Cutinase from Fusarium oxysporum catalyzes the acylation of tyrosol in an aqueous medium: Optimization and thermodynamic study of the reaction

Article abstract of DOI:10.1016/j.molcatb.2016.04.004

Recently, tyrosol has gained attention as a result of its many pharmacological properties and due to the fact that it can be isolated from cheap and abundant resources. Lipophilic tyrosyl esters, which are scarce in nature, have proven in certain cases to acquire improved biological activity compared to tyrosol itself, increasing their potential use in the food and cosmeceutical industries. The enzymatic approach for the synthesis of such esters has prevailed, as it is "green", compared to chemical practices. We hereby report the enzymatic synthesis of tyrosyl esters of various aliphatic fatty acids performed by a recombinant cutinase from Fusarium oxysporum (FoCut5a). The reaction system used consists of an aqueous phase saturated with the corresponding fatty-acid vinyl ester, which played the role of the acyl donor. We also proceeded to the study of several parameters on the yield of the tyrosyl butyrate ester synthesis. The maximum yield achieved was 60.7% after 4 h at 20 °C, in pH 7.0, with initial tyrosol concentration of 12.5 mM and using 5 μg FoCut5a mL^-1 reaction as catalyst. The optimum reaction conditions can be considered mild, highlighting the environmentally friendly nature of this reaction, along with the fact that there are not any harmful reagents involved. Additionally, the use of two thermodynamic models, Conductor-like Screening Model for Real Solvents (COSMO-RS) and UNIquac Functional-group Activity Coefficients (UNIFAC), were employed for the prediction of reactants' and products' solubilities and their distribution in the reaction biphasic system, aiming to correlate the reaction yields with these important thermodynamic quantities and understand the ability of this enzymatic reaction in synthesizing tyrosyl esters.

Full text of DOI:10.1016/j.molcatb.2016.04.004

Accepted Manuscript
Title: Cutinase from Fusarium oxysporum catalyzes the
acylation of tyrosol in an aqueous medium: optimization and
thermodynamic study of the reaction
Author: Efstratios Nikolaivits Giannis-Florjan Norra
Epaminondas Voutsas Evangelos Topakas
PII:
S1381-1177(16)30056-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.04.004
MOLCAB 3351
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Journal of Molecular Catalysis B: Enzymatic
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Accepted date:
6-2-2016
24-3-2016
6-4-2016
Please cite this article as: Efstratios Nikolaivits, Giannis-Florjan Norra,
Epaminondas Voutsas, Evangelos Topakas, Cutinase from Fusarium oxysporum
catalyzes the acylation of tyrosol in an aqueous medium: optimization and
thermodynamic study of the reaction, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2016.04.004
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Cutinase from Fusarium oxysporum catalyzes the acylation of
tyrosol in an aqueous medium: optimization and
thermodynamic study of the reaction
Efstratios Nikolaivits1, Giannis-Florjan Norra1, Epaminondas Voutsas2 and
Evangelos Topakas1*
1Biotechnology Laboratory, School of Chemical Engineering, National Technical
University of Athens, 9 Heroon Polytechniou Str., Zographou Campus, 15780 Athens,
Greece
2Laboratory of Thermodynamics and Transport Phenomena, School of Chemical
Engineering, National Technical University of Athens, 9 Heroon Polytechniou Str.,
Zographou Campus, 15780 Athens, Greece
*Correspondence to: E. Topakas. Tel: +30-210-7723264; fax: +30-210-7723163; e-mail:
vtopakas@chemeng.ntua.gr
1

Graphical abstract
Highlights
 FoCut5a cutinase is capable of synthesizing tyrosyl esters in an aqueous
medium
 A biphasic reaction system was optimized for optimal product yield
 Maximum conversion yield of 61% was achieved for the production of tyrosyl
butyrate
 Optimal reaction conditions were pH 7, 12.5mM tyrosol, 5 μgenz mL-1 for 4 h
at 20oC
 Reactant/product solubility and distribution in the biphasic system was
predicted
Abstract
Recently, tyrosol has gained attention as a result of its many pharmacological properties
and due to the fact that it can be isolated from cheap and abundant resources. Lipophilic
tyrosyl esters, which are scarce in nature, have proven in certain cases to acquire
2

