Journal of Engineering Science and Technology

Special Issue on SOMCHE 2014 & RSCE 2014 Conference, January (2015) 78 - 87
© School of Engineering, Taylor’s University

REACTION SYNTHESIS AND KINETIC MODELING OF

ISOAMYL ACETATE VIA ENZYMATIC ESTERIFICATION
IN SOLVENT-FREE SYSTEM

Y. A. NURHAZWANI, M. D. MASHITAH, R. A. S. SYAMSUL*

School of Chemical Engineering, Universiti Sains Malaysia, Engineering Campus, 14300

NibongTebal, SeberangPrai Selatan, Penang, Malaysia
*Corresponding Author: chsyamrizal@usm.my

Abstract
The aim of this study is to determine the reactions involved in esterification
reaction of isoamyl acetate, in solvent-free system (SFS), between acetic
anhydride and isoamyl alcohol in the presence of enzyme Candida antarctica
Lipase B (CALB) as catalyst. This study was done in a batch system using
stopped rubber conical shake flask. Results show that there is two main
reactions took place, which are: (i) between acetic anhydride and isoamyl
alcohol, and (ii) between acetic acid and isoamyl alcohol. Kinetic modeling was
also done using First Principle model and found that the kinetic constant for k1
and k2 value equals to -0.0135 and 0.2530 respectively.
Keywords: Esterification,Isoamyl acetate, Solvent-free system, Kinetic modeling.

1. Introduction
Esters are one of the most extensive of all naturally occurring organic compounds
which contain functional group, -COOR. Esters are widely used for various
purposes in chemical industry. For instance, ethyl acetate is commonly used as
solvent in extraction process [1], and dialkyl phthalates are used in plastic
industry to keep polymer from becoming brittle [2]. Many simple esters are
pleasant-smelling liquids and mainly used as fragrant odors of fruits and flowers.
For example, methyl butanoate is an element found in pineapple oil whereas
isoamyl acetate is an element of banana oil [3]. These esters are also naturally
present in animal fats and oil [4] and in many biologically important molecules.
Esters are ubiquitous and contain ‘nature-identical’ substance that can be used to
substitute natural flavors and fragrances.
Esterification is a process of combining an organic acid (R-COOH) with an

78
 Reaction Synthesis and Kinetic Modeling of Isoamyl Acetate via Enzymatic . . . . 79

Nomenclatures
C Concentration
i, j substrates
k Reaction rate constant
n Order of reaction
r Reaction rate

Greek Symbols
β1 Temperature
β2 Mass of enzyme
β3 Reaction time
β4 Reciprocal of substrates concentration

Abbreviations
CALB Candida antarctica Lipase-B
SFS Solvent-Free System
alcohol (R-OH) to form an ester (R-COO-R) and water (H2O).Esters have great
application which primarily used in food and cosmetic industries [5-7]and
biodiesel production[8]. Only minimum amount of esters are used as lubricant[7],
confectionary [9], and in pharmaceutical product [6]. Traditionally, esters can be
directly extracted from plant material or produced by chemical synthesis [10, 11].
However, the high handling cost and low product quantity makes it inappropriate
to be implemented for industrial application [12].
Researchers have come out with chemical route in the presence of a strong acid
catalyst at high temperature to produce esters at a very low cost. However, there are
many by-products and the removal of the catalyst from reaction medium is difficult.
In addition, the final product is not natural [10] as has been defined by U.S. Food
and Drug Administration in ‘Code of Federal Regulation’ [13]. Then,
biotechnological route of ester production was introduced by using free and
immobilized lipase from various sources in organic solvent to encounter the
problem in the chemical route[14]. Although higher conversion yields, solvent
toxicity is a problem for many applications. On top of that, some organic solvents
used are too expensive to allow profitable production in industrial scale [15, 16].
Additionally, a solvent-free system increased isoamyl acetate volumetric production
thus making the system preferred and attractive for industrial application [17].
Synthesis of isoamyl acetate in this study has been investigated by using acetic
anhydride and isoamyl alcohol catalysed by CALB in a solvent-free system.

