Introducing biobased ionic liquids as the nonaqueous media for enzymatic synthesis of phosphatidylserine

Article abstract of DOI:10.1021/jf505296k

Biobased ionic liquids with cholinium as the cation and amino acids as the anions, which could be prepared from renewable biomaterials by simple neutralization reactions, have recently been described as promising and green solvents. Herein, they were successfully used as the reaction media for enzyme-mediated transphosphatidylation of phosphatidylcholine with l-serine for phosphatidylserine synthesis for the first time. Our results indicated that the highest phosphatidylserine yield of 86.5% was achieved. Moreover, 75% original activity of the enzyme was maintained after being used for 10 batches. The present work could be considered an alternative enzymatic strategy for preparing phosphatidylserine. Additionally, the excellent results make the biobased ionic liquids more promising candidates for use as environmentally friendly solvents in biocatalysis fields.

Full text of DOI:10.1021/jf505296k

Article
pubs.acs.org/JAFC
Introducing Biobased Ionic Liquids as the Nonaqueous Media for
Enzymatic Synthesis of Phosphatidylserine
Yan-Hong Bi, Zhang-Qun Duan, Xiang-Qian Li, Zhao-Yu Wang,* and Xi-Rong Zhao†
†
‡
†,§
,†,§
†
School of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huai’an 223003, P. R. China
‡
Academy of State Administration of Grain, 11 Baiwanzhuang Street, Xicheng, Beijing 100037, P. R. China
§
Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huai’an 223005, P. R. China
ABSTRACT: Biobased ionic liquids with cholinium as the cation and amino acids as the anions, which could be prepared from
renewable biomaterials by simple neutralization reactions, have recently been described as promising and green solvents. Herein,
they were successfully used as the reaction media for enzyme-mediated transphosphatidylation of phosphatidylcholine with L-
serine for phosphatidylserine synthesis for the first time. Our results indicated that the highest phosphatidylserine yield of 86.5%
was achieved. Moreover, 75% original activity of the enzyme was maintained after being used for 10 batches. The present work
could be considered an alternative enzymatic strategy for preparing phosphatidylserine. Additionally, the excellent results make
the biobased ionic liquids more promising candidates for use as environmentally friendly solvents in biocatalysis fields.
KEYWORDS: biobased ionic liquids, phosphatidylserine, phospholipase D, transphosphatidylation
INTRODUCTION
preparation. As far as we know, this is the first description of
Ch][AA] ILs as the reaction media for enzymatic synthesis of
PS as well.
■
[
Phosphatidylserine (PS), which is a phospholipids component,
has many applications in functional food and pharmaceutical
1
,2
industries. Recent reports have shown that PS supplemented
in the diet plays an important role in preventing Alzheimer’s
dementia, improving memory, increasing vigilance and
EXPERIMENTAL PROCEDURES
■
Chemical and Biological Materials. PC (≥99%, from Soybean),
PS (≥97%, from Soybean), PA (98%, from Soybean), and choline
hydroxide ([Ch][OH], 46 wt % in H O) were purchased from Sigma-
3
−6
attention, relieving depression, and decreasing stress.
However, because of the minor content of PS in natural
form, the synthesis becomes more significant. The usual
approach for preparing PS is phospholipase D (PLD)-mediated
transphosphatidylation of phosphatidylcholine (PC) with L-
serine. Generally, the reaction is performed in the biphasic
system (i.e., water-immiscible organic solvent phase and
2
Aldrich (USA). L-Amino acids were obtained from Juyuan
Biotechnological Co. (Shanghai, China). PLD (from Streptomyces
−1
chromofuscus, 60 U mg , lyophilized powder. One unit was defined as
the amount of enzyme which catalyzed 1 μmol PC to form PS per
hour at 40 °C) was purchased from Asahi-kasei pharma corporation
(Japan). Triple-distilled water was used for the preparation of the
7
,8
9,10
aqueous phase)
or the purely aqueous system.
