Thermo-Kinetics of Lipase-Catalyzed Synthesis of 6-O-Glucosyldecanoate

Bioresource Technology 102 (2011)8727-8732
Thermo-kinetics of lipase-catalyzed synthesis of 6-O-glucosyldecanoate

A. M. Gumel,
1
M. S. M. Annuar,
1*
T. Heidelberg,
2
Y. Chisti
3

1
Institute of Biological Sciences,
2
Department of Chemistry,
Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

3
School of Engineering, PN 456, Massey University, Private Bag 11 222, Palmerston North,
New Zealand

*
Corresponding author:
Dr. M. Suffian M. Annuar
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia

e-mail: suffian_annuar@um.edu.my
Tel: +60379674003
Fax: +60379677182


Abstract
Lipase-catalyzed synthesis of 6-O-glucosyldecanoate from D-glucose and decanoic acid was
performed in dimethyl sulfoxide (DMSO), a mixture of DMSO and tert-butanol and tert-butanol
alone with a decreasing order of polarity. The highest conversion yield (>65%) of decanoic acid
was obtained in the blended solvent of intermediate polarity mainly because it could dissolve
relatively large amounts of both the reactants. The reaction obeyed Michaelis-Menten type of
kinetics. The affinity of the enzyme towards the limiting substrate (decanoic acid) was not
affected by the polarity of the solvent, but increased significantly with temperature. The
esterification reaction was endothermic with activation energy in the range of 60 to 67 kJ mol
1
.
Based on the Gibbs energy values, in the solvent blend of DMSO and tert-butanol the position of
the equilibrium was shifted more towards the products compared to the position in pure solvents.
Monoester of glucose was the main product of the reaction.

Keywords: 6-O-glucosyldecanoate; kinetics; lipase; esterification; water-activity;
thermodynamics.


1. Introduction
Sugar fatty acid esters such as 6-O-glucosyldecanote are an important class of biodegradable
nonionic surfactants. These esters are widely used in cosmetics, toiletries, detergents and other
applications (Ferrer et al., 2005; Queneau et al., 2008; Chang and Shaw, 2009; Furukawa et al.,
2010). Lipase-mediated synthesis of various sugar fatty acid esters has been reported (Coulon
and Ghoul, 1998; Adachi and Kobayashi, 2005; Divakar and Balaraman, 2007; Karmee, 2008;
Cauglia and Canepa, 2008; Chang and Shaw, 2009; Hernandez-Fernandez et al., 2010; Boulifi et
al., 2010; Sverac et al., 2011). Although lipase catalyzed synthesis of 6-O-glucosyldecanoate
has been reported (Anderson et al., 1998; Kirk et al., 1995; Kobayashi et al., 2010), to the best of
our knowledge, no specific information exists on the thermo-kinetics aspect of this reaction. This
work reports on the kinetics and thermodynamics of lipase-mediated synthesis of 6-O-
glucosyldecanoate for possible use in production of this potentially useful surfactant.
Immobilized lipase B of Candida antarctica (Anderson et al., 1998) is used for producing 6-O-
glucosyldecanoate from glucose and decanoic acid in solvents of different polarities. Effects of
the solvent polarity on the esterification reaction are discussed.
Lipase-mediated esterification occurs only in media which contain a low concentration of
water (Halling, 2000; Chamouleau et al., 2001; Sharma et al., 2001; Boulifi et al., 2010; Cauglia
& Canepa, 2008; Sverac et al., 2011; Singh et al., 2008; Valepyn et al., 2011). This is because
esterification is a reversible reaction; significant amount of water drives the reaction equilibrium
towards ester-bond hydrolysis instead of ester-bond synthesis. However, a certain amount of
water is necessary, for hydration of the lipase and to serve as nucleophile for its activity (Hudson
et al., 2005; Li et al., 2010). Enzymatic synthesis of sugar esters has been undertaken in mild

organic solvents such as dimethyl sulfoxide (DMSO)(Kaewprapan et al., 2011; Valepyn et al.,
2011), tert-butanol (Adnani et al., 2011), 2-methyl-2-butanol (Favrelle et al., 2011), hexane
(Adnani et al., 2010; Rahman et al., 2011) and ethanol (Neta et al., 2011). Some literatures
reported the synthesis in solvent blended mixtures (Ferrer et al., 2005; Fu & Vasudevan, 2010;
Plou et al., 2002; Valepyn et al., 2011). Compared to tertbutanol, DMSO is the most polar of the
solvents. A 1: 1 (by volume) mixture of DMSO and tert-butanol was used as a solvent of an
intermediate polarity compared to the other solvents mentioned. Ionic liquids (Chen et al., 2007;
De Diego et al., 2009; Fan & Qian, 2010; Ha et al., 2010; Lee et al., 2008; Moniruzzaman et al.,
2010; Muhammad et al., 2010) and supercritical fluids (Almeida et al., 1998; Fan & Qian, 2010;
Habulin et al., 2007; Keskin et al., 2007; Kumar, 2005; Liaw & Liu, 2010; Lozano et al., 2004;
Tai & Brunner, 2009) were also reported to be used as reaction media. Other researchers were
able to report the synthesis in solvent free process (Boulifi et al., 2010; Guillardeau, 1992; Shah
& Gupta, 2007; Ye et al., 2010).

