The pronounced effect of water activity on the positional selectivity of Novozym 435 during 1,3-diolein synthesis by esterification

Article abstract of DOI:10.1016/j.catcom.2009.10.030

We have investigated the effect of water activity on the positional selectivity of the immobilized lipase Novozym 435 during the esterification of oleic acid with glycerol for 1,3-diolein preparation. The highest preferential selectivity of Novozym 435 to

Full text of DOI:10.1016/j.catcom.2009.10.030

Catalysis Communications 11 (2010) 356–358
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Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
The pronounced effect of water activity on the positional selectivity of Novozym
435 during 1,3-diolein synthesis by esterification
*
*
Zhang-Qun Duan, Wei Du , De-Hua 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:
We have investigated the effect of water activity on the positional selectivity of the immobilized lipase
Novozym 435 during the esterification of oleic acid with glycerol for 1,3-diolein preparation. The highest
preferential selectivity of Novozym 435 to 1-position over 2-position of the glycerol molecular was
achieved when the water activity was 0.53. The mechanism by which the enzyme favors or disfavors
the selectivity at various water activities may be related to the changes in the internal flexibility of the
enzyme which depends on the effective hydration.
Received 23 September 2009
Received in revised form 22 October 2009
Accepted 28 October 2009
Available online 31 October 2009
Keywords:
Ó 2009 Elsevier B.V. All rights reserved.
Water activity
Positional selectivity
Novozym 435
1,3-Diolein
1. Introduction
sen for this study because it was an excellent solvent for this
reaction proved by our preliminary experiments.
The activity and selectivity of enzymes in organic media are
usually greatly affected by the level of water present in the med-
ium. As reported by many authors, water availability in organic
media cannot be quantified using its concentration because the
capacity of organic phase to dissolve water varies considerably
with the polarity of solvent. It is best analyzed in terms of water
activity since the hydration of the enzyme in different reaction
media is the same when the same water activity were controlled
[1]. And it has been widely accepted that each enzyme has the spe-
cific water activity requirements [2].
There are several reports about the effect of water activity on
the selectivity of enzymes [3–8], but some groups find that water
activity does not influence the selectivity of an enzyme [9,10]. So
it will be interesting to study the effect of water activity on the
positional selectivity of the immobilized lipase Novozym 435 dur-
ing 1,3-diolein synthesis by esterification of oleic acid with glyc-
erol. In view of this, we present here a systematic investigation
into the water activity effect on the positional selectivity of the en-
zyme from the points of view of kinetics, based on an earlier liter-
ature which described the effect of water activity on the
equilibrium and rate constants during triolein preparation cata-
lyzed by immobilized Mucor miehei lipase [11]. t-Butanol was cho-
2. Experimental
2.1. Biological and chemical materials
Novozym 435 (immobilized lipase from Candida antarctica, type
B) was a generous gift from Novozymes (Denmark). 1-Monoolein,
2-monoolein, 1,2-diolein, 1,3-diolein and triolein were purchased
from Sigma and were chromatographically pure. All other chemi-
cals and reagents were obtained commercially and were of analyt-
ical grade.
2.2. General procedure for 1,3-diolein synthesis at constant water
activity (aw)
The method of maintaining a constant water activity through-
out the lipase-catalyzed esterification proposed recently [8] was
adopted here. The reaction solution of oleic acid (0.15 M) and glyc-
erol (0.06 M) in t-butanol was prepared and used in all reactions.
The reaction solution and Novozym 435 were separately equili-
brated with aqueous saturated salt solution of known aw for 16 h
in two air tight desiccators (fitted with a rubber septum on the
lid). The following salts were used: LiCl (aw = 0.11), MgCl2 (aw
0.33), Mg(NO3)2 (aw = 0.53), NaCl (aw = 0.75) and K2SO4 (aw
0.97). The reaction was started by transferring 10 mL of the
equilibrated reaction solution with a syringe into the beaker
=
=
* Corresponding authors. Tel.: +86 10 62782654; fax: +86 10 62785475.
E-mail addresses: duwei@mail.tsinghua.edu.cn (W. Du), dhliu@mail.tsinghua.
edu.cn (D.-H. Liu).
1566-7367/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.catcom.2009.10.030

