Ionic liquid-assisted solubilization for improved enzymatic esterification of phenolic acids

Article abstract of DOI:10.1007/s11746-011-1989-3

Lipophilic derivatives of phenolic acids could greatly extend their applications in the lipophilic bio-environment and food processing, therefore, developing an efficient lipophilization reaction system constitutes an interesting topic of biocatalysis. Low solubility of phenolic acids in most enzyme-benign solvents represents the main reason for the inefficiency of enzymatic production of lipophilic phenolic derivatives. This work reports a novel approach to improve Candida antartica lipase B (Novozym 435) catalyzed lipophilization of phenolic acids by means of ionic liquids (IL), trioctylmethylammonium trifluoro-acetate (tOMA.TFA) assisted solubilization of the substrate. In this approach, the IL plays two major roles, namely, to dissolve phenolic acids at high concentration so as to create a homogeneous system with another substrate-1-octanol, and to be benign to the enzyme to keep the biocatalyst active; which is proved itself to be a correct strategy as improved conversion and volumetric productivity are obtained. The results showed that dosage of IL (denoted as the volume ratio of 1-octanol/tOMA.TFA), concentration of dihydrocaffeic acid (DHCA) and temperature are the key parameters governing the reaction efficiency. A maximum conversion of DHCA was achieved at the ratio of 1-octanol/tOMA.TFA 12:1 (v/v) (1-octanol/ DHCA, 38:1 (mol/mol)). A temperature of 70 °C was correct to obtain optimal conversion of DHCA. Even though the conversion of DHCA was higher at lower concentrations of DHCA, the overall volumetric productivity (reaction rate) was much higher when a high concentration of DHCA (1.6 M) was applied, due to IL-assisted solubilization of DHCA. Remarkable enhancement of the conversions of ferulic and caffeic acids were achieved, when the same reaction approach (tOMA.TFA assisted solubilization) was applied to these two phenolic acids, indicating the general applicability of this novel approach.

Full text of DOI:10.1007/s11746-011-1989-3

J Am Oil Chem Soc (2012) 89:1049–1055
DOI 10.1007/s11746-011-1989-3
ORIGINAL PAPER
Ionic Liquid-Assisted Solubilization for Improved Enzymatic
Esterification of Phenolic Acids
•
Zhiyong Yang Zheng Guo Xuebing Xu
•
Received: 20 July 2011 / Revised: 1 November 2011 / Accepted: 2 December 2011 / Published online: 17 January 2012
Ó AOCS 2012
Abstract Lipophilic derivatives of phenolic acids could
greatly extend their applications in the lipophilic bio-
environment and food processing, therefore, developing an
efficient lipophilization reaction system constitutes an
interesting topic of biocatalysis. Low solubility of phenolic
acids in most enzyme-benign solvents represents the main
reason for the inefficiency of enzymatic production of
lipophilic phenolic derivatives. This work reports a novel
approach to improve Candida antartica lipase B (Novozym
435) catalyzed lipophilization of phenolic acids by means
of ionic liquids (IL), trioctylmethylammonium trifluoro-
acetate (tOMA.TFA) assisted solubilization of the sub-
strate. In this approach, the IL plays two major roles,
namely, to dissolve phenolic acids at high concentration so
as to create a homogeneous system with another sub-
strate—1-octanol, and to be benign to the enzyme to keep
the biocatalyst active; which is proved itself to be a correct
strategy as improved conversion and volumetric produc-
tivity are obtained. The results showed that dosage of IL
(denoted as the volume ratio of 1-octanol/tOMA.TFA),
concentration of dihydrocaffeic acid (DHCA) and tem-
perature are the key parameters governing the reaction
efficiency. A maximum conversion of DHCA was achieved
at the ratio of 1-octanol/tOMA.TFA 12:1 (v/v) (1-octanol/
DHCA, 38:1 (mol/mol)). A temperature of 70 °C was
correct to obtain optimal conversion of DHCA. Even
though the conversion of DHCA was higher at lower
concentrations of DHCA, the overall volumetric produc-
tivity (reaction rate) was much higher when a high
concentration of DHCA (1.6 M) was applied, due to
IL-assisted solubilization of DHCA. Remarkable
enhancement of the conversions of ferulic and caffeic acids
were achieved, when the same reaction approach
(tOMA.TFA assisted solubilization) was applied to these
two phenolic acids, indicating the general applicability of
this novel approach.
