Biochemical Engineering Journal 71 (2013) 8996

Contents lists available at SciVerse ScienceDirect

Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Kinetic model of lipase-catalyzed conversion of ascorbic acid and oleic acid to

liposoluble vitamin C ester
Dejan Bezbradica a, , Marija Stojanovic a , Dusan Velickovic b , Aleksandra Dimitrijevic b , Milica Carevic a ,
Mladen Mihailovic a , Nenad Milosavic b
a
b

Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11000 Belgrade, Serbia
Department of Biochemistry, Faculty of Chemistry, Studentski trg 12, University of Belgrade, 11000 Belgrade, Serbia

a r t i c l e

i n f o

Article history:
Received 6 August 2012
Received in revised form 18 October 2012
Accepted 2 December 2012
Available online 10 December 2012
Keywords:
Ascorbyl oleate
Lipase
Candida antarctica
Kinetic parameters
Substrate inhibition
Production kinetics

a b s t r a c t
The kinetics of l-ascorbyl oleate synthesis catalyzed by immobilized lipase from Candida antarctica in acetone was investigated. Signicant inhibition of synthesis with an excess of ascorbic acid was observed.
Experimental data were successfully tted with a pingpong bibi kinetic model with substrate inhibition, and related kinetic constants were determined. The kinetic study was performed at optimum
experimental factors (temperature, initial water content, and enzyme concentration), which were determined using response surface methodology. Then, a model for predicting producttime progress curves
was developed by expanding the obtained pingpong model with terms describing ester hydrolysis.
Kinetic constants of the reverse reaction were determined, and good congruence between the model and
experimental data was achieved. Calculated kinetic constants revealed that lipase has the highest afnity for ascorbyl oleate, slightly lower activity with ascorbic acid, and the lowest activity with oleic acid.
The obtained results are valuable for elucidating the reaction mechanism and represent an important
contribution for reaction optimization and creating strategies to increase the productivity of vitamin C
ester synthesis.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Vitamin C is well known for its antioxidative properties. Nevertheless, the use of l-ascorbic acid in stabilizing fats and oils is
very scarce due to its hydrophilic nature [1]. On the contrary, fatty
acid ascorbyl esters are oil-soluble with the same or even enhanced
antioxidative properties compared to vitamin C. Mineral acids or
lipases can catalyze the esterication process between vitamin C
and fatty acid (or its methyl or vinyl ester). At the moment, despite
the numerous shortcomings, ascorbyl palmitate is being produced
industrially by chemical means [2]. On the other hand, there are
many advantages of the biosynthetic process, such as mild reaction
conditions, regioselectivity, and the possibility of using immobilized enzymes, resulting in simpler downstream processing [3].
Additionally, obtaining a product in such a way allows it to be
labeled as natural and have a higher market value [4]. Although
lipase-catalyzed synthesis of ascorbyl esters has already been
described by many authors, long reaction times, bio-incompatible
solvents, and high price of the enzyme are still some of the main
obstacles in the commercialization of the process [5]. Ascorbyl
esters derived from unsaturated fatty acids are superior compared

Corresponding author. Tel.: +381 11 3303727; fax: +381 11 3370387.

E-mail address: dbez@tmf.bg.ac.rs (D. Bezbradica).
1369-703X/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bej.2012.12.001

to those with saturated hydrocarbon chains in terms of solubility,

free radical scavenging capacity and benecial effects on human
nutrition [6]. Therefore, their biosynthesis, especially in GRAS (Generally Recognized as Safe) solvents, is of particular interest [5].
In regard to scale-up and process automation, it is necessary
to optimize operating parameters and establish adequate kinetic
models for the reactions. Additionally, the type of kinetic model
gives valuable information about the reaction mechanism and substrate inhibition which helps in organizing enzymatic processes
in such a way as to avoid intrinsic limitations at the molecular
level. There are just a few reports of ascorbyl oleate synthesis catalyzed by immobilized lipase from Candida antarctica [711]. On
the other hand, reports on the kinetics of aliphatic ester synthesis
are more frequent, but different kinetic models have been proposed. In general, esterication reactions catalyzed by immobilized
C. antarctica lipase preparation occurs via acylenzyme intermediates and are most commonly being described by models based
on a pingpong bibi or ordered bibi mechanism [12]. Additionally, inhibition by one or both substrates was reported on several
occasions [1317].
The main goals of this study were to obtain an adequate
kinetic model for the enzymatic synthesis of ascorbyl oleate
(Scheme 1) in acetone, determine key kinetic constants, and
compare afnity of substrates towards lipase. To investigate inhibition by excess of substrates, concentration was varied in a wide

