Study on acyl migration kinetics of partial glycerides: Dependence on temperature and water activity

Article abstract of DOI:10.1016/j.molcatb.2009.11.012

Acyl migration phenomenon was often observed during 1,3-positional specificity lipase-catalyzed reactions from triglycerides and partial glycerides, including acyl migration of 1,2-diglyceride (1,2-DG) to 1,3-diglyceride (1,3-DG) and 2-monoglyceride (2-MG) to 1-monoglyceride (1-MG). However, the acyl migration mechanism and kinetics were seldom studied despite of numerous researches on process optimization of 1,3-positional specificity lipase-catalyzed reaction. In this paper, the influence of related factors on acyl migration process as well as their influencing mechanism was further studied. It was found that temperature and water activity were two crucial factors that would influence acyl migration kinetics. Determination of the kinetic parameters under different temperatures revealed that the acyl migration reaction rates were greatly promoted by the increasing of temperature. The acyl migration rates of 1,2-diglyceride and 2-monoglyceride were quite different from each other, which was found to be due to the different activation energies. Further study of how would water influence the acyl migration process showed that water activity rather than water content was a key factor that influenced acyl migration and the acyl migration rate would decrease with the increase of water activity. It was further revealed that water activity influenced the charge dispersion of the transition state, which ultimately influenced the reaction activation energy and then influenced the acyl migration rate.

Full text of DOI:10.1016/j.molcatb.2009.11.012

Journal of Molecular Catalysis B: Enzymatic 63 (2010) 17–22
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Study on acyl migration kinetics of partial glycerides: Dependence on
temperature and water activity
∗
∗
Wei Li, Wei Du , Qiang Li, Ting Sun, Dehua Liu
Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 June 2009
Received in revised form
Acyl migration phenomenon was often observed during 1,3-positional specificity lipase-catalyzed reac-
tions from triglycerides and partial glycerides, including acyl migration of 1,2-diglyceride (1,2-DG) to
1
,3-diglyceride (1,3-DG) and 2-monoglyceride (2-MG) to 1-monoglyceride (1-MG). However, the acyl
2
9 November 2009
migration mechanism and kinetics were seldom studied despite of numerous researches on process
optimization of 1,3-positional specificity lipase-catalyzed reaction. In this paper, the influence of related
factors on acyl migration process as well as their influencing mechanism was further studied. It was
found that temperature and water activity were two crucial factors that would influence acyl migration
kinetics. Determination of the kinetic parameters under different temperatures revealed that the acyl
migration reaction rates were greatly promoted by the increasing of temperature. The acyl migration
rates of 1,2-diglyceride and 2-monoglyceride were quite different from each other, which was found to
be due to the different activation energies. Further study of how would water influence the acyl migra-
tion process showed that water activity rather than water content was a key factor that influenced acyl
migration and the acyl migration rate would decrease with the increase of water activity. It was further
revealed that water activity influenced the charge dispersion of the transition state, which ultimately
influenced the reaction activation energy and then influenced the acyl migration rate.
Accepted 30 November 2009
Available online 2 December 2009
Keywords:
Kinetics
Acyl migration
Partial glycerides
Water activity
©
2009 Elsevier B.V. All rights reserved.
1
. Introduction
erides [6–9]. During 1,3-positional specificity lipase-catalyzed
biodiesel production from triglyceride, partial glycerides of
1,2-diglyceride and 2-monoglyceride would accumulate in the
reaction medium and the theoretical methyl esters yield was
only 67%. While in the process of some 1,3-positional speci-
ficity lipase-catalyzed biodiesel production, the reported biodiesel
yield could research over 80% [6–9]. In our previous study
of Rhizopus oryzae (with intracellular 1,3-positional specificity
lipase)-mediated methanolysis of triolein, it was revealed that
higher practical methyl ester yield over theoretical yield was partly
due to acyl migration of 1,2-DG to 1,3-DG and 2-MG to 1-MG
(Scheme 1), which further progressed the methanolysis reaction.
So obviously acyl migration was important to the 1,3-positional
specificity lipase-catalyzed biodiesel production, since it is vital to
the biodiesel yield.
Acyl migration is also an important phenomenon during
1,3-positional specificity lipase-catalyzed production of partial
glycerides, which are useful in a broad range of food, cosmetic and
pharmaceutical applications, especially in the production of partial
glycerides in a particular isomeric form [10,11]. Acyl migration dur-
ing this process was also crucial to the ratio and purity of different
partial glyceride isomers such as 1,3-DG, 1,2-DG, 1-MG and 2-MG.
It was found that acyl migration could take place independent of
enzyme catalysis [14–16]. That means, acyl migration between 2-
Biodiesel, which is defined as methyl esters derived from plant
oils and animal fats, has attracted numerous attention as a clean
and renewable energy, since it is one of the possible solutions to
solve the world wide problem of energy crisis and environmental
protection [1–3]. Conventional chemical ways for biodiesel produc-
tion are well developed but has the disadvantage of high-energy
requirements, difficulties in the recovery of catalyst and glycerol,
as well as environmental pollution. On the contract, enzymatic
biodiesel production has the merits of mild reaction conditions, low
raw material requirements and easy product recovery compared to
chemical ways [4,5]. For long-term development, edible oils such as
vegetable oils and animal fats cannot be used preferably as the feed-
stocks for biodiesel production. Enzymatic approaches will have
great prospect since they can be applied to biodiesel production
based on relatively poor quality oil feedstocks [3–5].
Some lipases and whole cell catalysts for biodiesel production
were reported to have 1,3-specificity towards substrate triglyc-
∗ Corresponding authors. Tel.: +86 10 62772130; fax: +86 10 62772130.
E-mail addresses: duwei@tsinghua.edu.cn (W. Du), dhliu@tsinghua.edu.cn
(
D. Liu).
1
381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.11.012