Keywords Phenolic acids Á Dihydrocaffeic
acid (DHCA) Á Ionic liquid Á Candida antartica
lipase B (Novozym 435) Á Esterification
Introduction
Phenolic acids are biologically active antioxidants, which
find a variety of applications in the pharmaceutical, cos-
metic and food industries [1–3]. However, most of phenolic
acids are hydrophilic compounds and can mainly confer
their functional properties or health effects in aqueous
environments or water compartments [4]. Therefore, to
apply natural phenolic acids in lipophilic formulation and
processing, lipophilization is needed where enzymatic
esterification with alcohols has proved itself to be a
promising approach [5–8].
However, there exists a technical dilemma for creating
an efficient nonaqueous enzymatic reaction system: phe-
nolic acids have low solubility in most organic solvents
(such as hexane) where the enzyme is active; while in those
solvents (such as DMSO) in which phenolic acids have
good solubility the enzyme is deactivated or shows low
activity. Ionic liquids (ILs) are an interesting class of
tuneable designer solvents with essentially zero volatility,
which are claimed to be ‘‘green’’ media [9]. Ionic liquids
can act as solvents for diverse reactions and give different
selectivity and reactivity compared with conventional
Z. Yang Á Z. Guo Á X. Xu (&)
Department of Engineering, Aarhus University,
Gustav Wieds Vej 10, 8000 Aarhus C, Denmark
e-mail: xu@mb.au.dk
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J Am Oil Chem Soc (2012) 89:1049–1055
organic solvents because the ionic liquids are made of ions
rather than molecules [10]. This ionic nature entails ILs
complex and specific interactions with substrates and
enzyme, accounting for their different specificity and
reaction behaviors. In the past 10 years, ionic liquids had
been continued to attract attention as a replacement for
organic solvents for enzymatic transformation of various
compounds [11]. Katsoura et al. [12] investigated enzy-
matic esterification of several phenolic acids with short and
medium chain fatty alcohol in several ionic liquids, and
found out the enzymatic esterifications were very efficient
in ionic liquids. More recently, Chen et al. [13] reported
synthesis of oleyl ferulate in ionic liquid/iso-octane binary
system, which improved productivity was obtained.
conversion and volumetric productivity was observed in
comparison with the absence of the IL.
Materials and Methods
Materials
The ionic liquid tOMA.TFA (Trioctylmethylammonium
Trifluoroacetate) of 99.77% purity was purchased from
Merck KGaA (Darmstadt, Germany). The viscosity of
tOMA.TFA was 826 cP at 20 °C. Novozym 435 (Candida
antartica lipase B, immobilized on a macroporous polyac-
rylate resin, with a declared activity of approx. 10,000 PLU/g.
PLU is defined as the amount of propyl laurate formed, via
esterification of lauric acid with 1-propanol at 60 °C, per
minute catalyzed by the unit enzyme) was obtained from
Novozymes A/S (Bagsværd, Denmark). Selected phenolic
acids and 1-octanol (dried overnight over activated molec-
ular sieves before use) were bought from Sigma-Aldrich or
Fluka with the highest available purity. All other organic
solvents of analytical and HPLC grades were purchased
from Fisher Scientific Inc. (Slangerup, Denmark).