90

D. Bezbradica et al. / Biochemical Engineering Journal 71 (2013) 8996

Scheme 1. Formation of ascorbyl oleate in a reaction of ascorbic acid and oleic acid.

range from 0.05 to 1 M. Prior to kinetic studies, response surface

methodology (RSM) and 5-level-5-factor central composite rotatable design (CCRD) were employed to determine the effects of
key experimental parameters (initial water content, temperature,
ascorbic acid:oleic acid molar ratio, vitamin C concentration, and
enzyme content) on the initial rate of the vitamin C and oleic acid
esterication reaction, so the kinetic study was performed at optimum conditions. Finally, it was shown that reaction kinetics can be
tted throughout the reaction range with a model that also includes
the reverse reaction of ester hydrolysis, and the kinetic constants
of the reverse reaction were determined.

2. Materials and methods

2.1. Enzyme and chemicals
Novozym 435 (lipase from C. antarctica, type B immobilized
on acrylic resin) was purchased from Novozymes (Bagsvaerd,
Denmark). Substrates were l-ascorbic acid (purity 99.7%, Zorka,

Serbia) and oleic acid (Ph. Eur., NF pure purchased from

Sabac,
AppliChem, Darmstadt, Germany). Acetone was used as a reaction
medium (99.5%, JT Baker, USA). Substances used for the quantitative
HPLC analyses were methanol obtained from JT Baker (USA) and
phosphoric acid, purchased from SigmaAldrich (Chemie GmbH,
Steinheim, Germany); all were HPLC grade.

2.2. Procedure for the enzymatic synthesis

Experiments were carried out in 100 ml capped vials. The reaction mixture consisted of different amounts of ascorbic acid, oleic
acid, enzyme, water, and acetone (amounts specied for each
experiment separately), so that the total volume was 10 ml. The
reactions were conducted in a shaker at 250 rpm and at a temperature in the range from 40 to 60 C. All experiments were carried out
in duplicate, and average values are presented in Figs. All standard
deviations were less than 5%. Control samples (without enzyme)
were prepared by exposure to the same temperature treatment.
The product was not detected in control samples. In experiments

with the addition of molecular sieves, 80 mg of zeolite type sieves

were added per ml of the reaction medium.

2.3. Kinetic study

Kinetic studies were performed in accordance with experimental plans comprising of 64 experimental points of a matrix (8 8),
representing all possible pairs of ascorbic acid and oleic acid concentrations at following set of values: 0.05; 0.1; 0.15; 0.2; 0.3; 0.5;
0.75; 1.
The initial rate was determined as the slope of the reaction curve
tangent to the initial stage of the reaction. Because all experiments
were performed in duplicate, reaction curves were constructed
using average values of the reaction rate for each experimental
point. A linear portion of the reaction curve at various substrate
concentrations consisted of 46 experimental points, where the
number of experimental points included was determined by the
condition that correlation coefcients of the initial straight line
must be above 0.95.
2.4. HPLC analysis
For quantitative analysis of reactants and products, an Akta
Purier HPLC system was used. A reverse phase column (Waters
Spherisorb ODS 2-C18, 250 mm 4.6 mm, 5 m) was employed.
The injection volume of the reaction mixture, diluted fteen fold,
was 10 l. Methanol/H3 PO4 , 100/0.1 (v/v), was used as eluent with
a ow rate of 1 ml/min. The product was detected by a UV detector
at 235 nm.
3. Theory and calculations
3.1. Pingpong bibi model
The pingpong bibi mechanism illustrates alternate binding of
substrates and release of products in a bi-substrate reaction with
two products formed. It is the most frequently postulated reaction
mechanism in lipase-catalyzed esterications [1319]. The rst
stage of the reaction is the binding of the acyl-donor (Ol, oleic acid),
resulting in the formation of an acylenzyme complex. In the next
step, the rst product (water) is released. Then, the acyl acceptor
(AA, ascorbic acid) binds, and in the nal step, ester (AOl) is released
[18].