Z.-Q. Duan et al. / Catalysis Communications 11 (2010) 356–358
357
containing Novozym 435 via the rubber septum without opening
the lid. The reaction mixture was magnetically stirred.
1,3-DO, 1,2-DO, TO and Gly referred to oleic acid, l-monoolein, 2-
monoolein, 1,3-diolein, 1,2-diolein, triolein and glycerol,
respectively):
2.3. Analysis of the samples
d½OAꢂ
¼ k2½1-MOꢂ þ k10½2-MOꢂ þ k4½1; 3-DOꢂ þ ðk6 þ k12Þ½1; 2-DOꢂ
dt
The acylglycerol in the reaction mixture was analyzed by a Shi-
madzu 20A HPLC system (Shimadzu, Kyoto, Japan) with an ELSD-
LT II low temperature-evaporative light scattering detector. Two
microliters sample and 1 mL acetone were mixed thoroughly.
Twenty microliters of the aforementioned mixture was injected.
þ ðk8 þ k14Þ½TOꢂ ꢁ ððk1 þ k9Þ½Glyꢂ þ ðk3 þ k11Þ½1-MOꢂ
þ k5½2-MOꢂ þ k7½1; 2-DOꢂ þ k13½1; 3-DOꢂÞ½OAꢂ
d½1-MOꢂ
¼ k1½OAꢂ½Glyꢂ þ k4½1; 3-DOꢂ þ k12½1; 2-DOꢂ þ k16½2-MOꢂ
dt
The stationary and mobile phases were an C18 column (5 lm,
ꢁ ðk2 þ k5 þ ðk3 þ k11Þ½OAꢂÞ½1-MOꢂ
¼ k6½1; 2-DOꢂ þ k9½OAꢂ½Glyꢂ þ k15½1-MOꢂ
ꢁ ðk5½OAꢂ þ k10 þ k16Þ½2-MOꢂ
¼ k3½1-MOꢂ½OAꢂ þ k14½TOꢂ þ k18½1; 2-DOꢂ
ꢁ ðk4 þ k13½OAꢂ þ k17Þ½1; 3-DOꢂ
¼ ðk5½2-MOꢂ þ k11½1-MOꢂÞ½OAꢂ þ k8½TOꢂ þ k17½1; 3-DOꢂ
250 mm ꢀ 4.6 mm) (Dikma Technology, PLATISIL ODS, China) and
a gradient elution program (Table 1) by acetonitrile and dichloro-
methane at 1.5 mL minꢁ1, respectively. The column temperature
was 40 °C. The drift pipe temperature was 70 °C, and the nitrogen
pressure was 320 kPa. In addition, the oleic acid and glycerol con-
centration were detected using the methods described by Du [12].
d½2-MOꢂ
dt
d½1; 3-DOꢂ
dt
d½1; 2-DOꢂ
2.4. Rate constants identified
dt
For modeling the kinetic behaviors of 1,3-diolein preparation
catalyzed by Novozym 435 in t-butanol systems, we adopted the
synthetic scheme shown in Scheme 1 on the basis of the model
proposed previously [13,14]. The esterification reaction followed
second-order reversible reactions, while the acyl migration reac-
tions between 1-MO and 2-MO, 1,2-DO and 1,3-DO, respectively,
belonged to one-order reversible reactions; the mass transfer lim-
itation in the reaction system could be neglected. Based on the
above assumptions, the differential equations characterizing the
whole reaction process were listed as follows (OA, 1-MO, 2-MO,
ꢁ ðk6 þ k7½OAꢂ þ k12 þ k18Þ½1; 2-DOꢂ
¼ ðk7½1; 2-DOꢂ þ k13½1; 3-DOꢂÞ½OAꢂ ꢁ ðk8 þ k14Þ½TOꢂ
d½TOꢂ
dt
d½Glyꢂ
¼ k2½1-MOꢂ þ k10½2 ꢁ MOꢂ ꢁ ðk1 þ k9Þ½OAꢂ½Glyꢂ
dt
3. Results and discussion
A range of water activities (from 0.11 to 0.97) were selected to
investigate their effect on the positional selectivity of the immobi-
lized lipase Novozym 435 during the esterification of oleic acid
with glycerol carried out in t-butanol system. A detailed compari-
son on enzymatic 1,3-diolein synthesis with different water activ-
ity has been made in Table 2. It can be clearly seen that, the diolein
yield (entry 2) and the ratio of 1,3-diolein to 1,2-diolein (entry 3)
were distinctly dependent on the water activity. The diolein yield
was increased with the increasing water activity. This corre-
sponded to the activation of enzyme by hydration, involving a gen-
eral role of water in making the enzyme structure more flexible,
increasing the enzymatic rate [15,16]. But high water levels have
also been shown to reduce the rate of the lipase-catalyzed esterifi-
cation owing to increasing the hydrolysis rate. So, the trend was re-
versed when water activity reached a particular value (0.53),
further rise led to a drop in diolein yield. It was also worth noting
Table 1
Gradient elution program.
Time
(min)
Flow rate
Acetonitrile–acetic acid
(99.85:0.15) (V/V, %)
Dichloromethane
(V/V, %)
(mL minꢁ1
)
0
4
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.50
100
100
90
90
70
70
20
20
100
100
0
0
12
25
30
35
45
55
60
65
10
10
30
30
80
80
0
0
Scheme 1.