2. Materials and methods
2.1. Enzyme and chemicals
Candida antarctica lipase B (Anderson et al., 1998) immobilized on resin beads was purchased
as Novozyme 435 (Novozymes A/S, Bagsvaerd, Denmark). Decanoic acid, D(+)-glucose, tert-
butanol, dimethyl sulfoxide (DMSO), potassium hydroxide, acetic acid, sulfuric acid, molecular
sieve (4 ; Sigma-Aldrich catalog number 33430-8, CAS No. 70955-01-0), isopropanol,
chloroform, hexane and methanol were purchased from Sigma-Aldrich
(www.sigmaaldrich.com). All chemicals were of analytical grade.


2.2. General reaction procedure
Esterification was conducted in airtight 20 mL vials in triplicate. The reaction substrates and the
organic solvent had been pre-equilibrated to an initial water activity (a
w
) value of 0.08 by
contacting with saturated aqueous solution of KOH for 10 days (Greenspan, 1977). In different
reaction vials, the initial concentration of decanoic acid was varied from 0.1 mM to 0.5 mM, but
the initial concentration of glucose remained fixed at 0.5 mM. Each reaction vial contained 10
mg of the molecular sieve in 15 mL of one of the following solvents: tert-butanol, DMSO and a
1:1 (v/v) mixture of DMSO and tert-butanol. Lipase (15 mg) was added to start the reaction. The
sealed reaction vials were held in a shaker (120 rpm) incubator at various temperatures (293,
303, 313, 323 and 333 K) for 7-hours. An entire vial was harvested at regular time intervals for
various analyses. Control experiments were carried out without adding lipase to the reaction
vials.

2.3. Analyses
2.3.1 Concentration of decanoic acid
The concentration of the residual decanoic acid in the reaction mixture was determined by
computer-controlled thermometric titration (Metrohm 859
titrotherm; Metrohm AG, Herisau,

Switzerland) of a 10 mL sample with 0.1 M of standard potassium hydroxide in isopropanol in
the presence of paraformaldehyde as the titration end-point thermometric indicator (Carneiro et
al., 2002). This titration method is based on measurements of the heat released by the exothermic
reaction between OH
and H
+
species. The concentration of the product at any time was
estimated as the amount of decanoic acid that had been converted or consumed by that time.


2.3.2 Water concentration in the reaction sample
The water concentration in the reaction sample was determined by Karl-Fischer coulometric
titration (Metrohm 831
KF coloumeter; Metrohm AG, Herisau, Switzerland). Samples were

titrated against HYDRANAL
-coulomat AG-H reagent (Sigma-Aldrich; catalog number

34805-1L-R; www.sigmaaldrich.com).

2.3.3 Ester products
The formation of ester products was qualitatively detected by thin-layer chromatography (TLC)
performed on pre-prepared plates of activated silica gel 60 F
254
(Merck, Germany). A sample of
the reaction mixture was spotted onto a plate in the usual way. The developing solvent was a
mixture of chloroform, methanol, acetic acid and water in the volume ratio of 70:20:8:2. The
ester spots were visualized by spraying the developed plates with a mixture of concentrated
sulfuric acid, ethanol and water (2:90:8 by vol), and incubated at 110 C for 15 minutes.

2.3.4 NMR analysis
For measuring the NMR spectra, the ester was recovered as follows: a sample of the reaction
mixture was filtered through a Whatman
Grade No. 40 filter paper (www.whatman.com) to

remove the immobilized enzyme and molecular sieve. The filtrate was then quenched by mixing
with absolute ethanol in the volume ratio of 1:5, and the resulting mixture was concentrated to
5 mL using a rotary vacuum evaporator (LABO-ROTA C-311; Resona-technics, Switzerland) at
70 C. Hexane (10 mL) was added to concentrated filtrate to extract the residual fatty acids. The
resulting mixture was centrifuged (1537 g, 10 min) and the hexane phase was discarded. The
remaining pellet contained residual glucose and glucosyldecanoate esters. A 20% (w/v) NaCl

solution (10 mL) was added to the recovered pellet in a separating funnel to precipitate the ester
and dissolve the residual glucose. The upper layer of the mixture was recovered from the
separation funnel and dried in a vacuum oven (30 C, 48 h). The dry material was used in
measuring the NMR spectra.
1
H-NMR spectra were recorded on JEOL JNM-GSX 270 FT-NMR (JOEL Ltd, Tokyo,
Japan) at 250 MHz. Deutrated methanol (CD
3
OD) was used as solvent.

2.4. Apparent rate constant and thermo-kinetics parameters
First order apparent rate constant (k
1
) was determined based on a steady-state approximation and
the following reaction scheme:
(1)
In the above scheme: D is decanoic acid; G is glucose; I is an intermediate reaction complex of
glucose, decanoic acid and enzyme; E is enzyme; and P is the ester product.
When decanoic acid D is the limiting reactant (i.e. an excess of G and E) and k
1
>>k
1
, the
concentration of D at any time t can be expressed as follows:
(2)
Therefore,
(3)
where D
0
is the initial concentration of decanoic acid. Further, at steady state, a balance on the
concentration of the reaction intermediate I can be written as follows:
(4)
or,

(5)
If k
2
>>k
1
, Eq (5) simplifies to the following:
(6)
Based on the reaction scheme in Eq (1), the rate of product formation can be written as
follows:
(7)
Equations (6) and (7) can be combined to the following:
(8)
A substitution of Eq (3) in Eq (8) produces the following:
(9)
The above equation can be integrated as follows:
(10)
Therefore,
(11)
or,
(12)
Therefore,
(13)
A rearrangement of Eq (13) produces the following:
(14)
Equation (14) may be reformulated to the following:

(15)
Because ln e = 1, Eq (15) becomes:
(16)
Thus, the apparent rate constant k
1
can be calculated by using the measured values of the product
concentration P at various times t and the known value of D
0
. The k
1
value was determined using
the least square method, as the best fit parameter for the measured P(t) data. The product
concentration P at time t was estimated as being equivalent to the quantity of decanoic acid that
had been converted by time t.
The kinetic parameters of Michaelis-Menten model, i.e. the apparent maximum
volumetric reaction rate and the apparent constant , were determined using the double
reciprocal plot method (Shuler and Kargi, 2002). The data fitted a straight line with a correlation
coefficient (r
2
) value of 0.998.
To determine whether the diffusion of the reactant to the immobilized enzyme catalyst
particles limited the reaction, the apparent rate constant for a diffusion controlled system
was calculated, as follows:
(17)
In Eq (17), R is the gas constant, T (K) is absolute temperature and q
s
is the viscosity of the
reaction solvent. If k
1
<< , diffusion is unlikely to be limiting the reaction.
The viscosities of the solvents were measured at 251 C using Cannon-Fenske Opaque
(Reverse-Flow) viscometer No. 400 D283 (www.canoninstrument.com). Viscosities were
calculated using the following equation:
(18)

where
s
and
w
are the densities of the solvent and water, respectively; t
s
and t
w
are the times
time taken by the solvent and water to drop under the influence of gravity to a specified level in
the viscometer; and q
w
is the viscosity of water at 251 C. Densities were measured at 251 C,
using Anton-Paar DMA 35 Density Meter (www.anton-paar.com). The viscosities of tert-
butanol, DMSO and 1:1 (by vol) mixture of DMSO and tert-butanol were 0.034, 0.020 and 0.030
g cm
1
s
1
, respectively.
The thermodynamic activity coefficient
w
for water in the reaction system was calculated
from the data of coulometric titration, using Wilsons coefficients (Bell et al., 1997), as follows:
(19)
where A
ws
and A
sw
were calculated using Eq (20) and Eq (21), respectively, as follows:
(20)
(21)
In the above equations, V
w
is the molar volume (i.e. the molecular weight divided by density) of
water at 25 C; V
s
is the molar volume of the solvent; and are
Wilsons coefficients as cited by Bell et al. (1997); R is the molar gas constant (8.314 J mol
1

K
1
); x
w
and x
s
are the mole fractions of water and solvent in the reaction mixture, respectively.
The x
w
and x
s
values were calculated using the following equations:
(22)
(23)
where
w
is the volume fraction of water. The values of
w
(Eq 19) and x
w
(Eq 22) were used to
calculate the thermodynamic water activity a
w
, as follows:

(24)
The activation energy E
a
of the reaction was calculated using an Arrhenius plot. Thus, the
rate constant k
1
values measured at various temperatures T were plotted in the following form
and the slope was calculated as E
a
:
(25)
where A is Arrhenius parameter.
At equilibrium, the changes in apparent enthalpy AH

and entropy AS

of the reaction were
determined from the slope and intercept of a Vant Hoff plot:
(26)
The K
eq
value was calculated as follows:
(27)
The concentrations D, [H
2
O] and G for use in Eq (27) were measured at equilibrium. The
assumptions implicit in Vant Hoff analysis (i.e. a closed system at a constant pressure and
volume) were applicable to the esterification reaction system used. The Gibbs energy change AG
of the reaction was calculated as follows:
(28)

2.5. Log P for the solvents
The log P values for DMSO and tert-butanol were obtained from the literature (Lide, 2005). The
log P
mix
of the mixture of DMSO and tert-butanol was calculated as outlined by Fu and
Vasudevan (2010); thus,
(29)

where log P
DMSO
and log P
tBuOH
are the log P values of DMSO and tert-butanol, respectively,
and x
DMSO
and x
tBuOH
are the corresponding mole fractions of the two solvents in the reaction
mixture, respectively.

3. Results and discussion
3.1 Temperature and solvents
Temperature had a strong effect on both the apparent reaction rate constant k
1
and the percentage
conversion of decanoic acid to the product (Figure 1). Both the conversion yield and the rate
constant increased with increasing temperature, but in a nonlinear fashion, which depended on
the polarity of the solvent used. In tert-butanol, the least polar of the solvents used, the apparent
rate constant was least sensitive to temperature compared to the cases for the other. In contrast,
the decanoic acid conversion yield in tert-butanol was quite comparable to that in the most polar
solvent, DMSO. The solvent of intermediate polarity, i.e. a 1:1 mixture of DMSO and tert-
butanol, afforded the highest conversion yields at all temperatures (Figure 1). The absolute
values of k
1
and their dependence on temperature were quite similar for the most polar solvent
(i.e. DMSO) and the solvent of intermediate polarity (mixture of DMSO and tert-butanol).
The temperature affected the conversion both by influencing the solubilities of the two
reactants and by affecting the apparent rate constant k
1
. The solvent-associated differences in the
conversion values were attributed at least partly to differences in the solubilities of the reactants
in the solvents. Glucose was apparently most soluble in DMSO whereas decanoic acid was
observed to be more soluble in tert-butanol. In contrast, the solvent blend could dissolve both the
reactants reasonably well.