1
8
W. Li et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 17–22
Scheme 1. 1,3-Positional specificity lipase-catalyzed methanolysis with acyl migration.
MG and 1-MG as well as acyl migration between 1,2-DG and 1,3-DG
would occur with the absence of enzyme. And the acyl migration
was found to be first-order reversible reaction (Scheme 2).
Although acyl migration was always observed during the pro-
cess of 1,3-positional specificity enzyme-mediated process, few
reports were found on the study of mechanism and related kinetic
of acyl migration process alone. Murgia et al. [12] studied the
extent of acyl migration and hydrolysis phenomena in glycerol
monooleate/water-based systems and how they were influenced
by phase transition qualitatively. Laszlo et al. [13] have done
some research on the modeling of acyl migration kinetic of 1,2-
diacylglycerols. Fureby et al. [14], Vikbjerg et al. [15] and Oda
et al. [16] have done some research on the influence of reaction
parameters on acyl migration. However, their research was car-
ried out in the process of enzyme-catalyzed reaction. During these
processes, some reaction parameters might have effect on both
enzyme-catalyzed reaction and acyl migration. Therefore, it was
hard to decide how these factors would influence acyl migration
alone exactly.
migration. In this paper, the effect of temperature and water
activity on acyl migration process and kinetics alone was fur-
ther studied and the related mechanism was explored for the first
time.
2. Materials and methods
2.1. Materials
Triolein used as substrate was obtained locally. Palmitic acid,
stearic acid, oleic acid, linoleic acid, linolenic acid and heptade-
canoic acid as well as their methyl esters as GC standards and
1
-olein, 2-olein, 1,2-diolein, 1,3-diolein and triolein as HPLC stan-
dards were purchased from Sigma and were chromatographically
pure. Other chemicals were obtained commercially and were of
analytical grade. R. oryzae IFO4697 whole cell immobilized within
biomass support particles was cultivated and prepared in our lab-
oratory as described in our previous study [8].
Temperature and water activity were two crucial factors
that would influence enzyme-mediated process including acyl
2.2. Methanolysis of triolein
The methanolysis reactions were carried out in 50 ml screw-cap
◦
bottles at 35 C on a reciprocal shaker (130 oscillations/min). The
compositions of the reaction mixtures were as follows: 10 g triolein,
1
2 g tert-butanol, 1.46 g methanol, and 0.3 g 0.1 mmol/l phosphate
buffer (pH 6.8). 50 ␮l samples were taken from the reaction mixture
at specified time, centrifuged to obtain the upper layer and analyzed
by GC and HPLC. Each condition was done in duplicates.
The methyl esters contained in the reaction mixture were
analyzed using
a GC-14B gas chromatography (Shimadzu,
Kyoto, Japan) equipped with a CP-FFAP CB capillary column
(
25 m × 0.32 mm × 0.30 ␮m) supplied by Agilent. The aforemen-
tioned mixture (5 ␮l) and 300 ␮l of 1.4 mmol/l heptadecanoic acid
methyl ester (hexane as the solvent) which is served as the inter-
nal standard and ethanol (300 ␮l) were mixed thoroughly. The
column temperature was kept at 180 C for 0.5 min, heated to
2
◦
◦ ◦
50 C at 10 C/min, and then maintained for 6 min. The temper-
◦
◦
atures of the injector and detector were set at 245 C and 250 C,
respectively.
Scheme 2. Acyl migration between partial glycerides.

