An insight into the solvent effect on the positional selectivity of the immobilized lipase from Burkholderia cepacia in 1,3-diolein synthesis

Article abstract of DOI:10.1039/c5ra01218j

The solvent effect on the positional selectivity of the immobilized lipase from Burkholderia cepacia in 1,3-diolein synthesis was investigated for the first time. The results indicated that the preferential selectivity to sn-1 hydroxyl of the glycerol molecule over sn-2 hydroxyl was weaker in solvents with higher logP values.

Full text of DOI:10.1039/c5ra01218j

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An insight into the solvent effect on the positional
selectivity of the immobilized lipase from
Cite this: RSC Adv., 2015, 5, 23122
Burkholderia cepacia in 1,3-diolein synthesis†
Received 21st January 2015
Accepted 23rd February 2015
a
ac
b
a
Yan-Hong Bi, Zhao-Yu Wang,* Zhang-Qun Duan, Xiang-Jie Zhao,
a
a
Xiao-Ming Chen and Ling-Hong Nie
DOI: 10.1039/c5ra01218j
www.rsc.org/advances
The solvent effect on the positional selectivity of the immobilized
Different solvents with log P ranging from À0.23 to 4.5 were
lipase from Burkholderia cepacia in 1,3-diolein synthesis was investi- screened as the reaction media for PSL-C-catalyzed esterica-
gated for the first time. The results indicated that the preferential tion of oleic acid with glycerol with the aim of preparing
selectivity to sn-1 hydroxyl of the glycerol molecule over sn-2 hydroxyl 1,3-diolein. Diolein yield, including 1,3-diolein and 1,2-diolein,
was weaker in solvents with higher log P values.
was presented in Table 1 column 3. As can be seen, although the
reaction can proceed in all the solvents employed, the diolein
yield markedly depended on the reaction medium. The lowest
diolein yield (58.8%) was obtained in acetone the log P value of
which was the minimum (À0.23). The moderate diolein yields
were observed in the relatively hydrophilic solvents such as
tetrahydrofuran, t-butanol, 4-methyl-2-pentanone and chloro-
form. Six tested relatively hydrophobic solvents (toluene, tetra-
chloromethane, cyclohexane, n-hexane, n-heptane and
n-octane) showed higher diolein yields. The best results with
The consumption of 1,3-diacylglycerol oil has benecial effects
on suppressing the accumulation of body fat and preventing the
1–4
increase of body weight.
The enzyme-mediated 1,3-diac-
ylglycerol preparation has attracted more and more attention
because of its mild reaction conditions and safe products.
However, the positional isomer 1,2-diacylglycerol may be
generated simultaneously. Consequently, the positional selec-
tivity of the enzyme employed is important for high yield of
5–9
88.5% yield of diolein were achieved when n-heptane and
1,3-diacylglycerol.
n-octane were used as the reaction media. The higher enzyme
activity involved in this case might be attributable to the fact
that the solvents with higher log P value could retain the
microenvironment moisture around the catalytic active site of
PSL-C more, which can maintain the original conformation
of the enzyme, thus preventing its deactivation due to the loss of
Since the reaction medium could exert a profound inuence
on the enzyme selectivity, a great many research articles have
focused on the effect of the solvent physico-chemical properties
on the enzyme selectivity. It has been demonstrated that,
among all the reported physico-chemical properties, log P (the
logarithm of the partition coefficient of a solvent in the stan-
dard octanol–water two-phase system) was found to be more
16–20
essential water.
However, it was worth noting that, although the ratio values
of 1,3-diolein to 1,2-diolein which were listed in Table 1 column
10–15
inuential.
As far as we know, an insight into the inuence of log P of the
solvent on the positional selectivity of the immobilized lipase
from Burkholderia cepacia (PSL-C) during 1,3-diolein synthesis
by the esterication of oleic acid with glycerol has not been
reported. In light of this, we innovatively present here the study
of the effect of the reaction media on the positional selectivity of
PSL-C.
