Kinetics of Amycolatopsis mediterranei DSM 43304 lipase-mediated ...

Kinetics of Amycolatopsis mediterranei DSM 43304 lipase-mediated synthesis of
isoamyl acetate in n-hexane
Dharmendra S. Dheeman a , Jesús M. Frías a , Gary T.M. Henehan a
a School of Food Science & Environmental Health, Dublin Institute of Technology (DIT), Cathal Brugha
Street, Dublin 1, Ireland (dheeman@gmail.com, jesus.frias@dit.ie, gary.henehan@dit.ie)
ABSTRACT
Isoamyl acetate is one of the most employed esters in food industry because of its banana flavour property. A
number of commercial lipases have been employed to study kinetics of esterification and trans-esterification
in organic solvents to produce isoamyl acetate. However, there are few reports of application of noncommercial
lipases for the synthesis of isoamyl acetate by direct esterification. The present study is the first
report studying the kinetics of direct esterification reaction between a short-chain acid and isoamyl alcohol to
synthesize isoamyl acetate using a non-commercial Celite-immobilized lipase from Amycolatopsis
mediterranei DSM 43304. The objectives of the present wrok were to study the Celite-immobilized A.
mediterranei lipase-mediated kinetics and the effects of different parameters on the production of isoamyl
acetate through direct esterification in n-hexane. The effects of different operating parameters on molar
conversion and initial rates of reaction were studied in the absence of mass-transfer limitations. The reaction
rate was enhanced by increasing catalyst loading, concentration of acetic acid and the operating temperature;
however it was significantly decreased by an excess of acetic acid (>0.6 mol L -1 ) and addition of water (>1%,
v/v). The optimum parameters for maximum conversion (59%) were found to be an acid/alcohol molar-ratio
of 2, a catalyst concentration of 7.5% (w/v), and an initial addition of 1% (v/v) water at a reaction
temperature of 50 °C. A kinetic model based on a postulated multi-substrate Ping Pong Bi Bi mechanism was
proposed to describe the lipase-catalyzed direct esterification of isoamyl alcohol with acetic acid. Different
simplified kinetic models, derived from the classic Ping Pong Bi Bi mechanism, were fitted by non-linear
regression to the experimental data. Finally, incremental F-tests were performed to assess the simplest model
that was able to provide a statistically good fit throughout the entire time-course of the reaction.
Keywords: Amycolatopsis mediterranei; esterification; isoamyl acetate; kinetic modeling
INTRODUCTION
Isoamyl acetate is one of the most widely used esters in the food industry because of its characteristic banana
flavour. In the USA alone, more than 74 ton per annum of this ester is used [1]. Enzymatic synthesis of this
and other aroma active esters is very relevant to the food industry [2]. A number of commercial lipases have
been employed for direct esterification and trans-esterification in organic solvent to produce isoamyl acetate
[3-5]. However, few attempts have been made to synthesize isoamyl acetate using non-commercial lipases [6,
7]. Moreover, the ester yields obtained were rather low due to inhibitory effects on the enzymes by shortchain
acids employed [8]. In view of this, it is of interest to explore the application of newly isolated lipases
in the synthesis of isoamyl acetate by direct esterification in organic media. In order to identify optimum
conditions for performing ester synthesis, it is important to know reaction kinetics and pertinent rate
constants [9]. Since lipases catalyze multi-substrate and multi-product reactions, their kinetic models, based
on a single substrate Michaelis-Menten model, will be inadequate to provide accurate descriptions of
prevailing mechanistic phenomena and hence a two step reaction involving Ping Pong Bi Bi mechanism have
been proposed for lipase catalyzed reactions in organic media [10-12]. Despite the fact that several kinetic
studies are reported, the information required for process design and optimization of reaction under
investigation is limited [4]. Therefore, our aim in the present investigation was to test the performance of
Celite-immobilized A. mediterranei DSM 43304 lipase for the synthesis of isoamyl acetate in n-hexane and
model its kinetics. To this end, Celite-immobilized lipase from A. mediterranei DSM 43304 was employed
to catalyze the direct esterification of isoamyl alcohol using acetic acid as an acyl donor. The reactions were
performed in a batch stirred processing set-up using different reaction parameters to optimize initial rates and
conversion yields. Furthermore, the data were fitted by non-linear regression to increasingly simpler
mechanistic models (until a final model was selected), based on the assumptions underlying the Ping Pong Bi
Bi mechanism, and simulation curves were compared with the experimental results to validate the proposed
kinetic model.