W. Li et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 17–22
19
Table 1
Gradient elution program.
Time (min)
Acetonitrile–acetic acid
99.85:0.15) (V/V, %)
Dichloromethane
(V/V, %)
(
0
4
100
100
90
90
70
70
20
20
100
100
0
0
1
2
3
3
4
5
6
6
2
5
0
5
5
5
0
5
10
10
30
30
80
80
0
0
Note: Analysis is performed as a constant flow rate of 1.5 ml min−1.
2.3. Acyl migration study
1
ml reaction mixture of the aforementioned methanolysis
reactions was taken at 6 h’ reaction and used for acyl migra-
tion study. The acyl migration reactions were carried out in 5 ml
sealed tubes at specified temperature in a thermostatic container.
Samples of 50 ␮l were taken from the reaction mixture at spec-
ified time and analyzed by HPLC. Each condition was done in
duplicates.
◦
Fig. 1. Acyl migration of 1,2-DG to 1,3-DG at 30 C.
and 1-MG as well as acyl migration between 1,2-DG and 1,3-DG
would take place with the absence of the enzyme [14–16]. Here
the effect of temperature on acyl migration process alone was first
investigated through kinetic study of acyl migration under various
temperatures.
2.4. Water activity control
The reaction of R. oryzae-mediated methanolysis of triolein was
stopped at 6 h to obtain abundant 2-MG and 1,2(2,3)-DG. Then the
resultant mixture was kept at different temperatures and the con-
tent of 1,2-DG, 1,3-DG, 2-MG and 1-MG was analyzed with time
proceeding. Firstly the acyl migration kinetics of 1,2-DG to 1,3-DG
and 2-MG to 1-MG were studied and modeled under a first-order
reaction reversible scheme as Scheme 2 showed. The differential
equations characterizing the acyl migration reactions were as fol-
lows:
The substrates were first dried using 3 Å molecular sieves, and
then pre-equilibrated for at least 3 d in separate sealed contain-
ers enclosed with saturated salt solutions or solid adsorbent to
establish fixed water activities. The corresponding water contents
of different water activities were measured by Karl Fischer titra-
tion. Before the start of the acyl migration reaction, the substrates
were adjusted to specified water activities by adding correspond-
ing water content into the substrates, and then put into the
pre-equilibrated sealed containers to maintain the activities [17].
d[1, 2 − DG]
◦
Pre-equilibration was done at 25 C. The solid adsorbent was 3 Å
= −k [1, 2 − DG] + k [1, 3 − DG]
1
2
dt
d[1, 3 − DG]
dt
molecular sieves (aw < 0.01). The saturated salt solutions used were
(1)
d[1, 2 − DG]
prepared with MgCl2 (aw: 0.328), NaNO3 (aw: 0.743), K SO4 (aw:
= −
2
dt
0
.973) [17].
d[2 − MG]
=
k [2 − MG] − k [1 − MG]
3
4
dt
d[1 − MG]
dt
2
.5. Analysis of the samples
(2)
d[2 − MG]
=
−
The glyceride and partial glycerides in the reaction mixture
dt
were analyzed by a Shimadzu 20A HPLC system (Shimadzu Corp.,
Kyoto, Japan) with an ELSD-LTII low temperature-evaporative
light scattering detector. 2 ␮l sample and 1 ml acetone were
precisely measured and mixed thoroughly. The aforementioned
mixture (20 ␮l) was injected. The stationary and mobile phases
were an C18 column (5 ␮m, 250 mm × 4.6 mm) (Dikma Technology,
PLATISIL ODS, China) and a gradient elution program by acetonitrile
where [1,2-DG], [1,3-DG], [2-MG] and [1-MG] were the molar con-
centrations of 1,2-DG, 1,3-DG, 2-MG, and 1-MG, respectively.
The concentration change of each component was obtained
experimentally and the non-linear curve fitting software Matlab
was used for fitting the system of differential Eqs. (1) and (2) into
the experimental data. Comparison between modeled and exper-
imental data under 30 C was presented in Figs. 1 and 2. It could
be seen that a good agreement was obtained. During the reaction
process, 1,2-DG and 2-MG decreased gradually, while 1,3-DG and
◦
−1
and dichloromethane at 1.5 ml min
(Table 1) respectively. The
◦
column temperature was controlled at 40 C. The drift pipe tem-
◦
perature was controlled at 70 C, and the nitrogen pressure was
1
-MG increased gradually. Equilibrium between 1,2-DG and 1,3-
controlled at 320 kPa.
DG, as well as between 2-MG and 1-MG was reached after certain
time.
3
. Results and discussion
Comparison between acyl migration of DG and MG indicated
that the rates of migration of the 2-position fatty acid moiety in
2-MG were much higher than those in 1,2-DG, which was under-
standable because 2-MG apparently had two acyl acceptor, while
1,2-DG had only one, as could be seen in Scheme 2. Therefore, the
attacking chances of acceptor were twofold in 2-MG than that in
1,2-DG. However, due to this mechanism, the difference of acyl
migration rates could only be twofold of 2-MG to 1,2-DG. While the
experimental result of acyl migration rate constant of 2-MG under
3.1. Effect of temperature on acyl migration
During the process of Rhizopus oryzae (R. oryzae)-mediated
methanolysis for methyl ester synthesis, 1,2-DG and 2-MG were
found to accumulate in the reaction mixture at the initial reac-
tion period, which was mainly due to the positional specificity of
the lipase [16]. It was found that acyl migration between 2-MG