The relative solubilities of the reactants in the solvents were consistent with the literature
values or estimated values of solvent polarities (log P). Thus, DMSO ((CH
3
)
2
SO, log P = 1.35)
had a higher polarity than tert-butanol ((CH
3
)
3
COH, log P = 0.35) (Lide, 2005), and the blend of
DMSO and tert-butanol exhibited an intermediate polarity value (log P
mix
) of 0.62.
Irrespective of the solvent used, at any fixed temperature the reaction followed
Michaelis-Menten type of kinetics. Thus, the initial reaction rate v increased with increasing
initial concentration of decanoic acid in a hyperbolic manner (data not shown), thus confirming
the fatty acid to be the limiting reactant, as was assumed in the modeling of the kinetics. For
otherwise identical conditions, DMSO afforded the lowest initial reaction rate and the highest
rates were seen in the solvent blend of DMSO and tert-butanol. This was attributed to a higher
solubility of decanoic acid in the blended solvent compared to pure DMSO, as explained earlier.
To examine whether the solvents in their pure and blended forms affected the affinity of
the enzyme towards its limiting substrate (i.e. decanoic acid), the measured initial reaction rate
values at various initial concentrations of decanoic acid were plotted in the form of a
Lineweaver-Burk plot. This provided the values of the apparent maximum reaction rate and
the apparent Michaelis-Menten constant (Figure 2). is a relative measure of the affinity
of the enzyme towards the limiting substrate (Copeland, 2000). represents the substrate
concentration at which a half of the active sites of the enzyme in a reaction sample are occupied
by the substrate at steady state (Copeland, 2000). Thus, a lower relative value of indicates a
higher affinity of the enzyme towards the substrate.
The apparent maximum reaction rate increased with increasing temperature up to
about 313 K, but was unaffected by further increase in temperature within the range examined
(Figure 2). Both the temperature dependence of and its final value were highest in the

blended solvent compared to in the pure solvents. This, too, was a consequence of the better
dissolving capability of the blended solvent for both the reactants involved. In any given solvent,
the temperature independence of in the range of 313 < T s 333 K (Figure 2) suggests that
the enzyme was quite stable at the highest temperature used. Indeed, a higher temperature of up
to 343 K has been previously found to be satisfactory for the specific enzyme used in the present
work (Gumel et al., 2011).
The values were also found to varied with temperature as shown in Figure 2. The
nature of the solvent did not affect the values significantly, but the temperature did. The
observed independence of from solvent polarity was consistent with an earlier similar report
(Kobayashi et al., 2003) for the same enzyme as used in the present study. Thus, the affinity of
the enzyme towards decanoic acid was unaffected by the polarity of the reaction medium.
However, in all solvents, an increase in temperature of >310 K caused a precipitous decline in
the value of , i.e. the affinity of the enzyme towards the substrate increased. This decline in
may have been caused by an improved diffusion of the reactants to the active site of the
enzyme, but the k
1
values were always nearly 10
9
-folds lower than the values; therefore,
diffusion could not have limited the reaction. An increased temperature would have certainly
increased the constant wriggling motions that occur within an enzyme molecule and this may
have facilitated the binding of the substrates to the active site and also the detachment of the
product from it.

3.2 Water activity
In a batch reaction involving esterification, water is a co-product. Production of water increases
the water activity in the reaction medium. Furthermore, an accumulation of water tends to drive

the reaction equilibrium back towards the reactants. Notwithstanding this, a certain amount of
water needs to be present as being necessary for hydration of the enzyme and its activity
(Hudson et al., 2005; Salem et al., 2010).
The reaction rate and water activity profiles were monitored for 420 minutes during the
batch reaction. As shown in Figure 3, the water activity rose during the reaction in all solvents,
despite the presence of the molecular sieves in the reaction vials. This was because of the water
produced by the reaction. In the solvents of high polarity (i.e. DMSO) and intermediate polarity
(i.e. the solvent blend), the rate constant k
1
actually increased, at first slowly (reaction time of up
to about 200 min), but then more rapidly (reaction time of >200 min). After about 300 min, the
time no longer affected the rate constant (Figure 3). Later in the reaction, the k
1
values in the
least polar solvent (i.e. tert-butanol) were generally low compared to in the other two solvents
(Figure 3).
Apparently, the k
1
values were being affected by the water activity (a
w
). At water activity
values of <0.355, there may have been insufficient water for the enzyme to function
satisfactorily. In the water activity range of 0.355 < a
w
s 0.6, increasing water activity promoted
the reaction and enhanced k
1
(Figure 3). Once the water activity exceeded about 0.6, water
inhibited the reaction. In tert-butanol, the rapid initial reaction rate because of a high solubility of
decanoic acid led to a rapid rise in water activity. The initial slow rise in k
1
was not observed, as
the water activity was always well above 0.355.
In view of the results (Figure 1, Figure 2 and Figure 3), a good case exists for using the
blended solvent for this reaction. Other studies suggest that by modifying the polarity at the
enzyme active site, a solvent may contribute to stabilization of the charged transition states
during the reaction (Xu et al., 1994). Blended solvents have indeed been widely used to

favorably modify the polarity and ionization capacity of the reaction media in lipase-mediated
esterification reactions (Akoh and Mutua, 1994; Degn and Zimmermann, 2001; Jia et al., 2010).

3.3 Effect of molecular sieves
Molecular sieves were added to the reaction mixture in attempts to absorb the water produced by
the reaction and thus reduce the inhibitory effect of excessive water. Different concentrations of
the molecular sieves (020 g L
1
) were tested in reaction media, which contained equimolar
amounts (0.5 mM each) of glucose and decanoic acid. At a fixed temperature of 323 K, the initial
reaction rate in the various solvents depended on the concentration of the molecular sieves, as
shown in Figure 4. The initial reaction rate increased progressively with increasing concentration
of the sieves and peaked at a sieve concentration of about 13 g L
1
. A further increase in the
loading of the molecular sieves reduced the initial reaction rate. This effect occurred quite
consistently in all the solvents. The water activities of the reaction media with different
concentrations of the sieves present were measured to see if this might explain the behavior seen
in Figure 4.
In all media, an increasing concentration of the molecular sieves progressively reduced
the water activity as shown in Figure 4. At any fixed concentration of the molecular sieve, the
water activity was measured at various times during the reaction and Figure 4 provides the time-
averaged water activity. The plots in Figure 4 suggest that the optimal water activity for this
reaction was in the range of 0.45 to about 0.6, depending on the solvent. A molecular sieve
concentration of ~13 g L
1
was optimal, likely because at this concentration the water activity
was maintained within the requisite range for longer during the reaction. A higher concentration

of the sieves reduced water activity to values that were insufficient for enzyme hydration and/or
activity.