In a previous work, we have demonstrated the superior
performance of trioctylmethylammonium trifluoroacetate
(tOMA.TFA) over other types of IL in improving the con-
version and volumetric productivity of ascorbic acids [14].
tOMA.TFA is a type of IL identified in our group that is
specifically suitable for enzymatic reaction, because it has
the anion trifluoroacetate which is a good hydrogen bonding
acceptor to dissolve the compounds containing OH groups,
and a hydrophobic cation trioctylmethylammonium buf-
fering the polarity of the IL. It is therefore anticipated that
this ionic liquid may be applicable to the reaction system for
enzymatic conversion of phenolic acids with preferable
performance. In the current study, we investigated
tOMA.TFA-mediated enzymatic esterification of dihydro-
caffeic acid (DHCA) with 1-octanol (Scheme 1), where
1-octanol was selected as a model alcohol because octyl
phenolates show better antioxidation property according to
our results [15]. The effects of dosage of the IL (denoted as
ratio of tOMA.TFA/1-octanol), substrate concentration,
reaction temperature, the methods of water removal, etc.
were examined. To envisage its general applicability, this
system was also applied to enzymatic transformation of
ferulic and caffeic acids, where significant enhancement in
Enzymatic Esterification of Octyl Phenolic Acid Esters
All reactions were conducted in 50-ml jacketed reactors. In a
typical reaction, 1 mmol DHCA (0.182 g) was dissolved in
0.5 ml of ionic liquid tOMA.TFA first and then diluted with
2 ml of 1-octanol (1.648 g, 12.66 mmol). To avoid the
evaporation of 1-octanol at elevated temperatures, the
reaction vessel was closed with a stopper and fixed by
parafilm unless at a sampling moment. In the case of inves-
tigating the effect of volume ratio of 1-octanol/tOMA.TFA,
the amount of 1-octanol was increased from 2 to 8 ml
(6.592 g, 50.63 mmol) and the volume ratio between
1-octanol and ionic liquid tOMA.TFA was then increased
COOH
COO
Novozym 435
+
H2O
HO
+
HO
HO
OH
OH
1- Octanol
DHCA
1-Octyl dihydrocaffeate
Reaction scheme of DHCA with 1-octanol
F3C
C
O
N
O
Structure of ionic liquid tOMA·TFA
Scheme 1 Reaction scheme of phenolic acid with octanol by using dihydrocaffeic acid (DHCA) as a representative and showing the structure of
the ionic liquid tOMA.TFA
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J Am Oil Chem Soc (2012) 89:1049–1055
1051
from 4:1 to 16:1 correspondingly. A typical reaction was
carried out at a set temperature with magnetic agitation
(diamond shape bar 1 cm in length) at 300 rpm for up to
6 days, with or without the presence of activated molecular
liquid (tOMA.TFA) and then further diluted with 1-octanol
from 2 to 8 ml to give a volume ratio of 1-octanol/
tOMA.TFA at 4:1 to 16:1 (v/v). The reactions were carried
out at 80 °C and 300 rpm with 100 mg Novozym 435 as
biocatalyst and no molecular sieves applied. It should be
noted that the concentration of DHCA decreases while the
1-octanol dosage increases, which consequently leads to
increasing substrate ratio of 1-octanol/DHCA. As shown in
Fig. 1, the conversion of DHCA was around 60% in 5 days
at 1-octanol/tOMA.TFA 4:1 (v/v), and no significant
change in the conversion of DHCA observed when the
volume ratio was increased to 8:1. When the volume ratio
of 1-octanol/tOMA.TFA was increased to 12:1 (v/v), the
conversion increased to around 70%. However, further
increase to 16:1 (1-octanol/tOMA.TFA, v/v) did not further
increase the conversion of DHCA, even leading to a slight
decrease (Fig. 1). On the other hand, if the volume ratio of
1-octanol/tOMA.TFA was too low (below 2:1), little con-
version of DHCA was observed (data not shown), which is
probably due to the inhibiting effect of the ionic liquid
tOMA.TFA on the activity of the enzyme. There may be
two reasons accounting for increased conversion of DHCA
with increasing concentration of 1-octanol: (1) excessive
1-octanol as a reactant helps to shift the reaction equilib-
rium to the formation of octyl DHCA ester;(2) increasing
the dosage of 1-octanol increases the overall hydropho-
bicity of the system which is beneficial for the activity of
˚
sieves (3 A). The reaction was initiated by adding 100 mg of
Novozym 435, and monitored by periodically withdrawing
samples for HPLC analysis. For all the control reactions
without tOMA.TFA conducted in parallel, all other condi-
tions were identical except for the absence of the IL.