2
0
W. Li et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 17–22
Fig. 3. Arrhenius plots of rate constants.
Table 3
Comparison of first-order reaction constants and half-life values for acyl migration
of DG and MG under various water activities.
Rate constant and
half-life value
Water activity
◦
Fig. 2. Acyl migration of 2-MG to 1-MG at 30 C.
<
0.01
0.328
0.743
0.0151
0.973
0.0146
◦
0 C was nearly 7.4-fold of that of 1,2-DG. Therefore, the activation
energies of the acyl migration reaction of 2-MG and 1,2-DG must
be quite different from each other.
Acyl migration process was further modeled under various
temperatures, and the rate constants and half-life values were cal-
culated and listed in Table 2. Similar results and good agreements
of modeled data with experimental data were also obtained (data
not shown).
k1 (h−1)
0.0226
1.66
0.0194
1.65
3
k1/k2
1.64
1.65
k3 (h−1)
0.112
20.7
78.2
0.0980
20.6
94.6
0.0820
0.0681
k3/k4
20.5
136
8.85
20.7
176
10.8
t1/2DG (h)a
a
t1/2MG (h)
6.43
7.37
a
−1
−1
t1/2DG = (k1 + k2) ln 2, t1/2MG = (k3 + k4) ln 2.
MG together contributed to the lower acyl migration rate of 1,2-DG
to 1,3-DG than that of 2-MG to 1-MG. Yang [18] also reported the
effect of temperature on acyl migration and through temperature
programming, the suppression of acyl migration could be achieved
without loss of reaction yield.
From Table 2, it could be seen clearly that the acyl migration con-
stants of both 1,2-DG and 2-MG increased greatly along with the
increasing of temperature. The half-life values of 1,2-DG and 2-MG
◦
under 30 C were 132.53 h and 7.20 h, while they were only 13.80 h
◦
and 1.20 h under 55 C. The half-life values of 2-MG were always
much shorter than that of 1,2-DG under a certain temperature.
The equilibrium state of both DG and MG seemed to be constant
under different temperatures. The mole ratios of 1,2-DG and 2-MG
at equilibrium were found to be 0.38 and 0.05, respectively.
Based on data in Table 2, the Arrhenius plots of acyl migration
3.2. Effect of water activity on acyl migration
Besides temperature, water might be another important
parameter influencing acyl migration. Some researchers have
investigated the influence of water on acyl migration [14–16].
reaction were showed in Fig. 3. From the Arrhenius plots of k and
1
k3 values, the apparent activation energies were evaluated to be
7
3.8 and 59.4 kJ/mol, respectively.
Difference in activation energy between acyl migration of 1,2-
DG and that of 2-MG also illustrated their difference in acyl
migration rate constants. Therefore, higher apparent activation
energy of 1,2-DG and fewer acyl acceptors compared to that of 2-
Table 2
Comparison of first-order reaction constants and half-life values for acyl migration
of DG and MG under various temperatures.
Rate constants and
half-life value
Temperature
◦
◦
◦
◦
30 C
55
C
45
C
37
C
k1 (h−1)
k1/k2
0.128
1.65
0.581
20.8
13.8
1.20
0.0451
1.66
0.274
20.6
36.0
2.58
0.0252
1.64
0.152
20.6
76.3
4.70
0.0132
1.65
0.0975
20.7
132
7.20
k3 (h−1)
k3/k4
a
t1/2DG (h)
t1/2MG (h)a
a
−1
−1
t1/2DG = (k1 + k2) ln 2, t1/2MG = (k3 + k4) ln 2.
Fig. 4. Effect of water activity on acyl migration of 1,2-DG.