4
were also affected by the reaction media remarkably, the trend
was reversed compared with the one of the diolein yields with
the increasing log P of the solvent. The highest ratio values of
1,3-diolein to 1,2-diolein (31.1) was achieved in acetone which
was the most hydrophilic one among all the solvents used. And
then the ratio of 1,3-diolein to 1,2-diolein descended dramati-
cally along with the increase of the log P. The minimal ratio of
1
,3-diolein to 1,2-diolein was 7.1 which happened in the most
hydrophobic solvent n-octane. In particular, the ratio value of
,3-diolein to 1,2-diolein was still affected by the solvent
noticeably in six relatively hydrophobic solvents (toluene, tet-
a
School of Life Science and Chemical Engineering, Huaiyin Institute of Technology,
1
Huai'an 223003, P. R. China. E-mail: zhaoyu_wang@126.com
b
Academy of State Administration of Grain, Beijing 100037, P. R. China
c
Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process rachloromethane, cyclohexane, n-hexane, n-heptane and
Integration, Huai'an 223005, P. R. China
n-octane), in which the diolein yield was inuenced slightly. It is
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c5ra01218j
universally acknowledged that the ratio of 1,3-diolein to
1
23122 | RSC Adv., 2015, 5, 23122–23124
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Table 1 Solvent effect on the enzymatic 1,3-diolein synthesis
Solvent
log P
Diolein yield (%)
1,3-Diolein /1,2-diolein
k
1
k
3
k
9
k
11
k
1
/k
9
3
k /k11
À5
À4
Acetone
Tetrahydrofuran
t-Butanol
À0.23
0.49
0.8
1.31
2.0
2.5
3.0
3.2
3.5
58.8
75.3
81.1
82.5
85.0
87.3
87.9
88.2
88.3
88.5
88.5
31.1
25.8
22.6
17.8
12.1
10.2
8.9
8.7
8.1
7.6
7.1
0.691
1.004
1.142
1.156
1.212
1.307
1.395
1.485
1.553
1.571
1.692
0.754
1.036
1.251
1.384
1.423
1.466
1.701
1.932
1.994
2.016
2.268
0.048
0.097
0.121
0.145
0.161
0.229
0.311
0.476
0.632
0.846
0.918
4 Â 10
8 Â 10
0.003
0.008
0.016
0.024
0.034
0.041
0.046
0.049
0.059
14.40
10.35
9.44
7.97
7.53
5.71
4.49
3.12
2.46
1.86
1.84
18 850
1295
417.0
173.0
88.9
61.1
50.0
47.1
43.3
4
-Methyl-2-pentanone
Chloroform
Toluene
Tetrachloromethane
Cyclohexane
n-Hexane
n-Heptane
n-Octane
4.0
4.5
41.1
38.4
1
,2-diolein was subject to the preferential selectivity of PSL-C of k
1
9
were much smaller than the ones of k with the increasing
towards sn-1 hydroxyl over sn-2 hydroxyl of the glycerol molec- log P of the solvent. As a result, k
1
9
/k was decreased from 14.40
ular and the acyl-migration between sn-1 hydroxyl and sn-2 to 1.84. It was demonstrated that the preferential selectivity of
hydroxyl (which was neglected because of the little contribu- PSL-C to sn-1 hydroxyl over sn-2 hydroxyl decreased with the
tion, data not shown). As reported, the secondary structure of increase of the log P. While the solvent were n-heptane and
PSL-C varied with the organic solvents used, which could affect n-octane, k /k values were only 1.86 and 1.84, respectively.
1
9
19,20
the access of the substrates to the active site of PSL-C.