3.4 Thermodynamics of the reaction
The enthalpy of the reaction (AH), the entropy (AS) and the activation energy (E
a
) data for the
esterification reaction in the various solvents are shown in Table 1. The reaction is found to be
endothermic in view of the positive values of AH. Positive values of AH are of course consistent
with the observed increase in k
1
values with increasing temperature (Figure 1).
The activation energy values of the reaction in the different solvents were found to be
quite comparable. The nature of the solvent is not expected to significantly influence the E
a

values so long as the reaction occurs via the same general mechanism and transition state.
Similarly, the type of solvent did not significantly affect the entropy change for the reaction
system. This is expected so long as the solvent does not significantly affect the end products
(glucosyldecanoate and water in this case) being formed from a given set of reactants.
It is however, observed that an increase in reaction temperature reduced AG values in all
solvents (data not shown), indicating a shift of the reaction equilibrium towards the products
with increasing temperature. The values of AG were found to be lower in the solvent blend
compared to the values in the pure solvents. Therefore, the reaction equilibrium was most
favorable (i.e. tended towards glucosyldecanoate) in the blended solvent.
The values of AH, AS and E
a
in an esterification reaction depend on the specific reactants
involved. No data were found in the literature for the specific reaction examined in this study.


3.5 Product authentication
Formation of the expected product was confirmed by proton NMR spectroscopy. The
1
H
chemical shift for the terminal methyl (CH
3
) protons of the ester occurred at 0.890 ppm. The
chemical shift due to methylene (CH
2
) protons in the hydrocarbon chain of the glucose ester
occurred at 1.291 ppm. The two peaks at 1.602 ppm and 2.325 ppm were associated with the
methylene protons linked directly to the carbonyl (C=O) group of the ester. Occurrence of two
peaks suggested the formation of two different glucose esters. These were the decanoic acid
esters of -D-glucose and -D-glucose. Two esters formed because on dissolution D-glucose
existed in the reaction medium in its two isomeric forms (Molteni and Perrinello, 1998). The
peaks at 4.379 ppm and 5.071 ppm were attributed to -D-glucose ester and -D-glucose ester,
respectively. A possible di-ester peak was observed at 3.246 ppm. The monoesters peaks at 2.325
ppm and 1.602 ppm, strongly indicated that glucose monoesters of and forms were the major
esters produced. Indeed, the lipase used is known to favor the formation of monoesters with a
high specificity (Degn et al., 1999). Thus, from the obtained spectra, the fraction of the total
monoesters corresponding to o- and |-forms was estimated from the areas under the two
monoesters peaks. The o-from was found to constitute ~85% of the monoester whereas ~15% of
the monoesters were in the |-form.

4. Conclusions
Equi-volume blend of DMSO and tert-butanol with intermediate polarity proved to be the best
for esterification of D-glucose and decanoic acid as compared to the pure solvents themselves
with >65% conversion of decanoic acid to the product. The blend dissolved relatively high
concentrations of both reactants. The optimal a
w
for the reaction in the blended solvent was

about 0.6. In the batch reaction, the optimal loading of the molecular sieve was ~13 g L
-1
. The
reaction was endothermic and followed Michaelis-Menten kinetics. The affinity of the enzyme
towards decanoic acid was not affected by the polarity of the reaction medium.

Acknowledgements
The University of Malaya is acknowledged for funding this work through research grants
RG003/09AFR, RG165/11AFR and P0082/2010B.

References
Adachi, S., Kobayashi, T. 2005. Synthesis of esters by immobilized-lipase-catalyzed
condensation reaction of sugars and fatty acids in water-miscible organic solvent. Journal
of Bioscience and Bioengineering, 99(2), 87-94.
Adnani, A., Basri, M., Chaibakhsh, N., Ahangar, H.A., Salleh, A.B., Rahman, R.N.Z.R.A.,
Abdul Rahman, M.B. 2011. Chemometric analysis of lipase-catalyzed synthesis of xylitol
esters in a solvent-free system. Carbohydrate Research.
Adnani, A., Basri, M., Malek, E.A., Salleh, A.B., Abdul Rahman, M.B., Chaibakhsh, N.,
Rahman, R.N.Z.R.A. 2010. Optimization of lipase-catalyzed synthesis of xylitol ester by
Taguchi robust design method. Industrial Crops and Products, 31(2), 350-356.
Akoh, C.C., Mutua, L.M. 1994. Synthesis of alkyl glucoside fatty acid esters: effect of reaction
parameters and the incorporation of n-3 polyunsaturated fatty acids. Enzyme and
Microbial Technology, 16, 115-119.