Nevertheless, basic pingpong mechanisms can rarely adequately describe lipase-catalyzed esterications because inhibition
by excess of one of the substrates was frequently reported [1419].

D. Bezbradica et al. / Biochemical Engineering Journal 71 (2013) 8996

91

Table 1
Coded levels and corresponding actual values of variables.
Factors

Level

Water content, X1 (%, v/v)

Temperature, X2 ( C)
Enzyme amount, X3 (%, w/v)
Oleic acid/ascorbic acid molar ratio, X4
Vitamin C concentration, X5 (M)

0
40
0.2
1:3
0.02

0.05
45
0.4
1:6
0.07

0.1
50
0.6
1:9
0.12

0.15
55
0.8
1:12
0.17

0.2
60
1.0
1:15
0.22

Substrate inhibition occurs due to the formation of an inactive

dead-end complex. In a case of acyl donor inhibition, the inactive complex with two molecules of the acyl donor attached to the
enzyme is formed [15]. When inhibition by an excess of acyl acceptor occurs, such as in our reaction, an inactive enzyme-acceptor
complex is formed that cannot be subsequently transformed into a
product [14]. The pingpong bibi kinetic model with ascorbic acid
inhibition is described by Eq. (1).
es =

Vm,es [Ol][AA]
[Ol][AA] + KAA [Ol] + KOl [AA] + (KOl /Ki,AA )[AA]2

(1)

In Eq. (1), ves is the initial reaction rate; KAA and KOl are the
Michaelis constants of ascorbic and oleic acid, respectively; Vm,es
is the maximum reaction rate; and Ki,AA is the inhibition constant
for ascorbic acid. The term Vm,es is the product of the rate constant
(kcat ) of the nal reaction step and the initial enzyme concentration ([E]0 ). Kinetic constants were determined using a non-linear
regression t of the results obtained in the experiment described in
Section 2.3. Goodness of t was evaluated by calculating the correlation coefcient. Specicity constants for each of reaction species
were calculated by dividing kcat by the Michaelis constant of the
compound. The reaction progress was simulated, and parameters
of the reverse (hydrolysis) reaction were estimated using Matlab
software.

3.2. Experimental design and statistical analysis

A 5-level-5-factor CCRD, including 32 experimental points (16
factorial, 10 axial, and 6 center points), was employed in this study.
Table 1 shows the coded and actual variable levels. All 32 runs
were performed in random order so that systematic errors would
be avoided.
Experimental data were analyzed by a response surface regression (RSREG) method to t the second-order polynomial equation:

Y = k0 +

5

i=1

ki Xi +

5

i=1

kii Xi2 +

5
4 


kij Xi Xj

(2)

i=1 j=i+1

where Y is response (initial reaction rate), k0 , kt , ktt and kij

are constant regression coefcients, Xi and Xj are uncoded independent variables, and k is the number of single factors. The
least-squares method was employed for the response function
coefcients calculation and their statistical signicance evaluation.
Only the signicant terms (p 0.05) were considered for the nal
reduced model. Adequacy of the obtained model was determined
by the Fisher test. Student distribution was used to evaluate the
signicance of the coefcients. The signicance of all regression
coefcients was evaluated by comparing their calculated t-values
(Table 2) with the standard t-value of the degree of freedom of
applied experimental design, which was equal to 2.08.

4. Results and discussion

4.1. The effect of operating parameters on initial esterication
rate
The optimization study was performed in accordance with the
experimental design with 5-factors and 5-levels with 32 experimental points [20]. The adequacy of the model described by a
second-order polynomial equation (Eq. (2)) was analyzed using the
Fischer test, and the F-value was calculated to be 2.89, indicating
that the model properly describes experimental results. The Students t-test was employed to analyze the signicance of model
coefcients, and calculated t-values for each of the examined factors are listed in Table 2. A regression model (Eq. (3)) was developed
after eliminating insignicant coefcients, with the exception of
coefcients 4 and 44 because these are necessary for hierarchy
of the model due to the signicant interactive effect of factor x4 .
Y = 2.98 0.253X1 + 1.43X2 0.613X3 0.0829X4 + 0.19X12
+ 0.911X22 0.096X42 0.195X52 0.331X1 X3 + 0.297X2 X4
+ 0.191X2 X5 0.854X4 X5