improved biological activity compared to tyrosol itself, increasing their potential use in
the food and cosmeceutical industries. The enzymatic approach for the synthesis of such
esters has prevailed, as it is “green”, compared to chemical practices. We hereby report
the enzymatic synthesis of tyrosyl esters of various aliphatic fatty acids performed by a
recombinant cutinase from Fusarium oxysporum (FoCut5a). The reaction system used
consists of an aqueous phase saturated with the corresponding fatty-acid vinyl ester,
which played the role of the acyl donor. We also proceeded to the study of several
parameters on the yield of the tyrosyl butyrate ester synthesis. The maximum yield
ο
achieved was 60.7% after 4 h at 20 C, in pH 7.0, with initial tyrosol concentration of
12.5 mM and using 5 μg FoCut5a mL-1 reaction as catalyst. The optimum reaction
conditions can be considered mild, highlighting the environmentally friendly nature of
this reaction, along with the fact that there are not any harmful reagents involved.
Additionally, the use of two thermodynamic models, Conductor-like Screening Model
for Real Solvents (COSMO-RS) and UNIquac Functional-group Activity Coefficients
(UNIFAC), were employed for the prediction of reactants’ and products’ solubilities and
their distribution in the reaction biphasic system, aiming to correlate the reaction yields
with these important thermodynamic quantities and understand the ability of this
enzymatic reaction in synthesizing tyrosyl esters.
Keywords: Fusarium oxysporum; cutinase; tyrosyl esters; COSMO-RS; UNIFAC
3

1. Introduction
Tyrosol is a phenolic compound primarily occurring in olive oil [1], as well as olive
leaf extracts [2] and olive-mill wastewaters [3]. During the past decade many
pharmacological properties of tyrosol have been unveiled. Being an antioxidant [4],
tyrosol is considered to be cardioprotective, inhibiting LDL-oxidation, thus preventing
coronary heart disease [5], inducing survival and longevity proteins, which act as
myocardial protection against ischemia related stress [6] and showing antithrombotic
activity during platelet aggregation [7,8]. Some researchers have further proved that
tyrosol acts as a neuroprotective agent, preventing Alzheimer’s [9] and Parkinson’s
diseases [10] and also as a preventing agent of tumoral diseases [4,11]. Lipophilization
of natural antioxidants is a familiar practice (see review from Figueroa-Espinoza et al.
[12]) aiming to broaden their applications in oil-based food processing and cosmetics.
Acylating tyrosol with a lipophilic moiety makes it an amphiphilic molecule, which
could accumulate at oil-water or oil-air interfaces where oxidation is considered to
occur, increasing the oil protection. Synthesis of phenolic derivatives can be achieved
either chemically or enzymatically. Chemical procedures involve reagents, such as
N,N'-dicyclohexylcarbodiimide, tetrahydrofuran and dichloromethane [13], which may
prove harmful for the human health and are not environmentally friendly. Using
enzymes as catalysts has been demonstrated as a “green”, safe and efficient alternative
for food, pharmaceutical and cosmeceutical applications.
So far, there has been a plethora of reports for synthesis reactions performed by
hydrolytic enzymes. The water content in such kind of reactions is a very important
parameter. On one hand, water molecules bound on the enzyme ensure its catalytic
properties [14], while the use of miscible organic solvents causes the loss of the
enzyme’s essential surface water molecules affecting its catalytic activity [15]. On the
4

other hand, the excess of water triggers the enzyme’s hydrolytic activity at the expense
of its synthetic activity [16]. This type of reaction has also been carried out in
supercritical carbon dioxide [17–19] and in micro- [20] and mini- [21] emulsion
systems with 2% (v/v) and 50 or 80% (w/w) water content respectively. Furthermore,
some reactions of this sort have been reported to occur in diphasic systems: water-
heptane [22] or water-oil [23].
Cutinases are small members of the serine-hydrolase family that have shown to
hydrolyze esters, preferably with short-length chains (C2 or C4 being the optimum for
them) [24]. Their active site is accessible to the solvent, in contrary to the one of lipases
[25], therefore there is no need for interfacial activation. Cutinases, except from the
wide range of substrates that can hydrolyze, they are also capable of catalyzing
esterification and transesterification reactions. Fusarium solani pisi and Burkholderia
cepacia cutinases have been shown to synthesize flavor compounds such as short-chain
alkyl esters [21,26,27] in isooctane or miniemulsion systems. The esterification of
various natural phenolic acids with aliphatic alcohols has been performed by a
commercial F. solani cutinase, but with low conversion yields [28]. Furthermore,
esterification and transesterification of various phenolic acids or phenolic acid esters
with n-butanol or n-propanol, have been carried out in hexane-miniemulsion systems,
by different feruloyl esterases, with good yields [29–31].
In this study, the enzymatic synthesis of alkyl esters of tyrosol was carried out in
an aqueous system under mild reaction conditions, increasing the lipophilicity of
tyrosol for its potential use in cosmeceutical or oil-based foods. Their synthesis, so far,
has been performed with either chemical synthesis [32] or enzymatically with the use
of lipases [33]. In the present reaction system, the aqueous phase is saturated with the
acyl donor (vinyl ester) and the transesterification reaction is catalyzed by FoCut5a, a
5