2. Material and Methods

2.1. Equipment and materials
The experiments were carried out in a 100ml stopped rubber shake flask, which
was incubated in Incu-Shaker Mini (Benchmark Scientific, New Jersey) at
150rpm. Temperature was set at 30°C, 40°C and 50°C. Working pressure was at
ambient pressure condition.
Isoamyl alcohol used was supplied by Merck Co. (Malaysia), while acetic
anhydride (reagent grade, ≥98%) was supplied by Sigma Aldrich (Malaysia). All

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80 Y. A. Nurhazwani et al.

substrates were used without any pre-treatment. Immobilized enzyme from Candida
Antarctica, Novozyme 435 (specific activity≥ 10000/, recombinant, expressed
in Aspergillus Niger) supplied by Sigma Aldrich (Malaysia).

2.2. Synthesis procedure

Isoamyl acetate synthesis was carried out without any organic solvent in 100ml
stopped rubber shake flask with working volume of 15ml. Acetic anhydride and
isoamyl alcohol was added into the flask until alcohol to acid ratio equals to 0.1.
Then 1wt% of immobilized enzyme from Candida antarctica was inserted into
the mixture. The reaction mixture was then incubated in an Incu-shaker mini
(Benchmark) at 150 rpm for 360 minutes. Samples were withdrawn Every 10 min
and analysed with gas chromatograph until 6hr of reaction time. Experimental
procedures were repeated for different amount of enzyme and conditions as in Table 1.

2.3. Analysis
0.5ml of the reaction mixture was withdrawn every 10 min. Samples were
analysed by Agilent 7820A Gas Chromatograph supplied by Agilent
Technologies, equipped with a hydrogen flame ionization detector and a SGE
BP21 (FFAP) column (60m x 0.32mm x 0.25 µm). Helium was used as carrier
gas at flow rate of 5ml/min. After injection of samples, the temperature of oven
was kept at 100°C and linearly increased to 140°C. The rate of temperature
increase was set at 70°C/min, and was kept at 140°C for the remaining time of
analysis. Injector and detector temperatures were set at 200°C and 250°C,
respectively. Quantification of data was done by calibration with standards
samples. The peaks of the retention time are as follows: isoamyl acetate, 2.26min;
isoamyl alcohol, 2.38 min; acetic anhydride, 2.48min; and acetic acid, 3.21 min [18].

2.4. Experimental design with Response Surface Methodology (RSM)

Experimental design was conducted using Design of Experiment (DoE) software
(Design Expert 6.0.6), Start-Ease. A three-level and four-factor design was used
to determine the enzyme kinetic constants. Four experimental parameters
(operating temperature, T; mass of enzyme, m; reaction time, t; and reciprocal of
substrates concentration,1/[A]) were used in this study and 3-level indicated the
level of each range (-1, 0, +1).
Table 1. Experimental ranges and levels of variables.
Levels
Variables Coding Unit
-1 0 +1
Temperature  °C 30 40 50
Mass enzyme  wt% 4 8 12
Reaction time  h 2 4 6
Reciprocal of
substrates  l/mol 0.12 0.20 1.18
concentration

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 Reaction Synthesis and Kinetic Modeling of Isoamyl Acetate via Enzymatic . . . . 81

The list of parameters and levels studied in this experiment are shown in Table
1. Preliminary studies showed that all of the variables are statistically significant
and ignoring these variables may affect the design of experiment. Central
Composite Design (CCD) was chosen because it provides information on
experimental variable effects and overall experimental error in a minimum
number of runs. CCD also can be used under different experimental regions of
interest and operability. This CCD design consists of 2 parts: factorial points (-1,
+1), and center points (0, 0). Six replicate runs at the center point (0, 0) of the
design were performed to allow the estimation of pure error. All of the
experiments were carried out in the randomized order to minimize the
unexplained variability in the observed responses due to irrelevant factor.
A second order polynomial model was predicted by a multiple regression
procedure. This resulted in an empirical model related to the response by the equation:

 =  +     +    +      + , (1)

where  ,  , … ,  are the input variables, which influence the response Y;

  = 1,2, … !,   = 1,2, … , ; # = 1,2, … , !are unknown parameters; and 
is random error.