W. Li et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 17–22
21
Fig. 7. Activation energy of acyl migration.
0
.038 and 0.024 mol/l, respectively. The change of 1,2-DG and 2-
MG concentrations at different water activities were presented in
Figs. 4 and 5 (Fig. 5b was the initial part of Fig. 5a). It was indicated
by these figures that the acyl migration rates decreased with the
increase of water activity.
Further calculation of acyl migration rate constants and half-life
values presented in Table 3 confirmed the negative effect of water
on acyl migration. The half-life values of 1,2-DG and 2-MG were
1
75.93 h and 10.75 h at water activity of 0.973, and only 78.23 h
and 6.43 h under dry condition. These results also indicated that
the equilibrium states of both DG and MG were insensitive to water
activity of the medium.
3.3. Effect of water content on acyl migration
In terms of water content, the above studied water activities
were 0.22‰, 0.94‰, 1.84‰, 2.94‰, respectively, all of which in
trace amount. In order to disclose how water influences the acyl
migration reaction, water contents in larger amount were investi-
gated further (from 1% to 30% of the initial triolein weight).
It was found that the acyl migration of both 1,2-DG and 2-MG
did not vary much under such a wide range of water contents. Fur-
ther calculation of acyl migration reaction constants and half-life
values through kinetic study and modeling of first-order reversible
reaction by Matlab confirmed this conclusion (data not shown).
The results indicated that it was water activity rather than water
content that influenced the acyl migration process.
Water activity (aw) is a measurement of the energy status of
the water in a system. It is defined as the vapor pressure of water
divided by that of pure water at the same temperature. The highest
water activity of the reaction system was 1, so further increasing
the water content of the reaction system would not affect water
activity of the system after it had reached 1.
The mechanism of water activity influencing acyl migration was
further explored as follows. As presented in Fig. 6, acyl migration
belonged to SN2 nucleophilic substitution. For this kind of reaction,
charge would disperse at transition state, as presented in Fig. 6,
transition state 2. Fig. 7 explained the change of energy state during
the reaction process. Along with the increasing of water activity in
the reaction system, the polarity of the reaction medium increased,
which went against the dispersion of charge in the transition
Fig. 5. Effect of water activity on acyl migration of 2-MG (graph b is a “focus” of the
first 25 h of graph a).
However, their researches were carried out during the process of
enzyme-catalyzed reaction. Just as the aforementioned effect of
temperature, it was hard to tell the true effect of water on acyl
migration alone. Results reported by Fureby et al. [14] showed
that acyl migration favored a low water activity; while the result
reported by Vikbjerg et al. [15] and Oda et al. [16] claimed a reverse
conclusion. In this paper, the effect of water activity on acyl migra-
tion alone was first studied through modeling the acyl migration
process and calculating the kinetic parameters of acyl migration
under different water activities.
The reaction of R. oryzae-mediated methanolysis of triolein was
stopped at 6 h to obtain abundant 2-MGs and 1,2(2,3)-DGs. Then
the resultant mixture was kept at different water activities and the
content of 1,2-DG, 1,3-DG, 2-MG as well as 1-MG was analyzed
with time proceeding. The initial concentrations of 1,2-DG, 1,3-
DG, 2-MG, and 1-MG in the reaction mixture were 0.105, 0.029,
Fig. 6. Mechanism of acyl migration.

2
2
W. Li et al. / Journal of Molecular Catalysis B: Enzymatic 63 (2010) 17–22
state. Accordingly, the energy state of the transition state increased
Acknowledgments
ꢀ
(
(
from 2 to 2 in Fig. 7), which resulted in larger activation energy
Ea > Ea).
ꢀ
The authors express their thanks for the support from National
Natural Science Foundation of China (20706034), National 863 High
Tech Project (2006AA020203), 973 project (2007CB714302) as well
as Tsinghua Fundamental Research Funding.
The acyl migration rate at certain temperature was thus
decreased. While the energy state of reactant and product remain
unchanged, ꢀG of acyl migration reaction did not change, so the
equilibrium of acyl migration was not affected by water activity,
which was consistent with the result in Table 2.
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[
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