cutinase from Fusarium oxysporum. The synthesis of five tyrosyl esters was performed
and the effect of several parameters including temperature, pH, enzyme loading, and
substrate concentration on the transesterification reaction of tyrosol with vinyl butyrate
was studied. To increase the understanding of the reaction system, the use of activity
coefficient models was carried out, such as the quantum chemistry based Conductor-
like Screening Model for Real Solvents (COSMO-RS) that is the thermodynamic tool
for the prediction of reagents/products solubility or the group contribution UNIquac
Functional-group Activity Coefficients (UNIFAC) model.
2. Materials and methods
2.1 Enzyme and chemicals
The recombinant cutinase from F. oxysporum was heterologously produced from
Escherichia coli BL21 (DE3) and purified, as previously described [25]. Enzyme
concentration was calculated by UV spectrometry at 280 nm [34] using molar
extinction coefficient of 16180 M−1 cm−1 [25]. Vinyl esters (acetate, propionate,
butyrate, decanoate and laurate) and tyrosol were purchased from Sigma-Aldrich (St.
Louis, USA). All other chemicals used were of HPLC grade.
2.2 Acylation reactions and product analysis
Vinyl esters with different carbon-chain lengths (acetate-C2, propionate-C3,
butyrate-C4, decanoate-C10 and laurate-C12) were tested as acyl donors in order to
determine the specificity of FoCut5a in the transesterification reaction of tyrosol.
A typical tyrosyl ester synthesis reaction was carried out in a final volume of 0.5
mL, containing 3.5 mg tyrosol diluted in 0.375 mL phosphate-citrate buffer pH 7.0 (0.1
6

M), 0.1 mL vinyl ester and 25 μg FoCut5a diluted in 20 mM Tris-HCl pH 8.0 buffer.
Reactions were typically performed in 30 οC in an Eppendorf Thermomixer® Comfort
(Eppendorf, Germany) operated at 900 rpm.
Aliquots of reaction mixtures (10 μL) were analyzed by thin layer chromatography
(TLC) on aluminum sheets coated with Silica gel 60 F254 (Merck, Germany). An elution
system consisted of petroleum ether (40-60οC):ethyl acetate (4:1 v/v) was used for the
resolution of the acylated product. Tyrosol and its derivatives were developed through
evaporated iodine or detected under UV light (254 nm).
For the quantification of the reaction products, an HPLC method was constructed
and followed. Reaction aliquots (0.5 mL) were extracted with 1 mL (0.5-0.25-0.25 mL
gradually) of ethyl acetate prior to analysis. 0.2 mL of the extract was dried and then
diluted in 0.5 mL methanol (50% v/v) prior to analysis on a SHIMADZU LC-20AD
HPLC equipped with a SIL-20A autosampler. The column used is a C-18 reverse-phase
NUCLEOSIL® 100-5 (Macherey-Nagel, Germany) at a flow rate of 0.8 mL min-1.
Analysis of the samples was executed with a linear gradient method using double
distilled water (A) versus acetonitrile (B) as eluents. The total running time for samples
containing esters C2, C3 and C4 was 15 min during which the following proportions of
the solvent B were used: 0-5 min 10-30%, 5-8 min 30-90%, 8-13 min 90%, 13-15 min
90-10%. For medium-chain tyrosol derivatives (C10 and C12) the isocratic step (90% B)
lasted 20 min instead of 5 min. Detection took place with the photodiode array detector
Varian ProStar and the wavelength at which the absorption was recorded was 280 nm.
Τhe conversion yield for the synthesis of tyrosyl esters was calculated from the amount
of tyrosol having reacted compared to its initial quantity, using a reference curve
constructed by standard solutions of tyrosol.
7

2.3 Optimization of tyrosyl butyrate (TyC4) synthesis
The effect of several parameters was investigated in order to maximize the
conversion yield of tyrosol to its lipophilic derivative. Tyrosol was diluted in different
pH, using the following buffers (0.1 M salt concentration): phosphate-citrate for pH 3-
7, Tris-HCl for pH 8-9 and glycine-NaOH for pH 9-10. The effect of the enzyme
loading (1, 2.4, 5, 25 μg mL-1) and tyrosol concentration (5, 10, 12.5, 25, 50, 75 mM),
as well as temperature conditions (15, 20, 25, 30, 35 οC) was also investigated. Control
reactions in the absence of enzyme, were also realized, during all reaction sets.
2.4 Preparative-scale production and purification of tyrosyl butyrate
Tyrosol (69.1 mg) was mixed with 8 mL MOPS buffer pH 6.0 (0.1 M) saturated
with 2 mL vinyl butyrate. Reaction was initiated by addition of 0.5 mg enzyme and
incubated for 24 h at 30 οC under shaking. At the end of the reaction, the mixture was
gradually extracted 3 times with 10 mL ethyl acetate and the organic phases were
pooled. The product was purified by adsorption chromatography using a 500 mm×25
mm ID glass column packed with silica gel 60 (0.040–0.063 mm) in the elution solvent
(petroleum ether 40-60οC:ethyl acetate 4:1 v/v). The occurrence of ester was
systematically checked on each elution fraction by TLC.
The identification of TyC4 was done by 1H and 13C NMR spectroscopy, which was
performed in CD3OD, with a Bruker DRX spectrometer, equipped with broad band
probe at 400 MHz. 1H NMR (CDCl3), , ppm: 7.10 (2H, d, J = 8.1 Hz, H-1, H-5); 6.80
(2H, d, J = 8.3 Hz, H-2, H-6); 4.27 (2H, t, J = 7.1 Hz, H-8); 2.88 (2H, t, J = 7.0 Hz, H-
7); 2.29 (2H, t, J = 7.4 Hz, H-11); 1.65 (2H, m, H-12); 0.95 (3H, t, J = 7.4 Hz, H-13).
13C NMR, , ppm: 173.9 (C-10); 154.5 (C-4); 130.0 (C-3); 129.7 (C-1, C-5); 115.4 (C-
8