(3)

Values of model coefcients indicated a statistical signicance

of all varied factors, with quadratic terms of all variables except
enzyme amount, which was also included in the model. In regard
to the mutual effect of the parameters, a signicant interaction
was observed between the following variables: water content and
enzyme amount, temperature and substrate molar ratio, vitamin
C concentration and temperature, and substrate molar ratio and
Table 2
Coefcients values and results of Students t-test analysis.
Coefcient

Value

t-Value

0
1
2
3
4
5
11
22
33
44
55
12
13
14
15
23
24
25
34
35
45

2.98
0.253
1.43
0.613
0.0829
0.0905
0.194
0.911
0.141
0.0964
0.195
0.0521
0.331
0.0515
0.0138
0.0109
0.297
0.191
0.0233
0.126
0.854

20.9a
3.54a
20.0a
8.60a
1.16
1.27
2.92a
13.9a
1.94
1.58
2.99a
0.597
3.77a
0.590
0.158
0.125
3.39a
2.18a
0.266
1.44
9.74a

Signicant coefcients.

92

D. Bezbradica et al. / Biochemical Engineering Journal 71 (2013) 8996

Fig. 1. Contour plot of ascorbic acid and molar ratio effects on initial reaction rate. Values of other factors: x1 = 2; x2 = 2; x3 = 2.

ascorbic acid concentration. In Figs. 13, only the most interesting

ndings with impact on the following kinetic study are illustrated.
The effects of the substrate molar ratio and vitamin C concentration, factors which showed the most intensive interaction, are
illustrated in Fig. 1. Both experimental factors had signicant and
negative quadratic coefcients, so individual effects are described
by convex graphs with maximum values. Due to negative interactions, the position of the local maxima of individual factors shifts
towards lower values with increases of other factors (Fig. 1). As a
result, high initial velocities were achieved when experiments were
conducted at high substrate molar ratios and low ascorbic acid concentrations or vice versa. Such trends should be discussed bearing
in mind the previous ndings regarding mechanism of lipasecatalyzed esterications because it was univocally established that
the formation of the acylenzyme complex is the necessary rst
step [1416]. Consequently, at a low initial concentration of ascorbic acid, the local maximum of the initial rate was reached at the
highest oil/ascorbic acid molar ratios (even above 15), most likely
because the excess of oleic acid enables faster formation of the
acylenzyme complex. On the other hand, increases of ascorbic
acid concentrations led to the shift of local maxima toward lower
substrate ratios, even below 3 (Fig. 1). It is plausible that the main

cause of the initial reaction rate decrease is mass-transfer limitations, which usually occur at higher reaction medium viscosity [21],
and these are reached at the highest values of both factors, mostly
due to extremely high oleic acid concentration.
A strong negative interaction was also observed between the
enzyme amount and water content (Fig. 2). The lipase loading
(X3 ) effect was described with only a linear coefcient; hence,
the initial reaction rate continuously increased with the increase
of enzyme concentration in our study. But due to the negative
interaction, slopes varied with the initial water concentration. At
low water concentrations, initial reaction rates increased steeply
when lipase loading was increased and reached maximum values, while at higher water concentration, slope becomes almost
negligible. The results obtained in our experiment shows that
the addition of water was not necessary for achieving a maximum initial reaction rate, indicating that the solvent contains
a sufcient amount of water for keeping enzymes in the open
conformation essential for providing its activity. It seems that a
higher amount of water in the media represents the hindrance
for the mass transfer, leading to lower initial reaction rates; otherwise, the amount of water present in the solvent itself was
sufcient to provide lipases catalytic function. To investigate if

Fig. 2. Surface plot of the effects of enzyme and added water concentrations on initial reaction rates. Values of other factors: x2 = 2; x4 = 0; x5 = 0.