MATERIALS & METHODS
Biocatalyst
A. mediterranei DSM 43304 having high lipolytic activity was screened and optimized for lipase production
as described by Dheeman et al. [13]. The extracellular lipase from A. mediterranei DSM 43304 was
immobilized onto Celite matrix and the hydrolytic activity of the Celite-immobilized lipase was measured
titrimetrically using an olive oil emulsion in 50 mM Tris-HCl buffer (pH 8) at 37 °C [14]. With this assay,
the activity of Celite-immobilized lipase was 120 IU g -1 . The water content of the immobilized enzyme
particles, by comparison of weight before and after heating at 105 °C for 12 h, was determined to be 2.2%
(w/w). The same initial batch of immobilized lipase was used throughout the realization of this work.
Esterification reaction
Unless stated otherwise, synthesis of isoamyl acetate was carried out in a mechanically agitated glass reactor
with 50 mL capacity. The reactor containing 300 mM of isoamyl alcohol and 750 mg Celite-immobilized A.
mediterranei DSM 43304 lipase as a catalyst in n-hexane was placed in a thermostatic water bath providing a
constant temperature to within ±0.1 °C. When the reaction temperature reached the set value (37 °C), acetic
acid was added, to a final concentration of 300 mM, to initiate the reaction (total reaction volume 10 mL). A
reaction in the same conditions without enzyme was realized in parallel and was used as a control. All
experiments were performed in duplicate and replicated at least twice. The samples were analyzed on a gas
chromatograph (Perkin Elmer AutoSystem XL, MA, USA) connected to a DB-5 capillary column (30 m ×
0.25 mm, d f 0.25 µm, Agilent JW Scientific, CA, USA) and a flame ionization detector (FID).
Kinetic modeling
For kinetic modeling a total of 20 batch experiments were performed providing a total of 180 duplicate data
points. Initial reaction rates were obtained from the experimental concentration-time profiles by regression of
the linear portion of the kinetic data. The experimental concentration profiles were fitted to the kinetic
models using the Levenberg-Marquardt nonlinear regression algorithm available in the ODRPACK [15]. The
differential equations resulting from the different ester synthesis mechanism simplifications used in the
nonlinear regression was simulated using the ODEPACK library [16].
RESULTS & DISCUSSION
Effect of reaction parameters
The effect of varying the enzyme loading on the rate of reaction was investigated by gradually increasing the
mass of enzyme from 0.25 to 1.50 g of enzyme (i.e., from 0.83 to 5.0 g of enzyme mol -1 of the limiting
substrate) at a constant agitation of 120 rpm. The results showed that the reaction rate increased linearly with
increased enzyme loading. For the enzymatic synthesis of other esters, similar behaviour was found in the
literature [5, 17]. The curves overlapped for larger enzyme loadings (≥5.0 g mol -1 ), suggesting there was no
free substrate to bind with the excess of enzyme or external mass transfer resistance was limiting the rate.
Such a rate limitation was also reported for the enzymatic synthesis of ethyl palmitate, isoamyl acetate, and
octyl acetate [5, 18, 19]. Since there was no significant increase in rate and conversion with increased
enzyme loadings (>2.5 g mol -1 ), further experiments were performed using enzyme loadings of 2.5 g mol -1
(7.5%, w/v).
In the present study, preliminary experiments were performed to ensure the absence of external and internal
diffusion limitations in all experiments. The effect of speed of agitation on initial rate and conversion was
studied over the range of 80 to 250 rpm. It was found that both the initial rate and conversion increased with
agitation speeds from 80 to 200 rpm with no significant increase above 200 rpm, indicating the reaction rate
and conversion were no longer limited by mass transfer limitations of immobilized enzyme at 200 rpm.