2, C-6); 65.0 (C-8); 36.2 (C-7); 34.3 (C-11); 18.4 (C-12); 13.6 (C-13). Carbon
numbering corresponds to the product of acylated tyrosol shown in Figure 1.
2.5 Solubility and partition coefficient predictions with the COSMO-RS model
The COSMO-RS developed by Klamt and co-workers [35–37] is an efficient
method for the a priory prediction of thermophysical data of liquid mixtures on the basis
of unimolecular quantum chemical calculations for the individual molecules that
provide the necessary information for the evaluation of molecular interactions in
liquids. In the COSMO-RS model, a liquid is considered to be an ensemble of almost
closely packed ideally screened molecules. Each piece of surface is characterized by its
value of screening charge density (σi). The interaction energy of the ensemble is then
obtained by a statistically correct consideration of all possible pairs of pieces of surface.
The composition of the ensemble that is needed to apply this procedure is delivered by
the distribution function p(σ). This function that is called as the σ profile, describes the
amount of surface in the ensemble having a screening charge density between σ and
σ+dσ. The representative σ profile of a mixture is the concentration weighted average
of the pure σ profiles. The σ profile of a component needs to be calculated only once.
Aside from the electrostatic interactions, other intermolecular forces such as dispersive
and repulsive interactions and hydrogen bonds also occur in fluid mixtures. They are
merged in an energy concept within the COSMO-RS model. As a result, a COSMO-
RS calculation provides the chemical potential or the activity coefficient of component
i in the mixture. For further details, see the publications of Klamt and co-workers.
9

2.6 Computational details
The standard procedure that is also described in more details in [38,39] was applied
for COSMO-RS calculations. Quantum chemical COSMO calculations were performed
for all esters, except from vinyl acetate, which were not included in the database from
COSMOlogic GmbH & Co KG, Leverkusen, Germany. Vinyl acetate and water
conformers were obtained from the COSMOlogic database. The structures of the
various esters were designed using HyperChem Professional (Version 8.0) and were
initially optimized using the Geometry Optimization tool of the same program. The
quantum chemical COSMO calculations were then performed on the density functional
theory (DFT) level, utilizing the BP functional [40–42] with RI (resolution of identity)
approximation and a triple-ζ valence polarized basis set (TZVP) [43,44]. All structures
were fully optimized using the TURBOMOLE program package. Solubilities were
predicted at 30 oC with the COSMOtherm program (Version C2.1 Release 01.11).
Partition coefficients (K) of tyrosyl esters in the biphasic system of water and vinyl
ester were predicted with the following equation:
ꢃ
K ꢀ xꢁꢂ xꢁ (1)
⁄
w
where xp is the molar fraction of the product in aqueous phase (xp ) and in its
E
corresponding vinyl ester phase (xp ). Using the standard thermodynamic equation of
liquid-liquid equilibrium, and since the solubility of water in the vinyl ester phase and
vice versa is very low, the distribution ratios were approached by the ratio of the activity
coefficients at infinite dilution, which were calculated with COSMOtherm, according
to the following equation:
ꢉ,ꢌ
ꢇ
ꢈ,ꢌ
ꢊ
ꢇ
ꢈ
ꢉ
ꢇ
ꢊ
ꢆ
ꢊ
ꢇ
xꢁꢂγꢂꢁ ꢀ xꢁꢄγꢄꢁ ⇒ ꢅ ꢀ ꢀ ꢊ
ꢋ
(2)
ꢉ
ꢈ
ꢆ
ꢇ
ꢇ
10