D. Bezbradica et al. / Biochemical Engineering Journal 71 (2013) 8996

93

Fig. 3. The effect of temperature on the initial reaction rate. (a) Surface plot of ascorbic acid and temperature effects. Values of other factors: x1 = 2; x3 = 2; x4 = 0; (b) surface
plot of substrate molar ratio and temperature effects. Values of other factors: x1 = 2; x3 = 2; x5 = 2.

reaction rates can be increased with further reduction of water

concentration, two additional experiments were performed: one in
acetone previously dehydrated by molecular sieves and the other
in untreated acetone. Other experimental factors were kept at optimum conditions in both experiments. It was revealed that a higher
initial rate was achieved in acetone (15.8 mM h1 ) than in dehydrated acetone (14.2 mM h1 ), indicating that removal of water
present in acetone is not advantageous because it reduced lipase
activity.
The effect of temperature is depicted in Fig. 3. In previous studies, when the incubation temperature was optimized from the
aspect of ester yield, the positive effect of temperature increase
was reported [7]. Within the initial reaction rate optimization in our
research, the same inuence of reaction temperature was observed,
and therefore, the best results were achieved at 60 C. Slight positive interaction effects between ascorbic acid concentration and
temperature (25 = 0.191) caused a shift of local maxima from low
towards moderate ascorbic acid concentrations (Fig. 3a). At lower
temperatures, the rate of substrate interchange at the active site
and diffusion of substrates and products are signicantly lower,
so high ascorbic acid concentrations are futile. With the increase of
reaction temperature, the acceleration of interchange and diffusion

leads to higher rates at higher initial ascorbic acid concentrations.

A similar trend was observed with the effects of temperature and
substrate molar ratio, where positive interaction between factors
(24 = 0.297) led to a reduced decrease of the initial rate with the
increase of molar ratio at higher reaction temperatures (Fig. 3b).
This result could also be ascribed to the increased diffusion of
both substrates or to the reduction of reaction medium viscosity,
which becomes signicantly higher at a higher molar ratio due to
increased concentrations of viscous oleic acid. Notably, higher reaction temperatures could not be investigated because the boiling
point of the initial reaction mixture is slightly greater than 60 C,
even for high substrate(s) concentration mixtures, due to the high
volatility of acetone.
Findings, which had been obtained in a previous optimization
study, were applied in the subsequent kinetic study ndings. Further kinetic experiments were performed at 60 C, with 1% (w/v)
of immobilized enzyme and without addition of water to the reaction medium. Moreover, the goodness of the model prediction was
conrmed by an experiment performed at 0.22 M of ascorbic acid
and molar ratio 1:3 (and optimum values of other factors), where
the initial rate of 15.6 mM h1 was achieved, which is only a 3.2%
deviation from the predicted value.

94

D. Bezbradica et al. / Biochemical Engineering Journal 71 (2013) 8996

Fig. 4. The effects of both substrates on initial reaction rate. Reaction temperature 60 C; initial lipase concentration 1% (w/v); without addition of water.

4.2. Initial rate analysis

To correlate initial reaction rates with substrates concentrations in the kinetic model, a set of experiments was carried
out. Initial velocities for the ascorbyl oleate biosynthesis were
evaluated for the ascorbic and oleic acid concentrations in the
range of 0.051 mol/dm3 . The experimental data that represents
the mutual effect of ascorbic and oleic acid concentrations on
the initial reaction velocity are represented with a 3D graph in
Fig. 4. It was observed that the maximum value of the initial
rate (16.7 mmol h1 ) was achieved in an experiment with a high
oleic acid concentration (0.75 M) and a relatively low ascorbic acid
concentration (0.1 M). Signicant increases in the initial rate of
comparison with the experimental design could be ascribed to the
fact that three factors have extreme optimum values, and the experimental plan does not include the experiments with more than one
extreme value of experimental factors.
In graphic analyses of kinetic experiments, the effect of oleic acid
on the initial rate resembles a classic MichaelisMenten-shaped
curve (Fig. 5a). However, the effect of ascorbic acid is far more complex than this pattern because the increase of initial rates can be
observed only up to 0.2 M, and further increases in ascorbic acid
concentration led to the decrease of initial rates (Fig. 5b). Such
a trend indicates that inhibition with an excess of ascorbic acid
occurs, while inhibition with oleic acid does not. Therefore, the
results were tted with a pingpong bibi model with ascorbic acid
inhibition (Eq. (1)).
The goodness of t was very high (R2 = 0.979), and the 3Dcongruence between model and results is illustrated in Fig. 6;
tting of the effects of individual substrates at a xed concentration of other substrate is depicted in Fig. 5; and calculated kinetic
constants are listed in Table 3. The inhibition constant is signicantly higher than previously reported in similar kinetic models
[14,19], indicating that ascorbic acid is not a very strong lipase
inhibitor.
In previous studies of synthesis of esters with C. antarctica lipase,
various models with inhibition by different substrates or without
any substrate inhibition were postulated. For example, in a study
focused on citronellol laurate synthesis, an ordered bibi mechanism with acid inhibition was proposed [15]. An ordered bibi
kinetic model with competitive inhibition by both reactants and
products was reported by Garcia et al. for the isopropyl palmitate
synthesis in an organic solvent [16]. On the other hand, in sugar
ester synthesis, a pingpong bibi mechanism without inhibition
by any substrate was established [17].