However, above 200 rpm, there was no significant increase in reaction rate and conversion.
In order to assess the influence of initial concentration of substrates on the reaction kinetics, with the aim
ofoptimizing initial rate and conversion, experiments with different substrate molar ratios were examined.
The enzyme loading (2.5 g mol -1 ) and the concentration of isoamyl alcohol were kept constant in this set of
experiments. The initial rate of reaction and equilibrium conversion increased with increasing acetic acid
concentration up to a critical value (600 mM); however, further increase of acetic acid concentration above
600 mM, i.e, for an acetic acid/isoamyl alcohol molar ratio >2, resulted in decreased initial rate and
equilibrium conversion. The decreased initial rates and conversion may be due to the formation of ineffective
enzyme-substrate complexes at high substrate concentrations [12, 17]. Moreover, polar substrates tend to
accumulate in the micro-queous environment of the suspended enzyme and may reach concentration levels

sufficient to cause denaturation of the enzyme molecule [20]. The present results are consistent with the
reported inhibition of lipases by acetic acid during synthesis of esters [7, 21].
Water can have a dramatic influence on biocatalytic processes in non-aqueous media, depending upon the
nature of the organic solvent used as a suspension medium [22, 23] and the form of enzyme protein
employed [24]. Several mechanisms have been proposed for water-induced enzyme activation in nonaqueous
milieu. It may act as a molecular lubricant, increasing internal flexibility of the enzyme [25]
reducing unfavourable protein-protein interactions, responsible for structural distortions and aggregation or it
may increase the active site polarity [23, 24] leading to enhanced enzyme activity in non-aqueous solvents. In
the present study, the initial rate and equilibrium conversion were highest when 100 µL of water (1%, v/v)
was added to the reaction mixture under otherwise similar conditions. Water accumulation during the
esterification reaction possibly caused a decrease in the initial rate and equilibrium conversion, when the
amount of initially added water was more than 100 µL. Thus, due to its effect on the thermodynamic balance
of the reaction, an amount of water more than an optimum value (100 µL) resulted in decreased initial rates
and equilibrium conversion indicating that hydrolysis outweighed synthesis (esterification).
Reaction temperature has a profound effect on reaction rates, equilibrium conversion and enzyme stability
[19, 26]. However, the effect of temperature on equilibrium conversion and initial rate is difficult to predict
because it may influence the reaction efficiency depending on the source of the enzyme [26, 27], type of
immobilization [26] and ionization state of the enzyme active site [28]. In accordance with transition state
theory, increasing temperature would result in a positive effect on the rate constant; however, high
temperatures may disrupt enzyme tertiary structure resulting in loss of catalytic activity [5]. Therefore, in
view of the fact that at temperatures ≥60 °C the immobilized enzyme inactivates faster over the time period
of the reaction (72 h) and the boiling point of the reaction mixture occurred at 69 °C, the influence of the
temperature on the reaction kinetics was investigated in the temperature range of 30 to 50 °C. Since Celiteimmobilized
A. mediterranei DSM 43304 lipase is thermostable, ester synthesis was observed at all
temperatures from 30 to 50 °C and the initial rates and equilibrium conversions were found to increase with
the temperature with a maximum rate and equilibrium conversion obtained at 50 °C. This suggests the
predominance of kinetic effect in this temperature range [5, 17].
Esterification using optimal parameters
A sequential strategy of experimental design proved to be useful in determining the conditions for
maximizing the equilibrium conversion in n-hexane using Celite-immobilized A. mediterranei DSM 43304
lipase as a catalyst. Optimum conversion was obtained at an acetic acid/isoamyl alcohol molar ratio of 2,
initial addition of 1% (v/v) of water and 7.5% (w/v) of enzyme (i.e. 2.5 g of enzyme mol -1 of alcohol) at 50
°C. Under these conditions, a 12 h reaction time was sufficient to reach the equilibrium molar conversion of
59%; however under non-optimized operational conditions the equilibrium molar conversion reached was
21% after 36 h of reaction time (Figure 1). Widely different conversion yields of isoamyl acetate in organic
solvent systems have been reported in the literature. Hari Krishna et al. [29] used n- heptane for the synthesis
of isoamyl acetate and obtained a conversion yield of >80% in 72 h using Rhizomucor miehei Lipozyme IM-
20 at 6.7 g mol -1 of substrate, whereas a maximum conversion yield of 100% was reported by Romero et al.