Enzymatic esterifications of ferulic and caffeic acids
with 1-octanol were conducted in a similar manner. The
final concentrations of these two phenolic acids were 0.4 M
and the volume ratio of 1-octanol/IL was 4:1. The reactions
were conducted at 80 °C with the presence 100 mg Nov-
ozym 435 and 100 mg molecular sieves.
The initial reaction rate was calculated from the linear
part of conversion vs time (within 24 h). Each experiment
was conducted in two replicates. Means standard devi-
ations of the two determinations were used for result
evaluation.
HPLC Analysis
In all cases, a single product was detected by HPLC
analysis with a UV detector in the range of 200–325 nm
and the responses at 280 nm (DHCA and caffeic acid) and
325 nm (ferulic acid) were used for result evaluation.
HPLC analysis of samples was performed on a RP C18-
column (250 9 4.6 mm, 5 lm) connected to a Survival
Plus HPLC System equipped with a PDA (Photo Diode
Array) detector (Thermo Scientific, Denmark). Injection of
a 10-ll sample was isocratically eluted by 90% of solvent
A (methanol) and 10% of solvent B (water with 0.75% of
acetic acid) for 16 min [16].
80
70
60
50
40
30
20
10
0
HPLC–ESI–MS Analysis
HPLC–ESI–MS analyses were performed with an electro-
spray as an ionization source (ESI) coupled to a quadrupole
time-of-flight mass spectrometer (Bruker micrOTOF-Q,
Bremen, Germany). The column used and elution conditions
for HPLC part were the same as for HPLC analysis. Ioni-
zation was performed in the negative mode with an 8 L/min
nitrogen flow, 0.8 bar nebulizer pressure and a temperature
of 190 °C. The scan range was from 50 to 1,200 m/z [16].
0
1
2
3
4
5
Reaction time (Day)
Results and Discussion
Fig. 1 Effect of reaction volume ratio of 1-octanol/tOMA.TFA (v/v)
on the conversion of DHCA (circles 4:1; diamonds 8:1; triangles
12:1; squares 16:1). Reactions were conducted at 80 °C and catalyzed
by 100 mg of Novozym 435 without molecular sieves or vacuum. The
dosage of DHCA was kept constant at 1 mmol (0.182 g) for the four
tests; and the corresponding concentration of DHCA varies with
volume ratio of 1-octanol/tOMA.TFA
tOMA.TFA Dosage and Enzyme Activity
Firstly, we examined the effect of tOMA.TFA dosage.
First, 1 mmol of DHCA was dissolved in 0.5 ml ionic
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J Am Oil Chem Soc (2012) 89:1049–1055
the enzyme [13]. In short, as depicted in Fig. 1, in the test
range of volume ratio of 1-octanol/tOMA.TFA, the effect
of volume ratio change on reaction rate is not very sig-
nificant (1.2 mmol d-1 (g enzyme)-1 at 4:1 increased to
1.4 mmol d-1 (g enzyme)-1 at 12:1, estimated based on
the conversion in 5 days).