w
where γp is the activity coefficient of the product in water (γp ) and its corresponding
E
∞
vinyl ester (γp ) and γp is the activity coefficient of the product at infinite dilution in
water (γpw,∞) and the corresponding vinyl ester (γpE,∞). Initially, a liquid-liquid
equilibrium (LLE) calculation was performed for each water/acyl donor binary mixture
at 30 οC using COSMOtherm
2.7 Solubility and partition coefficient predictions with UNIFAC
For comparison purposes, the group contribution UNIFAC model [45] was also
applied in the prediction of solubilities and partition coefficients. The basis of UNIFAC
method is to consider molecules as an aggregation of functional groups. This approach
offers the great advantage of being able to describe a virtually infinite number of
different compounds with a small number of parameters. UNIFAC has two important
drawbacks. The first is that UNIFAC predictions are independent of the location of the
functional groups and cannot account for the effect that one group has to another if they
are close in the molecule (proximity effect). As a result, the identification of chemical
groups is independent of the intramolecular environment. Moreover, any kind of
contact between two groups in UNIFAC is associated with the same energy,
independent of their individual locations and directions. Despite these drawbacks
UNIFAC is widely applied due to its broad parameterization and its high calculation
speed.
In this work the UNIFAC-LLE model [46] has been used since it has been
especially developed for liquid-liquid equilibrium predictions. Solubility and partition
coefficient predictions with UNIFAC were performed in the same way as for COSMO-
RS.
11

2.8 Evaluation of the thermodynamic models by experimental data
For the evaluation of the predictions emanated from the thermodynamic models,
distribution experiments were performed for the binary (water-vinyl propionate, water-
vinyl butyrate) and tertiary systems (water-vinyl propionate-TyC3, water-vinyl
butyrate-TyC4). The amount of vinyl and tyrosyl esters was calculated by HPLC, using
reference curves constructed by standard concentrations of these esters. The amount of
water in the two vinyl esters was measured by the Karl-Fischer titration method, using
a TitroLine KF titrator.
3. Results
3.1 Effect of acyl donor chain length on the reaction yield
Except from acylating tyrosol with vinyl butyrate (C4), other vinyl esters were also
used as acyl donors in order to study the effect of the chain length on the reaction yield.
As shown in Figure 1, tyrosol was acylated with various fatty-acid vinyl esters in order
to determine the specificity of FoCut5a for the different acyl donors. The conversion of
short-chain vinyl esters (C2, C3, C4) is relatively high (over 30%) in contrast to the
medium-chain esters (C10, C12), which resulted to c.a. 1% tyrosyl ester production. The
highest yield was achieved using vinyl butyrate as acyl donor (41.8%), followed closely
by vinyl propionate (38.6%) (Table 1). To our knowledge, this is the first report so far
for the enzymatic synthesis of TyC4 that was preferentially synthesized by FoCut5a,
therefore we proceeded to the optimization of this esterification reaction.
12

3.2 Study of various parameters in the yield of TyC4 synthesis
3.2.1 Effect of enzyme concentration
In most industrial applications of biocatalysis, the enzymes prove to be a limiting
factor. Increasing the amount of enzyme means increase of initial rate and completion
of the reaction in a shorter period, while rising of the process cost. In the
transesterification reaction for the production of TyC4, while the initial reaction rate
(mM h-1) increases with the addition of more enzyme (data not shown), the initial rate
per enzyme amount (mM h-1 mgenzyme-1) reaches a peak for 2.4 μg enzyme mL-1 and is
low for 25 μg mL-1 (Fig. 2). In addition, the conversion yield for 5 μg enzyme mL-1 is
maximum (29.5% after 4 h), while the specific initial rate is not the highest. Considering
the conversion yield as the most crucial parameter, 5 μg FoCut5a mL-1 of reaction was
used for further experiments.
3.2.2 Effect of reaction pH
Typically, transesterification reactions take place in organic solvents and are
performed by immobilized enzymes. Since this synthetic reaction is catalyzed by a free
enzyme in an aqueous medium, it is necessary to investigate the effect of pH on the
acylation yield. Optimum reaction environment proved to be phosphate-citrate pH 7.0
buffer (Fig. 3A). The reaction seemed to be favored in alkaline pH, in which the control
reactions were also high. Cutinase could act in a wide range of pH (6-10) with a good
yield (more than 75% of the optimum) as seen in Fig. 3.
3.2.3 Effect of tyrosol concentration
Under standard reaction conditions (30 οC; pH 7.0; 5 μg enzyme mL-1) the effect
of tyrosol concentration on the initial reaction rate and the TyC4 formation was studied.
13