2.5. Validation of RSM model and determination of kinetic constant

RSM model developed needs to be validated in order to evaluate its efficiency.
The validation tests were done based on the conditions factor suggested by RSM
and the result is compared with the experimental values.
The obtained reaction rate model equation from RSM is then fitted to the
modified Michaelis-Menten equation using POLYMATH® software. By using
nonlinear equation solver, kinetic constants were determined.

3. Result and Discussion

3.1. Isoamyl acetate reaction synthesis
Theoretically, acetic anhydride possesses two acyl groups. In reaction with
isoamyl alcohol, one of the acyl from acetic anhydride will bind with isoamyl
alcohol and discharge one H+ to form isoamyl acetate and acetic acid. Then,
excess acyl (from acetic acid) will react with excess alcohol to form another
isoamyl acetate and water. The details of reaction scheme are as shown in Fig. 1:
Based on the chemical reaction scheme shown, esterification of isoamyl
acetate undergone two reactions, first is between the acetic anhydride and isoamyl
alcohol, producing acetic acid and isoamyl acetate, and the second reaction is
between the acetic acid and excess isoamyl alcohol, producing another isoamyl
acetate and water as by-product. The existence of water in the mixture by the
secondary reaction makes the overall esterification reactions more favorable
towards the hydrolysis reaction. This fits with the Le Chatelier’s Principle, where
the dynamic equilibrium of the reaction will be shifted towards the left-hand side
of the reversible reaction as the by-product increases. This condition was in agreement
with the researches done by Ghamgui et al. (2006) and Luhong et al. (2001).

Journal of Engineering Science and Technology Special Issue 8 1/2015

82 Y. A. Nurhazwani et al.

(a) Main reaction

(Isoamyl alcohol) (Acetic anhydride) (Isoamyl acetate) (Acetic acid)

(b) Secondary reaction

(isoamyl alcohol) (acetic acid) (isoamyl acetate) (water)

(c) Overall reaction

(isoamyl alcohol) (acetic anhydride) (isoamyl alcohol) (water)

Fig. 1. The details of reaction scheme.

3.2. Response surface analysis

The effect of temperature, mass of enzyme, reaction time and reciprocal of
substrates concentration on the enzymatic reaction rate were investigated using
response surface methodology. The results of the developed CCD are given in
Table 2. Multiple regression coefficients obtained from a least squares analysis
used to predict quadratic polynomial model for the reciprocal enzymatic reaction
rate are summarized in Table 3.
All coefficients obtained from the full quadratic polynomial model were
evaluated by regression analysis and tested for their significance. The
insignificant coefficients were eliminated on the basis of p values. It is well
known that smaller p values (<0.001) indicated significant value of model or
factor. From the result, it was found that coefficient for  ,  and   were
highly insignificant, hence the predicted polynomialmodel was rearranged by
eliminate terms which consist of  ,  and   in full quadratic model. The
coefficient of the modified quadratic model can be seen in Table 3. The
coefficient of determination for the modified quadratic model ( $ = 0.98) is
better than the full quadratic model ($ = 0.97 ) implies that the model was
satisfactory.
The final model for reciprocal of enzymatic reaction rate obtained from the
CCD analysis is:
 
)
= 7.0784 − 0.1867-! − 0.3193/! − 0.87951! + 10.0771 2[3] 5 +
4
0.0028- ! + 0.0239/ ! + 0.10601 ! − 0.0024-/! −
  
0.1341 6- 7[3] 89 − 0.0732 6/ 2[3] 59 − 0.1623 61 2[3] 59 (2)
 4 4

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 Reaction Synthesis and Kinetic Modeling of Isoamyl Acetate via Enzymatic . . . . 83

Table 2. Experimental results of the CCD.