conditions. However, there was almost no difference in the
conversion of DHCA when the vacuum was increased from
800 to 400 mbar. The addition of molecular sieves
(100 mg) seems to give a better performance than applying
vacuum. About 86% of conversion was achieved in 5 days
80
70
60
50
40
30
20
10
0
Effect of Temperature
Novozym 435 is a thermostable immobilized enzyme with a
maximum activity at 70–80 °C according to its product
sheet; and the recommended temperature is at 40–60 °C for
the sake of a longer half-life time [17]. However, in this IL-
assisted solubilization system, the DCHA is applied at high
concentration (0.4–1.6), and the IL tOMA.TFA with high
viscosity is introduced, both of which will contribute to an
increase in the overall viscosity of the system. Thus, to carry
out the reaction at elevated temperatures not only improves
the kinetic rate of enzymatic reaction but also increases
mass transfer due to a dramatic decrease in system viscosity
against the increase of temperature (The effect of temper-
ature on the viscosity of ILs could be estimated by the group
contribution method [18]). For example, when estimated by
the method of Gardas and Coutinho [18], the viscosity of
tOMA.TFA at 40 °C, is 228.7 cP which can be lowered by a
great extent to 25.6 cP when the temperature is increased to
80 °C; the viscosity at 80 °C is reduced by about ninefold
compared to that at 40 °C. Therefore, 60, 70 and 80 °C
were selected in this work to examine the effect of tem-
perature on the conversion of DHCA (Fig. 2). As depicted
in Fig. 2, the conversion of DHCA at 60 °C over 5 days was
around 50%; and the conversion at 70 °C was up to 70% for
the same reaction time period. However, when the reaction
was carried out at 80 °C, instead of an increase, the con-
version of DHCA in 5 days decreased to around 60%,
which is probably due to increasing deactivation of the
enzyme as temperature increases.
0
1
2
3
4
5
Reaction time (Day)
Fig. 2 Conversion of DHCA at different temperatures (diamonds
60 °C; squares 70 °C; triangles 80 °C). Reactions were conducted
without molecular sieves or vacuum. 1-Octanol/tOMA.TFA was fixed
at the ratio of 4:1 (v/v). The concentration of DHCA was 0.4 M, and
the mole ratio of 1-octanol/DHCA was 12.66:1. The enzyme load was
100 mg
70
60
50
40
30
20
10
0
100
90
80
70
60
50
Effect of Water Removal by Molecular Sieves
or Vacuum
Water is a byproduct from the esterification of DHCA and
1-octanol. Although Novozym 435 needs a certain amount
of water to maintain its catalytic activity, too much water
will reverse the esterification reaction. Moreover, too much
water in the reaction system might also lead to the aggre-
gation of the enzyme, which has a negative effect on the
reaction rate [19]. Therefore, the effect of water removal by
molecular sieves or vacuum was investigated in this work
(Fig. 3). As indicated in Fig. 2, when a vacuum was
applied to remove water from the reaction, conversion of
DHCA was increased by 20% compared to the reaction
without vacuum or molecular sieves under the same
A
B
C
D
E
Fig. 3 Comparison of reaction rates in the reaction systems using
different methods to remove water (A: no molecular sieves or
vacuum; B: 100 mg molecular sieves; C: 100 mg molecular sieves
and continued to supply 50 mg each day until reactions were stopped;
D: 800 mbar; E: 400 mbar). 1-Octanol/tOMA.TFA was fixed at the
ratio of 4:1 (v/v). The concentration of DHCA was 0.4 M, and the
mole ratio of 1-octanol/DHCA is 12.66:1. All reactions were
conducted at 80 °C and catalyzed by 100 mg of enzyme
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J Am Oil Chem Soc (2012) 89:1049–1055
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with the initial addition of 100 mg molecular sieves; and
the conversion can be further increased up to 90% if
thereafter the reaction system was continuously supplied
with 20 mg/ml of molecular sieves each day. In terms of
the initial reaction rate, there was no significant difference
between the different water removal methods, but all
showed remarkable improvement (from 37 to over
56 mM h-1 (g enzyme)-1) compared to no molecular
sieves addition or no vacuum application.