[5] using Candida antarctica Novozyme 435 at 13.8 g mol -1 of substrate in n-hexane. Liaquat and Owusu
Apenten [30] used n-hexane during esterification to obtain a conversion yield of 30% in 48 h with rapeseed
seedling lipase at 200 g mol -1 of substrate. Furthermore, Larios et al. [31] reported a conversion yield of 74%
for the synthesis of n-butyl acetate in n-hexane using C. antarctica lipase B at 50 g mol -1 of substrate. In the
present investigation a non-commercial Celite-immobilized lipase at 2.5 g mol -1 of substrate was employed to
achieve a conversion yield of 59% in 12 h, indicating a significant esterification at a lower enzyme
concentration.
Kinetic model based on time-course measurements
Several studies have reported the kinetic modeling of lipase mediated esterification [10, 12, 32] and transesterification
[17, 33, 34] reactions in organic media using commercially available immobilized lipases.
However no attempt has been made to develop a kinetic model for the direct esterification using a noncommercial
lipase. To the best of our knowledge the present study is the first report on the development of a
kinetic model for direct esterification reaction in organic solvent catalyzed by a non-commercial
actinomycete lipase. Esterification reactions of various organic acids with different alcohols by a variety of
commercial lipases are often modeled using the so called Ping Pong Bi Bi mechanism, a well known and
widely accepted mechanism for lipase-catalyzed reactions [9-11, 17]. In the present investigation, the Ping
Pong Bi Bi mechanism coupled with a competitive inhibition by one of the substrates was assumed as a basis

for the kinetic model building [10,11]. The description of this mechanism and the associated rate (r) is given
by Eq. (1) [35].
⎪⎧
[ ] [ ]
[ ΙΑΑc] × [ Η ] ⎪⎫
2Ο
ν
fν
r ⎨ Αc
× Η
2Ο
−
⎬
⎪⎩
keq
r =
⎪⎭
(1)
⎛
{ [ ] [ ]
[ ΙΑΑ]
⎞ ν
f
km,
Η Ο
ν
f
k
2
m,
ΙΑΑc
ν
⎜
⎟
f
km,
ΙΑΑ
Αc
+ ν
rkm,
Αc
ΙΑΑ × 1 + + [ ΙΑΑc] + [ Η
2Ο] + ν
f
[ Αc] × [ Η2Ο]
⎝ k'
ΙΑΑ ⎠ keq
keq
ν
f
km,
Η Ο
ν
f
ν
rk
2
m,
Αc
+ [ Αc] × [ ΙΑΑc] + [ ΙΑΑc] × [ Η2Ο] + [ ΙΑΑ] × [ Η
2Ο] }
k k
k
k
eq i,
Αc
eq
i,
Η 2Ο
where v f and v r are the maximal velocities for the forward and the reverse reactions, respectively, k eq is the
equilibrium constant, k m,IAA , k m,Ac , k m,H2O and k m,IAAc are the Michaelis-Menten constants for isoamyl alcohol
(IAA), acetic acid (Ac), water (H 2 O) and isoamyl acetate (IAAc), respectively, k’ IAA is the inhibition constant
for IAA, and k i,Ac and k i,H2O are the dissociation constants for Ac and H 2 O from the specific enzyme-inhibitor
complex, respectively.
Owing to the mathematical complexity of this full-model involving ten adjustable parameters with associated
problems of unidentifiability and indistinguishability of different parameters under normal experimental
situations, a strategy to study the effect of dropping out some parameter(s) in order to produce a simpler
model was implemented [11, 34]. Following the methodology of Paiva et al. [11], the Michaelis-Menten
dissociation constant terms for each of the compounds (k m,x , where ‘x’ represents either [Ac], [IAA], [IAAc]
or [H 2 O]) from the enzyme complex were considered for model reduction; the elimination of these
parameters from the denominator of model rate constant would mean that such parameters assume high
values in respect to the experimental data and there is no evidence of saturation in the concentration range
studied or there is no affinity of the enzyme to the compound in question. In order to study the adequacy of
this hypothesis, the model from Segel [35] was reformulated by eliminating the dissociation constants
yielding simpler models. These models were separately fitted to the experimental data and F-tests were
performed on the associated residual sum of squares with the aim of investigating the statistical likelihood of
such simplifications. The results obtained in this comparison are presented in Table 1.