The
aqueous solutions in the synthesis of ILs. All other chemicals and
reagents were of the highest purity commercially available. All the
reagents were anhydrous, and all the organic solvents were dehydrated
by 4 Å molecular sieves.
serious drawback of these systems is that they contain a large
quantity of water, which results in the undesirable hydrolysis of
PC and PS. Not only does the hydrolysis consume the substrate
and product, but also it gives rise to the accumulation of
considerable amounts of the undesirable byproduct, phospha-
tidic acid (PA). Biosynthesis can be further improved with a
rational selection of the reaction medium. In this sense, an
ideal choice would be to perform the enzymatic synthesis of PS
in a green, nontoxic, and nonaqueous system.
1
7,19
[Ch][AA] ILs Synthesis Procedure..
[Ch][OH] aqueous
solution was added dropwise to amino acid aqueous solution of slight
excess, with cooling. The mixture was stirred at about 3 °C for 48 h in
the dark. Water was then removed under vacuum at 55 °C. After this
step, acetonitrile/methanol (9:1, v/v) was added to precipitate the
unreacted amino acid. The mixture was stirred vigorously overnight
and then filtered through Celite. The solvents were evaporated under
reduced pressure. Finally, the purified ionic liquid was dried in vacuum
for 48 h at 70 °C and stored under moisture-free conditions until
utilization. The yields of all the desired products were more than 95%.
General Procedure for Enzymatic Synthesis of PS. In a typical
experiment, the enzyme-mediated reaction was performed in a batch
reactor in a water bath under magnetic stirring at 40 °C. The
compositions of the reaction mixtures were as follows: 3.0 mL of
11
Recent advances in ionic liquids (ILs) fields have inspired
numerous studies in exploiting ILs as reaction media for
1
2,13
biocatalysis.
Even so, the concerns have risen over the
potential toxicity as well as the low biodegradability of most of
the currently employed ILs. To overcome these drawbacks,
some ILs have already been synthesized from renewable,
14
1
5−19
nontoxic biosources (choline, amino acids, etc.).
[
Ch][AA] ILs, 0.05 mmol PC, 0.15 mmol L-serine, 60 U PLD, and
In the present work, biobased ILs with cholinium as the
cation and amino acids as the anions ([Ch][AA]) were
employed as the reaction media for PS synthesis via enzymatic
transphosphatidylation of PC with L-serine. The aim of this
work is to show that the introduction of biobased ILs may
develop an alternatively green and efficient scenario for PS
Received: November 3, 2014
Revised: January 15, 2015
Accepted: January 23, 2015
Published: January 23, 2015
©
2015 American Chemical Society
1558
DOI: 10.1021/jf505296k
J. Agric. Food Chem. 2015, 63, 1558−1561

Journal of Agricultural and Food Chemistry
Article
Table 1. Enzyme-Mediated PS Synthesis in Various [Ch][AA] ILs
a
b
Maximum yield. PA was not detected during the sample analysis by HPLC.
0
.5% water (based on the total weight of the reaction system). Samples
RESULTS AND DISCUSSION
■
(50 μL) were taken from the reaction mixture at specified time
In this work, 12 [Ch][AA] ILs, i.e., [Ch][Gly], [Ch][Ala],
Ch][Val], [Ch][Ser], [Ch][Thr], [Ch][Leu], [Ch][Ile],
[Ch][Pro], [Ch][Phe], [Ch][His], [Ch][Asp], and [Ch][Glu],
were successfully applied to enzymatic synthesis of PS. Table 1
shows that the prepared ILs had an obvious effect on the
enzymatic reaction for PS synthesis. The reaction initial rate
V0), PS yield, and the reaction time were measured to evaluate
the enzymatic reaction.
As can be seen from Table 1, the initial reaction rates were
remarkably influenced by the ILs employed. The minimum V0
intervals, centrifuged to obtain the upper layer, and analyzed by
HPLC.