Almeida, M.C., Ruivo, R., Maia, C., Freire, L., Corra de Sampaio, T., Barreiros, S. 1998.
Novozym 435 Activity in Compressed Gases. Water Activity and Temperature Effects.
Enzyme and Microbial Technology, 22(6), 494-499.
Anderson, E.M., Larsson, K.M., Kirk, O. 1998. One biocatalyst - Many applications: The use of
Candida antarctica B-lipase in organic synthesis. Biocatalysis and Biotransformation,
16(3), 181 - 204.
Bell, G., Janssen, A.E.M., Halling, P.J. 1997. Water activity fails to predict critical hydration
level for enzyme activity in polar organic solvents: Interconversion of water
concentrations and activities. Enzyme and Microbial Technology, 20(6), 471-477.
Boulifi, N.E., Aracil, J., Martnez, M. 2010. Lipase-catalyzed synthesis of isosorbide
monoricinoleate: Process optimization by response surface methodology. Bioresource
Technology, 101(22), 8520-8525.
Carneiro, M.J.D., Feres Jnior, M.A., Godinho, O.E.S. 2002. Determination of the acidity of oils
using paraformaldehyde as a thermometric end-point indicator. Journal of the Brazilian
Chemical Society, 13, 692-694.
Cauglia, F., Canepa, P. 2008. The enzymatic synthesis of glucosylmyristate as a reaction model
for general considerations on sugar esters' production. Bioresource Technology, 99(10),
4065-4072.
Chamouleau, F., Coulon, D., Girardin, M., Ghoul, M. 2001. Influence of water activity and water
content on sugar esters lipase-catalyzed synthesis in organic media. Journal of Molecular
Catalysis B: Enzymatic, 11(4-6), 949-954.

Chang, S.W., Shaw, J.F. 2009. Biocatalysis for the production of carbohydrate esters. New
Biotechnology, 26(3-4), 109-116.
Chen, Z., Zong, M., Gu, Z. 2007. Enzymatic synthesis of sugar esters in ionic liquids. Chinese
Journal of Organic Chemistry, 27, 1448-52.
Copeland, R.A. 2000. Enzymes: a practical introduction to structure, mechanism, and data
analysis. Wiley-Vch.
Coulon, D., Ghoul, M. 1998. The enzymatic synthesis of non-ionic surfactants: the sugar esters-
An overview. Agro food inducstry Hi-tech, 9, 22-26.
De Diego, T., Lozano, P., Abad, M.A., Steffensky, K., Vaultier, M., Iborra, J.L. 2009. On the
nature of ionic liquids and their effects on lipases that catalyze ester synthesis. Journal of
Biotechnology, 140(3-4), 234-241.
Degn, P., Pedersen, L.H., Duus J., Zimmermann, W. 1999. Lipase-catalysed synthesis of glucose
fatty acid esters in tertbutanol. Biotechnology Letters, 21, 275-280.
Degn, P., Zimmermann, W. 2001. Optimization of carbohydrate fatty acid ester synthesis in
organic media by a lipase from Candida antarctica. Biotechnology and Bioengineering,
74, 483-491.
Divakar, S., Balaraman, M. 2007. Uses of lipases in the industrial production of esters. in:
Industrial enzymes, (Ed.) M.A.P. Polaina J, Springer. London UK, pp. 283-300.
Fan, Y., Qian, J. 2010. Lipase catalysis in ionic liquids/supercritical carbon dioxide and its
applications. Journal of Molecular Catalysis B: Enzymatic, 66(1-2), 1-7.

Favrelle, A., Boyre, C., Laurent, P., Broze, G., Blecker, C., Paquot, M., Jrme, C., Debuigne,
A. 2011. Enzymatic synthesis and surface active properties of novel hemifluorinated
mannose esters. Carbohydrate Research.
Ferrer, M., Soliveri, J., Plou, F.J., Lpez-Corts, N., Reyes-Duarte, D., Christensen, M., Copa-
Patio, J.L., Ballesteros, A. 2005. Synthesis of sugar esters in solvent mixtures by lipases
from Thermomyces lanuginosus and Candida antarctica B, and their antimicrobial
properties. Enzyme and Microbial Technology, 36(4), 391-398.
Fu, B., Vasudevan, P.T. 2010. Effect of Solvent- Co-solvent Mixtures on Lipase-Catalyzed
Transesterification of Canola Oil. Energy & Fuels, 3975-3981.
Furukawa, S., Akiyoshi, Y., O'Toole, G.A., Ogihara, H., Morinaga, Y. 2010. Sugar fatty acid
esters inhibit biofilm formation by food-borne pathogenic bacteria. International Journal
of Food Microbiology, 138(1-2), 176-180.
Greenspan, L. 1977. Humidity fixed points of binary saturated aqueous solutions. Journal of
Research National Bureau of Standard, 81A(1), 89-96.
Guillardeau, L., Monte, T. D., Khaeld, N., Pina, M., Graille. 1992. Fructose caprylate
biosynthesis in a solvent free medium. Tenside, Surfactants, Detergents, 29, 342-344.
Gumel, A.M., Annuar, M.S.M., Heidelberg, T., Chisti, Y. 2011. Lipase mediated synthesis of
sugar fatty acid esters,. Submitted for publication.
Ha, S.H., Hiep, N.M., Lee, S.H., Koo, Y.M. 2010. Optimization of lipase-catalyzed glucose ester
synthesis in ionic liquids. Bioprocess and Biosystems Engineering, 33(1), 63-70.