Since sn-1 hydroxyl amount was twice sn-2 hydroxyl in the
Accordingly, the secondary structure changes of PSL-C may be glycerol molecular, when k /k value was close to 2.00, it can be
1
9
contributed to the positional selectivity variations based on considered that the selectivity of PSL-C to sn-1 hydroxyl and sn-2
medium engineering. Additionally, when six relatively hydro- hydroxyl was almost equivalent. It is of great benet to
phobic solvents served as the reaction media, diolein yields preparing 2-monoolein and 1,2-diolein which are usually used
were about the same, while the ratios of 1,3-diolein to 1,2-dio- as the emulsiers and surfactants in the food industry.
lein were changed signicantly. Especially, the ratios of
3
Moreover, although the increasing times of k were much
1,3-diolein to 1,2-diolein were higher in the solvents with ring smaller than the ones of k11 with the increasing log P of the
structure and lower in the hydrocarbon solvents with longer solvent, the values of k were still much bigger than the corre-
3
carbon chain. This observation suggested that the molecular sponding ones of k , which resulted in that k can be
1
1
11
constitution of the organic solvent may also play an important approximately ignored. It was indicated that the selectivity of
role in affecting the positional selectivity of PSL-C, which agreed PSL-C to sn-1 hydroxyl of the 1-monoolein molecular (the
with that reported by Liu et al., who demonstrated that the intermediate for 1,3-diolein synthesis) was much higher than
structures and functional groups of the employed solvents the one to sn-2 hydroxyl for each solvent. Additionally, an
19,20
exerted an inuence on the behavior of PSL-C as well.
3
interesting observation was that the values of k /k11 were much
The reaction kinetics was an efficient tool for understanding bigger than the ones of k /k in all the tested solvent. It could be
1
9
the inuence of solvent on the positional selectivity of lipase suggested that when one sn-1 hydroxyl of the glycerol molecular
14
which has been proved by our previous work. Hence, an was acylated forming 1-monoolein, the preferential selectivity of
interesting investigation on the solvent effect on the positional PSL-C to the other sn-1 hydroxyl over sn-2 hydroxyl would be
selectivity of PSL-C from the points of view of kinetics was increased markedly.
performed subsequently.
In view of all the results acquired above, it can be concluded
The enzymatic reaction rate constants were identied by that when the solvent with higher log P value served as the
solving the rate equations described in ESI with an adaptive reaction medium, the achieved diolein yield was higher, while
step-size Runge–Kutta method within a nonlinear regression the preferential selectivity of PSL-C to sn-1 hydroxyl of the
procedure using the Levenberg–Marquardt algorithm, with the glycerol molecular over sn-2 hydroxyl was weaker which led to
aim of obtaining the best t between the experimental data and the lower ratio of 1,3-diolein to 1,2-diolein. Therefore, the
14
the results calculated. The rate constants which were relevant relatively hydrophilic solvent was considered as the most suit-
to the positional selectivity of PSL-C were presented in Table 1. able reaction medium for PSL-C-catalyzed synthesis of 1,3-dio-
From Table 1 columns 5–8, it can be clearly seen that k
1
, k
3
,
lein aer scrutinizing the data tabulated in Table 1.
k
9
and k11 were increased with the increasing log P of the
Compared with the results obtained by Novozym 435 in our
14
solvent. It was account for that the diolein yield was enhanced previous work, the maximal diolein yield reduced by 2.7%
obviously along with the increase of the log P. k1 was much (88.5% vs. 91%) and the highest ratio value of 1,3-diolein to 1,2-
bigger than k for all the solvents used. It was indicated that the diolein was decreased by 3.7% (31.1 vs. 32.3). Although both
9
selectivity of PSL-C to sn-1 hydroxyl of the glycerol molecular values were a little lower, PSL-C was still considered as an
was much higher than the one to sn-2 hydroxyl in each solvent. excellent and promising biocatalyst which could be benecial
But we must pay attention to the fact that the increasing times for the rational production of 1,3-diolein.
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6
J. B. Kristensen, X. B. Xu and H. L. Mu, J. Agric. Food Chem.,
Conclusions
2005, 53, 7059.
In summary, this work presented an insight into the solvent
effect on the positional selectivity of PSL-C during 1,3-diolein
synthesis by esterication of oleic acid with glycerol. It may be
very helpful for investigating the inuence of the environment
on the enzyme selectivity in biocatalysis eld.
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