was reusable.
73
© Somnath D. Shinde, Institute of Chemical Technology (ICT), Mumbai.