Parameter Response
Exp Reaction
Temp Menz Time 1/[A](l/mo [ester] [alc] [anh]
No. rate
(°C) (%) (h) l) (mol/l) (mol/l) (mol/l)
(mol/l.min)
1 50 4 2 0.1490 1.8970 0.4000 2.6890 0.4310
2 40 8 4 0.2022 2.6480 1.4390 0.1350 0.6917
3 50 4 6 0.1490 2.3710 0.0860 2.3600 0.4882
4 50 4 2 1.1794 0.9700 5.6190 0.0060 0.1819
5 30 4 2 1.1794 0.9550 5.3800 0.0080 0.1256
6 30 12 2 0.1490 2.2320 0.1090 2.3590 0.5600
7 40 8 4 1.1794 1.0540 5.2330 0.0290 0.2493
8 30 12 6 0.1490 1.8240 0.0650 1.9150 0.6184
9 50 8 4 0.2022 2.6680 0.4530 0.3890 0.7016
10 30 4 6 0.1490 1.9270 0.1260 2.5430 0.4872
11 30 4 6 1.1794 1.0880 5.1200 0.0060 0.1449
12 30 12 2 1.1794 1.0390 5.0510 0.0070 0.1319
13 40 12 4 0.2022 2.7000 0.0680 1.9000 0.6920
14 30 12 6 1.1794 1.0980 5.2590 0.0070 0.1609
15 40 8 4 0.2022 2.6500 0.4360 0.1400 0.6920
16 50 12 6 0.1490 2.1600 0.0380 2.2980 0.5805
17 50 12 2 0.1490 2.0290 0.1710 2.6780 0.5224
18 40 4 4 0.2022 2.5530 0.7550 0.6610 0.6525
19 40 8 6 0.2022 2.8480 0.3540 0.0910 0.6940
20 30 8 4 0.2022 3.0180 0.3570 0.1000 0.7372
21 50 12 2 1.1794 1.1040 5.3940 0.0310 0.2560
22 50 4 6 1.1794 0.9850 0.9850 0.0060 0.2103
23 40 8 4 0.1490 3.0720 0.5800 0.2340 0.7339
24 40 8 2 0.2022 2.6040 0.6190 0.2580 0.6169
25 30 4 2 0.1490 1.8770 0.2690 2.9230 0.4350
26 40 8 4 0.2022 2.7000 0.4350 0.1370 0.6922
27 50 12 6 1.1794 0.9760 4.7230 0.0270 0.2856

Table 3. Regression coefficients of the quadratic model for CCD.

Full quadratic model Modified quadratic model
Factor
Prob> F Std. Error Prob> F Std. Error
Model < 0.0001 0.581429 < 0.0001 0.117647
:; 0.1083 0.120609 0.0807 0.111738
:< 0.2676 0.120609 0.2287 0.111738
:= 0.6098 0.120609 0.5800 0.111738
:> 0.3903 3.431322 < 0.0001 0.131503
:<; 0.2107 0.209439 0.1492 0.186699
:<< 0.0987 0.209439 0.0588 0.186699
:<= 0.0697 0.209439 0.0383 0.186699
:<> 0.9051 2.61164 - -
:;< 0.2677 0.083963 0.2288 0.077787
:;= 0.3775 0.083963 - -
:;> < 0.0001 0.152103 < 0.0001 0.140916
:<= 0.8902 0.083963 - -
:<> 0.0781 0.152103 0.0552 0.140916
:=> 0.0542 0.152103 0.0360 0.140916
?< 0.99 0.99
adjusted ?< 0.97 0.98

3.3. Validation of RSM model

Model developed in the above part has been validated by experimental data. The
model validation experiment was conducted at 40°C at 8wt% of mass enzyme for
6hrs. Figure 2 shows the comparison of isoamyl acetate reaction rate between
model developed by RSM and from experimental data.

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84 Y. A. Nurhazwani et al.

The continuous line indicates the actual result from the experimental data and
the dashed line indicates the result from model developed by RSM. The model
and experimental result shows a good agreement for reaction rate prediction with
$ value of 0.98. This indicates that the model can be used to find the kinetic
constant for this study.

Fig. 2. Reaction rate curve for Isoamyl Acetate

production for developed model and experimental data calculation.