The results (Fig. 4) were fitted to the Michaelis-Menten model using the GraphPad
Prism 5 software (R2=0.9989). The kinetic constants were calculated as follows:
Km=119.2±12.6 mM, kcat=134.6±9.8 s-1 and kcat/Km =1128.9±144.9 s-1 M-1 respectively.
The maximum conversion yield (40.6%) is achieved with 12.5 mM initial tyrosol
concentration.
3.2.4 Effect of reaction temperature
FoCut5a exhibits highest hydrolytic activity at 40 οC, however at that temperature,
intense enzyme deactivation from the first minutes takes place [25]. Under this view,
we carried out transesterification reactions at different temperatures in order to optimize
the tyrosol conversion yield. This set of reactions was performed under optimal
conditions (pH 7, 12.5 mM Ty concentration, 5 μg mL-1 enzyme loading). As seen in
Fig. 3B, the initial reaction rates at temperatures 15 οC and 20 οC are almost identical
and very low compared to higher temperatures. There is a great leap between
temperatures 20 οC and 25 οC, after which the initial reaction rate increases in a nearly
linear fashion. The maximum yield is observed at 20οC after 4 h (60.7%), which is very
close to the yield achieved at 15 οC after 5 h (58.4%).
3.3 Prediction of mutual solubilities of vinyl esters and water
The solubility of the acyl donor in the water phase and vice versa may play a very
crucial part in the reaction yield, as the enzyme’s accessibility at this reactant may
depend on it. In Table 1, the solubilities of the acyl donors in water and vice versa, as
predicted from the COSMO-RS and UNIFAC models are presented. It can be observed
that increase of the vinyl ester’s carbon-chain length decreases its solubility in water
with medium-chain vinyl esters (decanoate and laurate) showing very low solubility, in
14

the order of 10-8 or 10-9. Similarly, increase of the vinyl ester’s carbon-chain length
decreases the solubility of water in it. However, this effect is much weaker than the one
observed for the solubility of the vinyl esters in water (Table 1). COSMO-RS and
UNIFAC yield similar and satisfactory mutual solubility predictions, with UNIFAC to
be slightly better than COSMO-RS.
3.4 Prediction of tyrosyl esters’ distribution in the reaction medium
The mode of action of FoCut5a in this particular reaction system may be explained
by understanding the behavior of the esters produced in each case. Both thermodynamic
models (COSMO-RS and UNIFAC) were used for the prediction of the partition
coefficients of all tyrosyl esters produced experimentally. The comparison of the
models’ predictions with the experimentally measured values for TyC3 and TyC4
indicates that COSMO-RS yields satisfactory results, while UNIFAC strongly
underestimates the experimental data.
As seen in Table 2, products are distributed in the acyl donor phase in all cases
(K<1). Additionally, the increase of the tyrosyl ester’s chain-length increases its
presence in the acyl donor phase, making it scarcer in the aqueous phase. Exception to
this is the COSMO-RS prediction for the TyC3, which shows the highest distribution in
the aqueous phase compared to the other tyrosyl esters. UNIFAC model predicts the
decrease of the distribution of products (with increasing chain-length) almost linearly.
4. Discussion
Tyrosol is the main phenolic compound found in olive oil, exhibiting biological
activity. It is also present in the olive oil production by-products (leaves and mill
wastewaters [2,3]) making it a low-cost, easy to access antioxidant, especially in
15

countries with high olive oil production such as Spain, Italy and Greece (97% of the
2014-2015 EU production according to the International Olive Oil Council-IOOC or
77% of the 1997-2000 world-wide production [47]). It has been suggested that
hydrophobic derivatives of natural antioxidants exhibit higher antioxidant activity
against LDL oxidation than their respective precursors [20,48]. Tyrosol’s
monoacetylated derivatives are more effective inhibitors of rabbit platelet aggregation
(which causes atherosclerosis), compared to tyrosol itself, rendering them a more potent
protection agent against cardiovascular disease [7]. Moreover, although tyrosol does
not display any antimicrobial activity, medium-chain tyrosyl esters (TyC8, TyC10,
TyC12) inhibit the growth of several bacterial strains (Staphyloccocus aureus,
Staphyloccocus xylosus, Bacillus cereus, Bacillus flavum) and two leishmanial strains
(Leishmania. major and Leishmania infantum) [33].
Fatty acid tyrosyl esters have shown to possess sufficient surfactant properties,
which adds them to the short list of surfactant-antioxidants [49]. Being such, they can
be used in the food industry as antioxidants, in oil-water emulsions or oil matrices.
Lipoic acid tyrosyl ester was tested as antioxidant in fish-oil water emulsion system,
resulting in 50-60% inhibition of oxidation [50]. On the contrary, tyrosol appeared to
be a more adequate antioxidant in olive oil, than its esters that were slightly less
effective [51]. This can be explained by the so called “polar paradox”, which states that
hydrophilic molecules are more effective as antioxidants in the bulk of oil, while
lipophilic are in oil-water systems [52].
FoCut5a was able to catalyze the transesterification of tyrosol with various fatty
acid vinyl esters in a water medium saturated with the corresponding vinyl ester.
Kremnický et al. (2004) were the ones who discovered a water-based reaction system,
16