Pingpong bibi models with alcohol inhibition, such as the one

proposed in our study, were previously reported for the synthesis of tetrahydrofurfuryl butyrate in heptane and butyl isobutyrate
biosynthesis in n-hexane [13,14]. The inhibition mechanism with
an excess of alcohol was most thoroughly elaborated by Yadav and
Lathi [14]. Because it is generally accepted that the formation of
the acylenzyme complex is the rst step of the reaction, it seems

Fig. 5. The representative kinetic model curves at xed concentrations of one substrate: (a) 0.3 M ascorbic acid; (b) 1 M oleic acid.

D. Bezbradica et al. / Biochemical Engineering Journal 71 (2013) 8996

95

Fig. 6. The surface plot of the obtained pingpong bibi model with inhibition by ascorbic acid. Reaction temperature 60 C; initial lipase concentration 1% (w/v); without
addition of water.

that alcohol inhibition occurs due to the formation of inactive dead

end complex between alcohol (in our reaction, this role is vitamin
C) and enzyme. Discrepancies between reported kinetic models
indicate that size, branching and abundance of hydroxy groups in
the side chains of both substrates strongly affect the reaction mechanism. C. antarctica B has been previously classied into the group
of lipases with a funnel-like active site, which is the main reason
for their higher afnity towards short- and medium-chain acid substrates and steep decreases in afnity for C14 acids [22]. Therefore,
inhibition by an excess of vitamin C could be ascribed to an easier
approach of vitamin C than the sterically hindered approach of oleic
acid, which resulted in the more frequent formation of an inactive
complex than produces the acylenzyme complex. A signicantly
higher (approximately 28 times) specicity constant for ascorbic
acid (Table 3) conrms this higher afnity.

[12], the discrepancy between the modeled and the experimental results can be attributed to the onset of intensive hydrolysis of
the formed ester. This hypothesis was tested by adding molecular
sieves after 2 h of reaction because the rate of hydrolysis is inuenced by the concentration of free water in a reaction mixture [23].
The results (indicated by crosses in Fig. 7) show that in the presence
of sieves, the linear increase of product concentration is prolonged
until approximately the 4th hour of the reaction, indicating that the
onset of hydrolysis is delayed by the removal of water. Nevertheless, hydrolysis occurred most likely when the adsorbent capacity
had been exceeded.

4.3. Model for progress curve

Therefore, it seems unavoidable to include hydrolysis in a model

that describes a complete producttime curve (Eq. (4)). Ester
hydrolysis was t with a lumped rate expression that resembles
a MichaelisMenten equation (Eq. (5)) because it has been widely
applied for lipase-catalyzed hydrolysis [24]. In Eq. (5), Vm,h represents the maximum initial rate of hydrolysis and KAOl the Michaelis
constant of ascorbyl oleate.
The determined values of unknown parameters in Eq. (5) are
listed in Table 3, and the simulation of the reaction described by
Eq. (4) is depicted in Fig. 7 (solid line). Strong congruence between
ascorbyl oleate synthesis and the model were observed (R2 = 0.943),
indicating that the kinetic model established at initial conditions,
in addition to elucidating the reaction mechanism, can also be
applied for estimating the reaction course after introducing a term

To develop an efcient enzymatic process of ester synthesis and

adequate reactor design, it is crucial to obtain a good t between
the kinetic model and (product concentration)(reaction time)
curve throughout the reaction range. Therefore, the kinetic model
obtained in this study, which is based on initial reaction rates,
was tested on reaction curves and was further adjusted to obtain
the best t for the experimental results. Typical results, obtained
at an initial concentration of 0.1 M vitamin C and a molar ratio
of 1:5, are depicted in Fig. 7. It can be seen that the simulation
of the model (dotted line) ts the experimental results (circles)
only during the initial 90 min of the reaction. Because obtaining a
kinetic model is suitable only if the reverse reaction does not occur

d[AOl]
= es h
dt
h =

(4)