3.4. Kinetic constant

Based on the general reaction rate equation,
@ = AB (3)
where @ is the reaction rate for product j, k is the reaction rate constant, A is the
concentration of product j, and n is the order of reaction.
Isoamyl acetate enzymatic synthesis between acetic anhydride and isoamyl
alcohol having forward and backward reaction, hence Equation 3 becomes;
@ =  A
B
− A
B
(4)
where  CDE are rate constants for forward and backward reaction respectively,
A FGHA are concentration for substrates i and product j respectively, and n is the
order number of the reaction.
Overall reaction for isoamyl acetate esterification from acetic anhydride and
isoamyl alcohol is:
LM
2IJ IJIJ !IJ IJ KJ + IJ IK! K N 2IJ IKKIJ IJ !IJIJ + J K (5)
OP

Based on Eq. (4), there were few reaction rate equations that are possible for this
isoamyl acetate enzymatic esterification reaction, therefore all of the reaction rate
constants for all equation developed were find out using nonlinear equation solver
in POLYMATH and the regression of each equation were compared. All of the
possible reaction rate equation, constant developed and regression analysis were
tabulated in Table 4.
Table 4 shown that the significant reaction rate equation was for the third
reaction rate with $ = 0.9385and FH#. $ = 0.9360. The kinetic constant for
 CDE equals to -0.0135 and 0.2530 respectively with order number of 1.

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 Reaction Synthesis and Kinetic Modeling of Isoamyl Acetate via Enzymatic . . . . 85

Table 4. Possible reaction rate equation,

constant developed and regression analysis.
No. Reaction rate equation Q; Q< ?< RST. ?<
1 @ =   AU ! +  AV ! 0.0461 0.3984 0.5705 0.5533
2 @ =    AU ! +  AW ! 0.0078 0.2426 0.9121 0.9086
3 @ =    AX ! +  AW ! -0.0135 0.2530 0.9385 0.9360
4 @ =    AU ! + Y AW Z 0.0013 0.2447 0.9109 0.9073
5 @ =    AU ! +  AW ! 0.0187 0.0912 0.8111 0.8035
6 @ =    AX ! +  AW ! 0.0032 0.0961 0.7864 0.7778
7 @ =    AX ! +  AW ! 0.0172 0.0951 0.7890 0.7806
8 @ =    AU AX ! +  AW ! 0.1912 0.0909 0.7936 0.7853
9 @ =    AU AX ! +  AW ! -0.0147 0.2474 0.9109 0.9073

3.5. Validation of kinetic model

To certify the validity of kinetic constant developed by POLYMATH in above
section, comparison of reaction rate from developed kinetic constant and actual
reaction rate from experimental data was done. Figure 3 shows the comparison of
isoamyl acetate reaction rate calculated by using kinetic constant developed by
POLYMATH and reaction rate from experimental data.
The continuous line indicates the actual result from the experimental data
and the dashed line indicates the result from model developed by RSM. The
model and experimental data shows a good agreement with $ value of 0.94.
This indicates that the developed kinetic constants are valid to be use in the
study of enzymatic esterification of isoamyl acetate from acetic anhydride in
solvent free system.
Therefore, the final reaction rate equation for enzymatic esterification reaction
from acetic anhydride and isoamyl acetate is;
@ =  −0.0135AX + 0.2530AW (6)
where AX is the concentration of isoamyl alcohol, and AW is the concentration of ester.

Fig. 3. Reaction rate curve for Isoamyl Acetate production

from developed kinetic constant and experimental data calculation.

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86 Y. A. Nurhazwani et al.

4. Conclusion
Synthesis of isoamyl acetate was carried out by reacting acetic anhydride with
isoamyl alcohol in a solvent-free system. Candida Antarctica lipase-B (CALB)
was used to enhance the production rate of ester. There are two chemical
reactions involved in the esterification of isoamyl acetate from acetic anhydride
and isoamyl alcohol. The main reaction is between acetic anhydride and isoamyl
alcohol, and the second reaction is between produced acetic acid with the excess
isoamyl alcohol. The modelling and optimization process of this enzymatic
esterification process were done using RSM based model. A reciprocal of reaction
rate model was developed and the ANOVA test implied that the model was
satisfactorily representing the real relationship of the main reaction parameters
with R2 (0.98). Kinetic constants of the kinetic equation were obtained from
POLYMATH software by using the experimental data and reaction rate model
developed before by RSM. The final model resulted  and value equals
to−0.0135[G\ and 0.2350 [G\ respectively. Validation test were done by
doing graphical comparison between the kinetic model developed and
experimental data, it shows that the kinetic model developed is having a good
agreement with the actual experimental data by R2 value of 0.94.

Acknowledgement
This study has been supported by a Fundamental Research Grant Scheme (FRGS)
(PJKIMIA/6071255).

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