which allowed a crude lyophilized preparation of Trichoderma reesei, exhibiting acetyl
esterase activity, to catalyze the acetylation of methyl β-ᴅ-xylopyranoside, using vinyl
acetate as acetylating donor [53]. That also proved to be an equilibrium-controlled
reaction. The same reaction system has been then used for acetylation of mono-, di- and
oligosaccharides by several purified acetyl esterases [54,55]. To the authors’
knowledge, this is the first time where an aqueous biphasic reaction system was
employed for the acylation of a biological active compound catalyzed by a cutinase,
while describing the thermodynamic distribution of reactants and products.
In our study, we firstly investigated the effect of the acyl donor’s (vinyl ester)
chain-length on the production yield. The maximum conversion was observed for vinyl
butyrate, followed closely by vinyl propionate. Even though cutinases usually show
maximum hydrolytic efficiency for the C2 or C4 esters [24], little is known about the
effect of the carbon-chain length on the synthesis kinetics. F. solani pisi cutinase shows
to best utilize butyric acid (C4) for the synthesis of ethyl esters [27] and similarly B.
cepacia cutinase prefers the C4 alcohol for the esterification of butyric acid [26]. In both
cases substrates with three carbon atoms have not been tested. Concerning the
hydrolytic activity, FoCut5a shows maximum efficiency (kcat/Km) for the hydrolysis of
C4 esters [25], however, there is no data reported for C3 substrates. As noted from the
experimental data, medium carbon-chain vinyl esters (C10 and C12) were difficult for
FoCut5a to utilize in this system. This can be attributed to the fact that cutinases show
low efficiency for the medium carbon-chain esters both in hydrolysis [24] and synthesis
[26,27]. Moreover, according to the COSMO-RS predictions, vinyl decanoate and
laurate have almost zero solubility in water, where the enzyme has access and probably
this is an additional reason why the production yields for the respective tyrosyl esters
are low.
17

As previously reported [25], although FoCut5a shows its highest hydrolytic
activity at 40 οC, the highest yield for the transesterification reaction was achieved at
20 οC. This could be possibly explained by the low thermal stability of this enzyme at
temperatures exceeding 30 οC. While maximum conversion was obtained at 20 οC, the
highest initial reaction rate was achieved at 35 οC. The room-temperature esterification
reaction mediated by FoCut5a, results in an environmentally friendly process with
minimal requirements in terms of energy consumption. In addition, the optimal enzyme
loading was found to be 5 μg mL-1, which is very low, while being very efficient at a
low temperature, meaning that the cost of the enzyme does not constitute a limiting
factor to the process.
Commercial F. solani cutinase (30 mg mL-1) has also been used in lipophilization
ο
reactions of various phenolic acids, which were esterified with 1-octanol at 45 C in
tert-butanol. At that temperature the initial reaction rate was 15.2 μmol h-1 genzyme-1 [28],
more than 105 times lower compared to the one of FoCut5a at 20 οC, which resulted to
maximum conversion of 29% after 12 days. The same enzyme expressed in S.
cerevisiae and lyophilized (1.4 mg mL-1), synthesized ethyl esters at 30οC in isooctane.
-1
The maximum initial rate observed was 1.15 μmol min-1 mgenzyme for ethyl butyrate
and the maximum conversion was 97% for ethyl valerate after 6 h [27]. The initial rate
succeeded in our reaction system under optimal conditions was 28 times higher than
the one observed for that enzyme. Wild-type B. cepacia cutinase (2.6 mg mL-1) was
used for the synthesis of alkyl esters in isooctane. Butyl butyrate synthesis reached the
maximum conversion yield (95%) after 12 h at 37 οC [26].
When it comes to the synthesis of lipophilic tyrosyl esters several studies were
previously reported all of them performed in organic solvents, exploiting immobilized
18