Vm,h [AOl]
= es h
KAOl + [AOl]

(5)

Table 3
Determined values of the model kinetic constants.
Esterication

Hydrolysis

Kinetic constant (unit)

Calculated value

Kinetic constant (unit)

Calculated value

Vm,es (mmol h1 dm3 )

KAA (mol dm3 )
KOl (mol dm3 )
Ki,AA (mol dm3 )
KS,AA (dm3 h1 g1 )
KS,Ol (dm3 h1 g1 )

21.9
0.0111
0.309
0.864
0.197
0.00709

KAOl (mol dm3 )

Vm,h (mmol h1 dm3 )
KS,AOl (dm3 h1 g1 )

0.00963
16.9
0.175

96

D. Bezbradica et al. / Biochemical Engineering Journal 71 (2013) 8996

Fig. 7. Fitting of experimental results with different kinetic models. Initial concentrations were: 0.1 M ascorbic acid and 0.5 M oleic acid. Circles: results of experiment without
molecular sieve addition; crosses: results of experiment with molecular sieve addition; dotted line: the simulation of pingpong bibi model with ascorbic acid inhibition
(Eq. (3)); solid line: the simulation of model including ester hydrolysis (Eq. (4)).

describing the reverse reaction. Additionally, the determined specicity constant for ester as a substrate (KS,AOl ) is approximately 25
times higher than the specicity constant for oleic acid, indicating
that the removal of at least one of the reaction products (water or
ester) is necessary for reaching high product yields.
5. Conclusions
The aim of this study was to thoroughly examine the kinetics
of ascorbyl oleate synthesis, to describe the reaction mechanism
and establish a model that accurately describes the reaction ow.
It was revealed that the pingpong bibi model with inhibition
by excess of ascorbic acid tted experimental results with high
accuracy. Comparing the obtained values of specicity constants
indicates that lipase has a signicantly stronger afnity towards
ascorbic acid. These ndings were also valuable for explaining the
inhibition mechanism because inhibition by an acyl acceptor is usually based on the formation of an inactive complex, which is most
likely promoted with a strong afnity towards ascorbic acid.
After extending the pingpong bibi model with a term
describing the reverse hydrolysis of ester and determining the
corresponding kinetic constants, an adequate model for predicting product concentration was obtained. Good prediction of the
reach of the reaction is of utmost importance for further enzymatic
process development, especially when performed in conditions
previously optimized by RSM, as was the case in this study. In
summary, the results of this study may present an important contribution for elucidating the mechanism of lipase-catalyzed synthesis
of vitamin C esters and a novel, simplied approach to the development of a model that successfully predicts the dependence of
formed product concentration and reaction time.
Acknowledgement
The authors are grateful for nancial support from the Serbian
Ministry of Science (projects III 46010 and 451-03-00605/201216/51).
References
[1] L. Cao, A. Fischer, U.T. Bornscheuer, R.D. Schmid, Lipase-catalyzed solid phase
preparation of sugar fatty acid esters, Biocatal. Biotransform. 14 (1997)
269283.
[2] Y. Yan, U.T. Bornscheuer, R.D. Schmid, Lipase-catalyzed synthesis of vitamin C
fatty acid esters, Biotechnol. Lett. 21 (1999) 10511054.