Habulin, M., Sabeder, S., Paljevac, M., Primozic, M., Knez, Z. 2007. Lipase-catalyzed
esterification of citronellol with lauric acid in supercritical carbon dioxide/co-solvent
media. Journal of Supercritical Fluids, 43(2), 199-203.
Halling, P.J. 2000. Biocatalysis in low-water media: understanding effects of reaction conditions.
Current Opinion in Chemical Biology, 4(1), 74-80.
Hernandez-Fernandez, F.J., de los Rios, A.P., Lozano-Blanco, L.J., Godinez, C. 2010.
Biocatalytic ester synthesis in ionic liquid media. Journal of Chemical Technology and
Biotechnology, 85(11), 1423-1435.
Hudson, E.P., Eppler, R.K., Clark, D.S. 2005. Biocatalysis in semi-aqueous and nearly
anhydrous conditions. Current Opinion in Biotechnology, 16(6), 637-643.
Jia, C., Zhao, J., Feng, B., Zhang, X., Xia, W. 2010. A simple approach for the selective
enzymatic synthesis of dilauroyl maltose in organic media. Journal of Molecular
Catalysis B: Enzymatic, 62(3-4), 265-269.
Kaewprapan, K., Wongkongkatep, J., Panbangred, W., Phinyocheep, P., Marie, E., Durand, A.,
Inprakhon, P. 2011. Lipase-catalyzed synthesis of hydrophobically modified dextrans:
Activity and regioselectivity of lipase from Candida rugosa. Journal of Bioscience and
Bioengineering, In Press, Corrected Proof.
Karmee, S.K. 2008. Lipase catalyzed synthesis of ester based surfactants from biomass
derivatives. Biofuels, Bioproducts and Biorefining, 2(2), 144-154.
Keskin, S., Kayrak-Talay, D., Akman, U., Hortasu, . 2007. A review of ionic liquids towards
supercritical fluid applications. Journal of Supercritical Fluids, 43(1), 150-180.

Kirk, O., Christensen, M.W., Beck, F., Damhus, T. 1995. Lipase-Catalyzed Regioselective
Acylation and Deacylation of Glucose Derivatives. Biocatalysis and Biotransformation,
12(2), 91-97.
Kobayashi, T., Adachi, S., Matsuno, R. 2003. Kinetic analysis of the immobilized-lipase-
catalyzed synthesis of octanoyl octyl glucoside in acetonitrile. Biochemical Engineering
Journal, 16(3), 323-328.
Kumar, R., Modak, J. and Madras, G. 2005. Effect of the chain length of the acid on the
enzymatic synthesis of flavours in supercritical carbon di oxide. Biochemical
Engineering Journal, 23, 199-202.
Lee, S.H., Ha, S.H., Hiep, N.M., Chang, W.-J., Koo, Y.-M. 2008. Lipase-catalyzed synthesis of
glucose fatty acid ester using ionic liquids mixtures. Journal of Biotechnology, 133(4),
486-489.
Li, C., Tan, T., Zhang, H., Feng, W. 2010. Analysis of the Conformational Stability and Activity
of Candida antarctica Lipase B in Organic Solvents. Journal of Biological Chemistry,
285(37), 28434.
Liaw, E.-T., Liu, K.-J. 2010. Synthesis of terpinyl acetate by lipase-catalyzed esterification in
supercritical carbon dioxide. Bioresource Technology, 101(10), 3320-3324.
Lide, D.R. 2005. CRC Handbook of Chemistry and Physics.
Lozano, P., Villora, G., Gomez, D., Gayo, A.B., Snchez-Conesa, J.A., Rubio, M., Iborra, J.L.
2004. Membrane reactor with immobilized Candida antarctica lipase B for ester
synthesis in supercritical carbon dioxide. Journal of Supercritical Fluids, 29(1-2), 121-
128.

Molteni, C., Parrinello, M. 1998. Glucose in Aqueous Solution by First Principles Molecular
Dynamics. Journal of the American Chemical Society, 120(9), 2168-2171.
Moniruzzaman, M., Nakashima, K., Kamiya, N., Goto, M. 2010. Recent advances of enzymatic
reactions in ionic liquids. Biochemical Engineering Journal, 48(3), 295-314.
Muhammad, M., Kazunori N., Noriho K., Masahiro G. 2010. Recent advances of enzymatic
reactions in ionic liquids. Biochemical Engineering Journal, 48, 295-314.
Neta, N.S., Peres, A.M., Teixeira, J.A., Rodrigues, L.R. 2011. Maximization of fructose esters
synthesis by response surface methodology. New Biotechnology, 28(4), 349-355.
Plou, F.J., Cruces, M.A., Ferrer, M., Fuentes, G., Pastor, E., Bernab, M., Christensen, M.,
Comelles, F., Parra, J.L., Ballesteros, A. 2002. Enzymatic acylation of di- and
trisaccharides with fatty acids: choosing the appropriate enzyme, support and solvent.
Journal of Biotechnology, 96(1), 55-66.
Queneau, Y., Chambert, S., Besset, C., Cheaib, R. 2008. Recent progress in the synthesis of
carbohydrate-based amphiphilic materials: the examples of sucrose and isomaltulose.
Carbohydrate Research, 343(12), 1999-2009.
Rahman, N.F.A., Basri, M., Rahman, M.B.A., Rahman, R.N.Z.R.A., Salleh, A.B. 2011. High
yield lipase-catalyzed synthesis of Engkabang fat esters for the cosmetic industry.
Bioresource Technology, 102(3), 2168-2176.
Salem, J.H., Humeau, C., Chevalot, I., Harscoat-Schiavo, C., Vanderesse, R., Blanchard, F.,
Fick, M. 2010. Effect of acyl donor chain length on isoquercitrin acylation and biological
activities of corresponding esters. Process Biochemistry, 45(3), 382-389.