lipases as catalysts. For example, the transesterification of tyrosol with ethyl acetate in
ο
hexane using a non-commercial lipase from Staphylococcus xylosus, (54 C, 500 UI
lipase) was employed, which led to 95% yield after 48 h [56]. The direct esterification
of tyrosol with several fatty acids has also been investigated, catalyzed by immobilized
non-commercial lipases from Rhizopus oryzae and S. xylosus (1000 UI) and by the
commercial lipase Novozyme 435® (5 mg mL-1) [33,57]. The reactions were carried
out at 45 οC in 2-methyl-2-propanol/hexane for 120 h (non-commercial enzymes) or 72
h (Novozyme 435®). R. oryzae lipase exhibited maximum conversion yield for the
tyrosyl laurate ester (70%), while S. xylosus and C. antarctica lipases for the tyrosyl
acetate (95 and 99.7%, respectively). The novel reaction system proposed in this study
does not result in such high production yields, although the employed enzyme amount
is much less (1000 times compared to Novozyme 435®) and the reaction is completed
in 4 hours. According to our knowledge, tyrosyl butyrate (TyC4) had not been
synthesized enzymatically before.
Concerning the thermodynamic studies, it was observed that the reaction yields can
be generally well correlated with the solubilities of the vinyl esters in water, i.e. the
decrease in the solubility leads to decrease in the reaction yield, as well as with the
distribution ratios of the products (tyrosol esters) in the biphasic reaction medium, i.e.
the decrease in the distribution ratio leads also to decrease in the reaction yield. The
solubilities of vinyl esters in water can be satisfactorily predicted by both UNIFAC and
COSMO-RS, while for prediction of the distribution ratios only COSMO-RS gives
satisfactory results.
In the presence of water an amount of the ester produced inevitably gets
hydrolyzed. We propose that the reaction takes place in the aqueous phase or the water-
19

oil interphase, where the enzyme has access. According to our thermodynamic study
the majority of the product is transferred in the acyl donor phase (vinyl ester)
immediately after its production, due to its low solubility in water, so it gets protected
from further hydrolysis. We believe this is the main reason why this reaction keeps
moving towards the synthesis, until equilibrium is reached. Reaction mixtures were
monitored for 24 h in preliminary experiments (data not shown), but it was observed
that the reactants and products reached an equilibrium state after only a few hours (4
h).
Overall, this novel reaction system presented for the production of lipophilic
tyrosyl esters, especially tyrosyl butyrate, can prove attractive for industrial utilization,
since it is an environmental friendly process compared to the existing synthetic
reactions, employing mild conditions in an aqueous medium. In addition, the cost of
the enzyme that usually proves to be a limitation in biocatalytic reactions, would be
marginal due to low amount needed. Furthermore, no heating or cooling steps are
needed prior to the reaction as it is practically performed at room temperature. Another
advantage is that the product could be easily collected from the acyl-donor phase free
of enzyme. The major shortcoming of this process is the use of activated donors
requiring a chemical synthetic step to synthesize the corresponding vinyl esters prior to
the enzymatic transesterification reaction. Moreover, the use of an immobilized
cutinase may reduce the cost of the enzyme through the recycling of biocatalyst,
therefore the immobilization of FoCut5a will be further explored.
5. Conclusions
The broad applications of tyrosyl esters demand a sustainable and environmentally
friendly production that could be provided by Biocatalysis. The discovery of novel
20

biocatalysts is fundamental, aiming in a decrease of bioconversion cost utilizing mild
reaction conditions, whereas diminishing the reaction byproducts and waste. In the
present investigation, the potential of F. oxysporum cutinase for the transacylation of
tyrosol in an aqueous medium was studied, while the reaction conditions were
optimized. For the synthesis of short-chain fatty-acid tyrosyl esters, a reaction system
primarily consisting of water and a vinyl ester as acyl donor was suggested. The
enzymatic-mediated reaction reached a satisfactory yield of 61% in 4 h, employing a
miniscule amount of biocatalyst (5 μg FoCut5a mL-1 of reaction) under mild conditions
of pH and temperature (7.0 and 20 oC, respectively). Due to its high water content the
reaction system used is very attractive, as the enzyme and the corresponding product
are protected in the aqueous and vinyl ester phase, respectively. The reactants’ and
product’s solubilities and their distribution in the reaction biphasic system was
investigated by the use of COSMO-RS and UNIFAC thermodynamic models, aiming
in understanding the synthetic behavior of this reaction system catalyzed by FoCut5a
cutinase.
Acknowledgements
The authors are grateful to Dr. Nikolaos Fokialakis (Faculty of Pharmacy, National and
Kapodistrian University of Athens) for the NMR runs.
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Figure captions
Fig. 1 Schematic depiction of the transesterification reaction of tyrosol with various
vinyl esters differing in their carbon-chain length
Fig. 2 Effect of the concentration of added enzyme on conversion yield (●) and initial
rate (○). Reactions were performed in phosphate-citrate pH 7 buffer, with 50 mM
tyrosol for 4 h at 30 οC.
Fig. 3 Effect of reaction pH (A) on conversion yield after 24 h and temperature (B) on
conversion yield (●) and initial rate (○). Reactions were performed in phosphate-citrate
pH 7 buffer, 12.5 mM tyrosol with 5 μg enzyme mL-1 for 4 h (optimal conditions)..
Fig. 4 Effect of tyrosol concentration and on the conversion yield (●) and initial rate
(○). Reactions were performed in phosphate-citrate pH 7 buffer, with 5 μg enzyme mL-
1 for 3 h at 30 οC.
27

Fig 1
Fig 2
28

Fig 3
Fig 4
29