[3] C.H. Wong, G.M. Whitesides, Enzyme in Synthetic Organic Chemistry, Pergamon Press, Oxford, 1994.
[4] M. Guillen, M.D. Benaiges, F. Valero, Biosynthesis of ethyl butyrate by immobilized recombinant Rhyzopus oryzae lipase expressed in Pichia pastoris, Biochem.
Eng. J. 65 (2012) 19.
[5] S.K. Karmee, Biocatalytic synthesis of ascorbyl esters and their biotechnological
applications, Appl. Microbiol. Biotechnol. 81 (2009) 10131022.
[6] Q.X. Song, Y. Zhao, W.Q. Xu, W.Y. Zhou, D.Z. Wei, Enzymatic synthesis of
l-ascorbyl linoleate in organic media, Bioprocess Biosyst. Eng. 28 (2005)
211215.
[7] Q.X. Song, D.Z. Wei, Study of vitamin C ester synthesis by immobilized lipase
from Candida sp., J. Mol. Catal. B: Enzym. 18 (2002) 261266.
[8] M. Adamczak, U.T. Bornscheuer, W. Bednarski, Synthesis of ascorbyl oleate
by immobilized Candida antarctica lipases, Process Biochem. 40 (2005)
31773180.
[9] M. Adamczak, U.T. Bornscheuer, Improving ascorbyl oleate synthesis catalyzed
by Candida antarctica lipase B in ionic liquids and water activity control by salt
hydrates, Process Biochem. 44 (2009) 257261.
[10] H.J. Hsieh, J.W. Chen, R. Giridhar, W.T. Wu, Synthesis of mixed esters of ascorbic
acid using methyl esters of palm and soybean oils, Prep. Biochem. Biotechnol.
35 (2005) 113118.
[11] K. Kuwabara, Y. Watanabe, S. Adachi, K. Nakanishi, R. Matsuno, Synthesis of
6-O-unsaturated acyl l-ascorbates by immobilized lipases in acetone in the
presence of molecular sieve, Biochem. Eng. J. 16 (2003) 1722.
[12] I.H. Segel, Enzyme Kinetic, Wiley/Interscience, New York, 1975.
[13] T. Garcia, A. Coteron, M. Martinez, J. Aracil, Kinetic modeling of esterication reactions catalyzed by immobilized lipases, Chem. Eng. Sci. 51 (1996)
28412846.
[14] G.D. Yadav, P.S. Lathi, Kinetics and Mechanism of synthesis of butyl isobutyrate
over immobilized lipases, Biochem. Eng. J. 16 (2003) 245252.
[15] G.D. Yadav, P.S. Lathi, Synthesis of citronellol laurate in organic media catalyzed by immobilized lipase: kinetic studies, J. Mol. Catal. B: Enzym. 27 (2004)
113119.
[16] T. Garcia, N. Sanchez, M. Martinez, J. Aracil, Enzymatic synthesis of fatty esters.
Part I. Kinetic approach, Enzyme Microb. Technol. 25 (1999) 584590.
[17] J.A. Arcos, C.G. Hill, C. Otero, Kinetics of the lipase-catalyzed synthesis of the
glucose esters in acetone, Biotechnol. Bioeng. 73 (2001) 104110.
[18] A.E.M. Janssen, B.J. Sjursnes, A.V. Vakurov, P.J. Halling, Kinetics of lipasecatalyzed esterication in organic media: correct model and solvent effects
on parameters, Enzyme Microb. Technol. 24 (1999) 463470.

Z. Knezevic,
The Candida rugosa
c,
[19] D. Bezbradica, D. Mijin, S. Siler-Marinkovi
lipase catalyzed synthesis of amyl isobutyrate in organic solvent and solventfree system: a kinetic study, J. Mol. Catal. B: Enzym. 38 (2006) 1116.

D. Bezbradica, Z . Jakovljevic,
S. Brankovic-Dimitrijevi

D.
c,
c,
[20] Z. Knezevic-Jugovi
Mijin, Lipase catalyzed synthesis of avor esters in non-aqueous media: optimization of the yield of pentyl 2-methylpropanoate by statistical analysis, J.
Serb. Chem. Soc. 73 (2008) 11391151.
[21] M.J. Jimnez, L. Esteban, A. Robles, E. Hita, P.A. Gonzlez, M.M. Muno, E. Molina,
Production of triacylglycerols rich in palmitic acid at sn-2 position by lipasecatalyzed acidolysis, Biochem. Eng. J. 51 (2010) 172179.
[22] J. Pleiss, M. Fischer, R.D. Schmid, Anatomy of lipase binding sites: the scissile
fatty acid binding site, Chem. Phys. Lipids 93 (1998) 6780.
[23] F. Viklund, J. Alander, K. Hult, Antioxidative properties and enzymatic synthesis
of ascorbyl FA esters, J. Am. Oil Chem. Soc. 80 (2003) 795799.
[24] A.L. Paiva, V.M. Balcao, F.X. Malcata, Kinetics and mechanisms of reactions catalyzed by immobilized lipases, Enzyme Microb. Technol. 27 (2000)
187204.