Table 1. F-test results for model nesting
Source model SSQ parameters
Model
(NP)
df RSS a df
Extra
(σ) b
Mean
square
(s 2 ) c F
ratio d
P
value
Full model vs. Nested-1
Eq- 1 793.49 10 53 – – 14.97 – –
Eq-2 792.97 4 59 -0.52 6 -0.09 -0.01 0.99
a Residual sum of squares is the difference in the SSQ of models, b Extra degrees of freedom is the difference in the
number of model parameters. c Mean square is the residual sum of squares divided by degrees of freedom (df). d F ratio is
the full model (f) mean square divided by the mean square of the nested model (n),
It can be concluded that, at p < 0.05 level of statistical significance, the resulting rate expression can be
described by Eq. (2).
⎪⎧
[ ΙΑΑc] × [ Η ] ⎪⎫
2Ο
{( k
cat f
[ Et
]) × ( k
cat r
[ Et
])} × ⎨[ Αc] × [ ΙΑΑ]
−
⎬
⎪⎩
k
eq ⎪⎭
r =
(2)
( k
f
[ Et
])
( k [ ])[ Αc] × [ ΙΑΑ] cat
cat r
Et
+ [ ΙΑΑc] × [ Η
2Ο]
k
The experimental concentration profiles were modeled using Eq. (2). Experimental points and the simulated
curves obtained with the developed model are shown in Figure 1. The simulated curves follow reasonably
well the experimental points, showing the kinetic model using Eq. (2) is able to describe the entire reaction
progress under conditions of equimolar initial substrate concentrations as well as excess of one of the
substrates (see Figure 1 for prediction and Table 2 for estimated parameters).
eq

Concentration [mmol L -1 ]
300
280
260
240
Non-optimized condition
100
95
90
85
80
60
20
40
15
10
20
5
0
0 20 40 60
0
80
Conversion [%]
Concentration [mmol L -1 ]
Optimized condition
300
250
100
80
200
60
150
100
40
50
20
0
0 20 40 60
0
80
Conversion [%]
Time [h]
Time [h]
Figure 1. Experimental and simulated ester production profiles. Enzyme loadings: 250 mg (х,▼), 500 mg (∆,▲), 750 mg
(○,●), 1500 mg (□,■). Open symbol: isoamyl alcohol, Filled symbol: isoamyl acetate.
Table 1. Parameter estimates of Eq-2 modelling using the kinetic data.
Model parameter Value 95% confidence interval
k
cat , f
[mol h g-1] 6.91×10-1 5.97×10-1 to 7.84×10-1
k
cat , r
[mol h g-1] 2.57×10-1 1.27×10-1 to 3.87×10-1
k
eq
[-] 7.02×10-2 6.37×10-2 to 7.68×10-2
RSS* [(mol L-1)2] 7.92×102
CONCLUSION
R 2 adj 0.99
The present study investigated the direct esterification of isoamyl alcohol with acetic acid, in n-hexane, using
a non-commercial Celite-immobilized A. mediterranei lipase. The selection of various reaction parameters
that maximize equilibrium conversion yield at relatively low enzyme concentration was studied in detail.
Optimized conditions for the synthesis of isoamyl acetate were 7.5% (w/v) of Celite-immobilized lipase, an
acid/alcohol molar ratio of 2 with an initial addition of 1% (v/v) water at 50 °C and 200 rpm. Under these
conditions the equilibrium conversion yield obtained was 59% in 12 h. A simplified model, based on a
postulated Ping Pong Bi Bi mechanism, adequately described the kinetics of Celite-immobilized lipase
catalyzed direct esterification of isoamyl alcohol with acetic acid.
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