358
Z.-Q. Duan et al. / Catalysis Communications 11 (2010) 356–358
Table 2
the preferential selectivity to the other 1-position over 2-position
would be enhanced strongly.
Influence of water activity on the enzymatic 1,3-diolein synthesis.
Based on the above results, it was concluded that the water
activity has a pronounced influence on the preferential selectivity
of Novozym 435 to 1-position over 2-position of the glycerol
molecular during the 1,3-diolein synthesis by esterification. The
mechanism by which the enzyme favors or disfavors the selectivity
at various water activities may be related to the changes in the
internal flexibility of the enzyme which depends on the effective
hydration. Recently, the molecular modeling revealed that there
was a possibility for the water molecular to bind in the stereo-
specificity pocket of C. antarctica lipase B specifically and reduced
its size, enhancing the difference in binding of the substituents
on the secondary alcohols molecular, and then increased the
enantioselectivity [4,5]. So, the molecular modeling may be a help-
ful tool for exploring the molecular mechanism of the positional
selectivity of Novozym 435 during 1,3-diolein synthesis by
esterification of oleic acid with glycerol.
Water activity (aw
Diolein yield (%)
1,3-Diolein/1,2-diolein
k1
k3
k9
k11
k1/k9
k3/k11
)
0.11
66
0.33
78
20.5
0.901
1.148
0.097
0.0027
9.30
0.53
84
0.75
80
0.97
75
13.2
0.535
0.846
0.083
0.0025
6.42
344
24.1
1.231
1.347
0.126
0.0030
9.73
480
22.6
1.112
1.267
0.115
0.0028
9.54
450
18.7
0.082
1.056
0.091
0.0025
8.03
426
380
that, the ratio of 1,3-diolein to 1,2-diolein increased to 24.1 and
then decreased along with the increase of the water activity. As
we all known, a bigger ratio of 1,3-diolein to 1,2-diolein could be
attributed to a higher preferential selectivity of Novozym 435 to-
wards 1-position over 2-position of the glycerol molecular. So, to
get a better understanding of the phenomena involved in the
changes in the ratio of 1,3-diolein to 1,2-diolein, an insight into
the influence of water activity on the positional selectivity of Nov-
ozym 435 from the points of view of kinetics could be of great
interest.
4. Conclusion
The rate constants were identified by solving the differential
equations described previously with an adaptive step-size Run-
ge–Kutta method within a nonlinear regression procedure, using
the Levenberg–Marquardt algorithm, so as to obtain the best fit be-
tween the experimental data and the results calculated. And some
rate constants which were relevant to the positional selectivity of
Novozym 435 were listed in Table 2.
As shown from Table 2 (entries 4–7), when the water activity
was increased from 0.11 to 0.53, k1, k3, k9 and k11 increased. It
could be interpreted that an increase in the internal flexibility of
the enzyme caused by increasing water activity in general, resulted
in the increase of enzymatic activity [15,16]. Among the increase of
these four rate constants, k1 and k3 increased noticeably; k9, and
k11 increased mildly. As a result, k1/k9 and k3/k11 increased with
the increasing water activity. It was demonstrated that the prefer-
ential selectivity of Novozym 435 to 1-position over 2-position of
the glycerol molecular was raised with the increase of water activ-
ity. The reason may be that the increase of the water activity re-
sulted in more water molecular binding near the active site of
the enzyme and made it more flexible. Accordingly, the selectivity
of the enzyme increased with the increasing water activity [9,17–
19].
In summary, the influence of water activity on enzymatic selec-
tivity is complex, but essential for the biocatalysis. The results of
this work allowed a better understanding of the pronounced effect
of water activity on the positional selectivity of the immobilized li-
pase Novozym 435 during 1,3-diolein synthesis by esterification of
oleic acid with glycerol. The molecular modeling may be a promis-
ing tool for elucidating how the enzyme serves the task of distin-
guishing between the isomers behind water activity effect.
Acknowledgments
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.
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Interestingly, it was also observed that k3/k11 was much bigger
than k1/k9 for each water activity. A speculative explanation could
be that after one 1-position of the glycerol molecular was acylated,