70
60
50
40
30
20
10
0
A
Effect of Substrate Concentration
The effect of the concentration of DHCA on conversion
was also studied by varying the concentration of DHCA
from 0.4 to 1.6 M. It was observed using ionic liquid
tOMA.TFA as an assistant solvent is able to increase the
solubility of DHCA, especially at higher concentrations (to
give total dissolution of DHCA up to 1.6 M), and thus
increase the conversion of DHCA greatly. When the con-
centration of DHCA was increased to 1.2 M, the conver-
sion of DHCA in 5 days was 62% with the presence of
ionic liquid tOMA.TFA, which is about 8% higher than the
system where ionic liquid tOMA.TFA was not used as an
assistant solvent (Fig. 4a).
0
1
2
3
4
5
Reaction time (Day)
250
200
150
100
50
B
As indicated in Fig. 4b, the reaction rate is increased as
substrate concentration increases before the enzyme is satu-
rated and a maximum reaction rate is reached. As shown in
Fig. 4b, the reaction rate was 68 mM h-1 (g enzyme)-1 for
the IL-assisted solubilization system when the concentration
of DHCA was 0.4 M; and the reaction rate was gradually
increased to 244 mM h-1 (g enzyme)-1 when the concen-
tration of dihydrocaffeic acid was increased to 1.6 M. In
general, this observation agreed with the previous report
concerning the relationship of concentration and reaction rate
with p-coumaric acid and cinnamic acid as substrates [6].
Interestingly, the initial reaction rate was 178 mM h-1
(g enzyme)-1 when the reaction was carried out in the
reaction system without ionic liquid and the concentration
of DHCA was 0.4 M, then the reaction rate decreased to
127 mM h-1 (g enzyme)-1 when the concentration was
increased to 0.8 M. Afterwards, the initial reaction rate was
increased again and reached 212 mM h-1 (g enzyme)-1
when the concentration was increased to 1.6 M. The pos-
sible explanation for this phenomenon is, the solubility of
DHCA in 1-octanol is lower than in ionic liquid
tOMA.TFA, and the enzyme activity may be inhibited by
mass-transfer limitation when the concentration of DHCA
was increased to the level beyond the solubility of DHCA in
1-octanol. On the other hand, as an ionic solvent, the
introduction of tOMA.TFA, to some extent, will increase
the overall polarity and viscosity of the reaction system,
which may influence enzyme activity and mass transfer
[20]. At the same time, DHCA is completely soluble in
0
0,4
0,8
1,2
1,6
Concentration of DHCA (M)
Fig. 4 Conversions of DHCA at a concentration of 1.2 M with
(diamonds) or without tOMA.TFA (squares) (a); and comparison of
the reaction rates at different DHCA concentrations with (diamonds)
or without tOMA.TFA (squares) (b). In the system where tOMA.TFA
was added, the mixture volume ratio of 1-octanol/tOMA.TFA was 4:1
(v/v). Reactions were catalyzed by 100 mg enzyme at 80 °C with the
presence of 100 mg molecular sieves
1-octanol at 0.4 M at 80 °C even without tOMA.TFA. This
may explain why the initial reaction rate was much higher at
the 0.4 M concentration of DHCA in the reaction system
without ionic liquid tOMA.TFA than the reaction with the
IL. Nevertheless, adding ionic liquid tOMA.TFA as an
assistant solvent can increase the solubility of DHCA
greatly and result in higher initial reaction rates at high
substrate concentrations, which leads to slightly improved
volumetric productivity of the desired products as displayed
in Fig. 4.
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Esterification of Other Selected Phenolic Acids
90
80
70
60
50
40
30
20
10
0
A
In previous studies [5–8, 21–24], different phenolic acids
from cinnamic series have been used for esterification in
various media. It was found esterification was possible for
the phenolic acids which the aromatic cycle was not
parahydroxylated and the side chain was saturated. Ferulic
acid was reported to be esterified with fatty alcohols with
more than eight carbons, but in low yields [3]. In a recent
study [12], various cinnamic acids were chosen for con-
jugation with short and media chain fatty alcohols (C1–
C10) in different ionic liquids. Most of them achieved a
relatively high yield in a short time, but the yields of the
esterification of ferulic acid and caffeic acid with 1-octanol
were still relatively low.