[
2
0
Analysis of the Samples. The sample was analyzed by a Waters
e2695 HPLC system (Waters, Milford, U.S.A.) with an evaporative
light scattering detector (ELSD). External standards of PC, PS, and PA
were used to prepare calibration solutions at five different
concentrations. Two microliter sample and 1 mL hexane−isopropane
(
(
1/1, v/v) were precisely measured and mixed thoroughly. Ten
microliters of the aforementioned mixture was injected. HPLC
separation was on a Lichrospher 100 Diol column (5 μm, 250 mm
(
[
0.36 μmol PS/min) was obtained in [Ch][Asp] and
×
4.6 mm, Merck). Mobile phase A was n-hexane/2-propanol/
methanol (38:1:1, v/v). Mobile phase B was 2-propanol/methanol
2:3, v/v). Each mobile phase contained 1 mM ammonium acetate.
Ch][Glu], while the maximum V (0.64 μmol PS/min) was
0
achieved when [Ch][Gly] was used as the reaction medium. It
(
is clear that the V values varied with the viscosity of the
0
−1
The flow rate was 1.5 mL min , and the gradient was as follows: 0−
8.7% B from 0 to 20 min, 18.7−100% B from 20 to 20.2 min, 100% B
from 20.2 to 25 min, 100−0% B from 25 to 25.1 min, and 0% B from
5.1 to 30 min. The column temperature and drift pipe temperature
[
Ch][AA] ILs used. Generally, the viscosity is one of the most
1
important factors affecting the applications of the solvent for
catalysis. It is well-known that an increase in solvent viscosity
can augment the diffusion resistance of substrates, thus
enhancing the mass transfer limitations and impairing the
interactions between enzyme particles and substrates. Accord-
ingly, the increase in the viscosity of the [Ch][AA] ILs resulted
2
were controlled at 25 and 65 °C, respectively, and the nitrogen
pressure was controlled at 40 psi. The retention times were 2.52, 2.95,
and 23.83 min for PA, PS, and PC, respectively. PS yield was calculated
from the HPLC data. The average error for this assay is less than 0.5%.
All the reported data are averages of experiments performed at least in
duplicate.
Operational Stability (Reusability) of PLD in [Ch][Glu]. The
operational stabilities of PLD in [Ch][Glu] during batch reactions
were evaluated. After each batch reaction, PLD was separated by
filtration using Whatman #1 filter paper, washed with isopropyl
alcohol three times, and then added into the fresh reaction mixtures
for the next batch.
in the decrease of the V value with the exception of [Ch][Pro].
0
Also, the V values showed a clear dependence on the anion
0
structures of the [Ch][AA] ILs. An increase in the number of
carbon atoms in the side chain of amino acid generally resulted
in a lower V value (Table 1, entries 1, 2, 3, 6, and 7). Of the
0
ILs tested, [Ch][Gly], with the anion being the simplest amino
acid, displayed the highest V value (0.64 μmol PS/min). In
0
addition to the molecular size of the anion, the introduction of
extra hydroxyl, phenyl ring, or carboxylic acid group
1
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DOI: 10.1021/jf505296k
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Journal of Agricultural and Food Chemistry
Article
significantly decreased the V value (Table 1, entries 4, 5, 8−
0
12). In the [Ch][AA] ILs containing hydroxyl group in the side
chain of amino acid ([Ch][Ser] and [Ch][Thr]), the V values
0
were slightly lower, while the ILs with aromatic group in the
anions displayed relatively lower V values (i.e., 0.45, 0.50, and
0
0.49 μmol PS/min for [Ch][Pro], [Ch][Phe], and [Ch][His],
respectively). Unfortunately, the lowest V values of 0.36 μmol
0
PS/min were recorded for [Ch][Asp] and [Ch][Glu] in which
the acidic amino acids served as the anions. A reason for the
substantial correlation between V0 values and the anion
structures of the [Ch][AA] ILs may be that the efficiency of
the enzyme action during PS synthesis was affected by the
solvent used. Particularly, the intermolecular forces of the ILs
such as van der Waals, hydrogen bond, and π-stacking
interactions might contribute to this effect.