Sverac, E., Galy, O., Turon, F., Monsan, P., Marty, A. 2011. Continuous lipase-catalyzed
production of esters from crude high-oleic sunflower oil. Bioresource Technology.
Shah, S., Gupta, M.N. 2007. Lipase catalyzed preparation of biodiesel from Jatropha oil in a
solvent free system. Process Biochemistry, 42(3), 409-414.
Sharma, R., Chisti, Y., Banerjee, U.C. 2001. Production, purification, characterization, and
applications of lipases. Biotechnology Advances, 19(8), 627-662.
Shuler, M.L., Kargi, F. 2002. Bioprocess Engineering. Prentice Hall.
Singh, M., Singh, S., Singh, R.S., Chisti, Y., Banerjee, U.C. 2008. Transesterification of primary
and secondary alcohols using Pseudomonas aeruginosa lipase. Bioresource Technology,
99(7), 2116-2120.
Tai, H.P., Brunner, G. 2009. Sugar fatty acid ester synthesis in high-pressure acetone-CO2
system. Journal of Supercritical Fluids, 48(1), 36-40.
Valepyn, E., Nys, J., Richel, A., Laurent, P., Berezina, N., Talon, O., Paquot, M. 2011. Lipase-
catalyzed synthesis of l-cysteine glucosyl esters in organic media. Biocatalysis and
Biotransformation(0), 1-6.
Xu, Z.F., Affleck, R., Wangikar, P., Suzawa, V., Dordick, J.S. and Clark, D.S. 1994. Transition
state stabilization of subtilisin in organic media. Biotechnology and Bioengineering, 43,
515-520.
Ye, R., Pyo, S.H., Hayes, D.G. 2010. Lipase-Catalyzed Synthesis of SaccharideFatty Acid
Esters Using Suspensions of Saccharide Crystals in Solvent-Free Media. Journal of the
American Oil Chemists' Society, 87(3), 281-293.



Figure captions
Figure 1. Apparent rate constant k
1
and decanoic acid conversion as a function of temperature.
Reaction conditions: 0.5 mM decanoic acid, 0.5 mM glucose, 10 mg molecular sieve, 15 mg
lipase, in 15 mL solvent. Maximum standard deviation of the data points was s2%.

Figure 2. and as a function of reaction temperature. Reaction conditions: 0.5 mM
glucose, 10 mg molecular sieve, 15 mg lipase, in 15 mL solvent. Maximum standard deviation of
the data points was s2%.

Figure 3. Apparent rate constant k
1
and water activity a
w
as a function of reaction time. Reaction
conditions: Glucose 0.5 mM, 10 mg molecular sieve, 15 mg lipase, in 15 mL solvent at 323 K.
Maximum standard deviation of the data points was s2%.

Figure 4. Initial rate v and water activity (a
w
) as a function of molecular sieve loading. Reaction
conditions: 0.5 mM decanoic acid, 0.5 mM glucose, 15 mg lipase, in 15 mL solvent at 323 K.
Maximum standard deviation of the data points was s2%.


Table 1. Activation energy (E
a
), enthalpy (H) and entropy change (S) for the solvents used.

Solvent H
(kJ mol
1
)
E
a

(kJ mol
1
)
S
(J mol
1
K
1
)
tert-Butanol 80.3 67.3 221
DMSO 60.7 62.0 157
DMSO: tert-butanol (1:1 v/v) 62.2 60.2 159


Temperature (K)
290 300 310 320 330 340
k
1

(
x
1
0
4

m
i
n
1
)
0
2
4
6
8
10
m
a
x
i
m
u
m

d
e
c
a
n
o
i
c

a
c
i
d

c
o
n
v
e
r
s
i
o
n

(
%
)
20
30
40
50
60
70
tert-butanol
DMSO
DMSO:tert-butanol
tert-butanol
DMSO
DMSO:tert-butanol
k
1
conversion

Figure 1


Temp (K)
290 300 310 320 330 340
V
m
a
x

a
p
p

x

1
0
-
6
(
m
o
l

L
-
1

m
i
n
-
1
)
2.5
3.0
3.5
4.0
4.5
5.0
K
M
a
p
p
0.00
0.02
0.04
0.06
0.08
0.10
tert-butanol
DMSO
DMSO:tert-butanol
tert-butanol
DMSO
DMSO:tert-butanol
V
max

app
K
M
app

Figure 2

Time (min)
0 100 200 300 400 500
k
1

(
x

1
0
-
4

m
i
n
-
1
)
0
5
10
15
20
25
a
w
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
tert-butanol
tert-butanol
DMSO
DMSO
DMSO:tert-butanol
DMSO:tert-butanol
k
1

a
w

Figure 3

molecular sieve (g L
-1
)
0 2 4 6 8 10 12 14 16 18 20 22
v

(
m
o
l

L
-
1

m
i
n
-
1
)
0
1
2
3
4
5
a
w
0.1
0.2
0.3
0.4
0.5
0.6
0.7
tert-butanol
DMSO
DMSO:tert-butanol
tert-butanol
DMSO
DMSO:tert-butanol
v
a
w
excess molecular sieve

Figure 4

Thermo-Kinetics of Lipase-Catalyzed Synthesis of 6-O-Glucosyldecanoate

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Thermo-Kinetics of Lipase-Catalyzed Synthesis of 6-O-Glucosyldecanoate

Uploaded by

Copyright:

Available Formats

Bioresource Technology 102 (2011)8727-8732

Thermo-kinetics of lipase-catalyzed synthesis of 6-O-glucosyldecanoate

titrotherm; Metrohm AG, Herisau,

KF coloumeter; Metrohm AG, Herisau, Switzerland). Samples were

-coulomat AG-H reagent (Sigma-Aldrich; catalog number

Grade No. 40 filter paper (www.whatman.com) to

You might also like