In this study, ferulic acid and caffeic acid were also
chosen for evaluation with this IL-assisted solubilization
system. The reactions were conducted either with or
without the presence of tOMA.TFA (Fig. 5). As shown in
Fig. 5, the conversion of ferulic acid can reach 86% after
16 days of reaction in the presence of tOMA.TFA, but only
58% was achieved without tOMA.TFA. More interestingly,
the conversion of caffeic acid reached 60% after 12 days of
reaction with tOMA.TFA assisted solubilization, whereas
only 14% was obtained when no ionic liquid applied in the
system. The results indicate that conversion of these two
cinnamic acids can be significantly increased by increasing
their solubility with the assistance of tOMA.TFA. Inspec-
tion of Fig. 4 and 5 reveals that, the presence of
tOMA.TFA has a more significant effect in enhancing
conversion of ferulic acid and caffeic acid than DHCA.
This is because their different solubilities in 1-octanol,
which at elevated temperature (80 °C) DHCA was almost
completely soluble in 1-octanol at 0.4 M as experimentally
observed; whereas little solubility of ferulic acid and caf-
feic acid in 1-octanol was observed. It not surprising that a
lower reaction rate of tOMA.TFA-mediated esterification
of DHCA at lower concentration than without tOMA.TFA
was obtained, because at this moment the IL plays only a
minor role in improving the solubility of DHCA but the
introduction of tOMA.TFA leads to slight decrease in
enzyme activity due to the ionic nature of ILs. The con-
tribution of increasing substrate solubility by adding
tOMA.TFA to the increase of overall reaction rate appears
obvious at high concentrations of DHCA (Fig. 4); and is
more remarkable for ferulic acid and caffeic acid (Fig. 5).
0
2
4
6
8
10
12
14
16
Reaction time (day)
70
60
50
40
30
20
10
0
B
0
2
4
6
8
10
12
Reaction time (day)
Fig. 5 Conversion of ferulic acid (a) and caffeic acid (b) in the
reaction system with ionic liquid tOMA.TFA (diamonds) or without
ionic liquid tOMA.TFA (squares). 1-Octanol and ionic liquid
tOMA.TFA was mixed in the volume ratio of 4:1 (v/v). The
concentrations of both phenolic acids are 0.4 M, and mole ratio of
1-octanol/ferulic acid (or caffeic acid) is 12.66:1 (mol:mol). Reac-
tions were catalyzed with 100 mg of enzyme with the presence of
100 mg molecular sieves at 80 °C
was found that the solubility of selected phenolic acids
(dihydrocaffeic acid, ferulic acid, and caffeic acid) can be
greatly improved by being dissolved in tOMA.TFA. The
changes of volume ratio of 1-octanol/tOMA.TFA and
temperature can profoundly affect the conversion of
DHCA, and optimum yields can be achieved at a 1-octanol/
tOMA.TFA ratio of 1/12 and at a temperature of 70 °C.
Conclusion
This work has demonstrated a novel approach to improve
enzymatic synthesis of lipophilic cinnamic derivatives by
using ionic liquid (tOMA.TFA) assisted solubilization. It
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Addition of molecular sieves is a good method to remove
water generated from the reaction and increases the con-
version significantly. The conversion of DHCA decreased
as its concentration increased, however, the overall reac-
tion rate increased with increasing concentration of DHCA,
which resulted in increased volumetric productivity. The
application of this IL-assisted solubilization system was
shown to be generally valid by applying it to enzymatic
syntheses of ferulic and caffeic acid esters. Conversions of
both ferulic and caffeic acids were significantly higher than
the control systems (without tOMA.TFA), and also higher
than previously reported data, indicating the general value
of this new approach.
Acknowledgments This work was supported by the Strategic Food
and Health Program (FoSu) as well as the grant from Graduate School
of Science, Aarhus University.
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