Moreover, the maximal PS yields of the PLD-mediated
reaction and the time they consumed in all the [Ch][AA] ILs
employed are presented in Table 1 columns 6 and 7,
respectively. As can be observed, although the PS yield value
in [Ch][Gly] was only 78.1%, the reaction time (12 h) it
needed was the shortest. By contrast, the reaction in [Ch][Glu]
proceeded slowest but constantly increased up to the highest
PS yield value of 86.5% after 60 h. Obviously, the lowest PS
yield (45.2%) was found in [Ch][Phe].
Figure 1. Operational stability of PLD in [Ch][Glu]. Reaction
conditions: 3.0 mL of [Ch][Glu], 0.05 mmol PC, 0.15 mmol L-serine,
6
0 U PLD, 0.5% water (based on the total weight of the reaction
system), 40 °C.
taken as 100%. It was worth noting that PLD displayed
excellent operational stability, and 75% of its original activity
was maintained after being used for 10 batches, highlighting the
presumable cost-effectiveness of the enzyme.
In general, the whole synthesis process for PS is highlighted
by the application of eco-friendly solvents [Ch][AA] ILs as the
reaction media. An efficient, cost-effective, and easy-to-use
“green chemistry” method for the enzymatic PS preparation
was provided. Additionally, the excellent results make the
biobased ionic liquids promising candidates for use as
environmentally friendly solvents in biocatalysis applications.
In Table 1, we found that the PS yield has no apparent
dependence on the viscosity of the [Ch][AA] ILs used.
Similarly, it was shown that the PS yields showed a close
correlation to the anion structures of the [Ch][AA] ILs as well.
When the side chain of amino acid was alkyl group, elongation
of the side chain generally led to lower PS yield (Table 1,
entries 1, 2, 3, 6, and 7). The [Ch][AA] ILs containing basic
amino acids as the anions displayed relatively lower PS yields,
i.e., 73% and 71% for [Ch][Ser] and [Ch][Thr], respectively.
The introduction of an additional carboxylic acid group to the
side chain of the amino acid substantially increased the PS
yields (Table 1, entries 12 and 13). Higher PS yields of 84%
and 86% were exhibited in [Ch][Asp] and [Ch][Glu],
respectively. To be noted was that the introduction of a phenyl
ring exerted drastic effect on the PS yields (Table 1, entries 8−
AUTHOR INFORMATION
Corresponding Author
*Tel.: +86-517-83591044. Fax: +86-517-83591165. E-mail:
zhaoyu_wang@126.com.
■
Funding
10). The PS yields obtained in three [Ch][AA] ILs with
The authors express their thanks for the support from Qing Lan
Project of Jiangsu Province, Natural Science Foundation of
Jiangsu Province (No. BK2012243), Natural Science Research
Project of Higher Education of Jiangsu Province (No.
10KJB530001), and National Natural Science Foundation of
China (No. 21102027).
aromatic group were much lower. In particular, the lowest PS
yield (45%) was found in [Ch][Phe]. As we all know, the
maximal PS yield value was attributable to the thermodynamic
equilibrium of the enzymatic transphosphatidylation of PC with
L-serine for PS synthesis. Consequently, as far as the
dependence of PS yield on the [Ch][AA] ILs was concerned,
a speculative explanation could be that the thermodynamic
equilibrium of the enzyme-mediated reaction may be affected
by the ILs used. Furthermore, the [Ch][AA] ILs effects on the
thermodynamic equilibrium of the reaction may be due to their
specific structures, especially the anions structures.
From a practical viewpoint, the reusability of the biocatalyst
is one of essential factors for the cost-efficiency. If the
biocatalyst can be reused, the economical sustainability is
increased. Encouraged by the excellent experiment results
above, the operational stability of PLD in [Ch][Glu] was
investigated to further examine the potential of the enzyme as a
catalyst for PS production. After each batch reaction, the
enzyme was recovered by filtration, and the next batch was
carried out with fresh substrates. Figure 1 presents the plot of
the activity of the enzyme as a function of the number of
reaction batches. Batch 0 corresponds to the activity of the
fresh enzyme employed in the first reaction trial which was
Notes
The authors declare no competing financial interest.
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