Laccase-catalyzed dimerization of ferulic acid amplifies antioxidant activity

Article abstract of DOI:10.1016/j.molcatb.2011.08.010

Enzymatic modification can be used to enhance the bioactive properties of phenolic compounds. The present study employed laccase from Trametes pubescens to catalyze the modification of ferulic acid in a monophasic or biphasic system, as a way of enhancing

Full text of DOI:10.1016/j.molcatb.2011.08.010

Journal of Molecular Catalysis B: Enzymatic 74 (2012) 29–35
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Laccase-catalyzed dimerization of ferulic acid amplifies antioxidant activity
Oluyemisi E. Adelakuna , Tukayi Kudangaa,∗ , Ayesha Parkera , Ivan R. Greenb , Marilize le Roes-Hilla ,
Stephanie G. Burtona
a Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, Bellville Campus, Symphony Way, P.O. Box 1906, Bellville 7535, Cape Town, South
Africa
b Chemistry Department, University of the Western Cape, Modderdam Road, Private Bag X17, Bellville 7530, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2011
Received in revised form 2 August 2011
Accepted 26 August 2011
Available online 6 September 2011
Enzymatic modification can be used to enhance the bioactive properties of phenolic compounds. The
present study employed laccase from Trametes pubescens to catalyze the modification of ferulic acid in
a monophasic or biphasic system, as a way of enhancing its antioxidant capacity. Two dimeric products
(m/z 385.1) were purified and characterized as the ˇ-5 and ˇ–ˇ dimers. In the monophasic system, the
ˇ-5 dimer was preferentially formed in dioxane while the ˇ–ˇ dimer formation was enriched in ethanol
as co-solvent. In the biphasic system, formation of the dimers increased as the concentration of ethyl
acetate was increased from 80% to 95%. The ˇ-5 dimer showed higher antioxidant capacity than the
substrate as demonstrated by standard antioxidant assays (DPPH and TEAC). These results demonstrate
that alteration of reaction conditions influences the laccase-mediated oxidation of ferulic acid to form
dimers with higher antioxidant capacity than the substrate.
Keywords:
Ferulic acid
Laccase
Antioxidant activity
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
been reported to also increase sperm viability, lower cholesterol in
serum and liver and protect against angina, hypertensive and coro-
Plant phenols and phenolic acids are increasingly becoming
a subject of intensive research due to their bioactive properties
which include antioxidant, anti-mutagenic, anti-viral and anti-
inflammatory activities [1–4].
nary diseases [19]. However, it has been most widely investigated
for its antioxidant properties which are important in the preven-
tion of lipid oxidation in food and also in the putative prevention of
free-radical-induced diseases such as cancer and atherosclerosis,
or aging caused by oxidative tissue degeneration [20,21]. Antioxi-
dant properties are ascribed to its structure (Fig. 1) and specifically
to its phenolic nucleus coupled to an extended conjugated side
chain which facilitate the formation of a resonance-stabilized phe-
noxy radical [22]. Other important structural properties include
electron-donating groups on the benzene ring (3-methoxyl and,
more importantly, 4-hydroxyl) which give the additional prop-
erty of terminating free radical chain reactions; the carboxylic acid
group with an adjacent unsaturated C–C double bond which can
provide additional attack sites for free radicals thus preventing
them from attacking membranes; and the carboxylic acid group
which acts as an anchor group which binds it to the lipid bilayer,
providing some protection against lipid peroxidation [20]. Despite
having these attractive properties, the antioxidant capacity of fer-
ulic acid is generally low compared to conventional antioxidants
and other hydroxyl-cinnamic acids [23,24]. Sánchez-Moreno et al.
[25] also reported that ferulic acid has a low free radical scavenging
activity, but showed higher activity in inhibition of lipid oxidation
and in the protection against hydroxyl and peroxyl radical oxida-
tion in synaptosomal and neuronal cell culture systems in vitro [26].
Recently there have been a number of attempts to enzymatically
modify phenolic molecules as a way of improving antioxidant prop-
Among the phenolic acids, ferulic acid, 3-(4-hydroxy-3-
methoxy-phenyl)-acrylic acid, is the most abundant hydroxycin-
namic acid in the plant world [5], constituting 5 g kg−1 in wheat
bran, 9 g kg−1 in sugar beet pulp [6,7], 15–28 g kg−1 of rice bran
oil [8] and 25 g kg−1 in corn kernel [9]. It is one of the major
phenolic lignin monomers found in woods and grasses, and is
widely distributed in cereals, fruits and vegetables [10,11]. Some
of these sources are used to provide ferulic acid as a substrate for
conversion into value added chemicals such as guaiacol, vanillin,
vanillic acid and protocatechuic acid [12]. Ferulic acid is found
as the free acid, low molecular weight conjugates, esters with
cell wall heteroxylans, or covalently bound to lignin and other
biopolymers. Ferulic acid is one of the active ingredients of many
Chinese traditional medicines used in the prevention and treat-
ment of various diseases [13–15]. It shows many physiological
functions, including antioxidant, antimicrobial, anti-inflammatory,
anti-thrombosis, and anti-cancer activities [16,17] and can be eas-
ily absorbed and metabolized in the human body [18]. It has
∗
Corresponding author. Tel.: +27 21 953 8497; fax: +27 21 953 8494.
E-mail addresses: tikudanga@yahoo.co.uk, kudangat@cput.ac.za (T. Kudanga).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.08.010

30
O.E. Adelakun et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 29–35
Fig. 1. Structure of ferulic acid showing the important features for antioxidant activity.
erties. Laccases are among the enzymes that are currently being
investigated.
reaction mixture contained ferulic acid (10 mM), laccase (10 U) in
100 mM sodium acetate buffer (pH 5.0) and ethyl acetate at various
concentrations (80, 85, 90, 95 and 96%, v/v). For the monopha-
sic system the miscible solvents were used at 80% (previously
determined as the optimum for product yield and reduced side
reactions). The reactions were carried out for 24 h at 28 ◦C with
shaking on an orbital shaker at 180 rpm. The reactions were moni-
tored by Thin Layer Chromatography (TLC) and High Performance
Liquid Chromatography (HPLC) as described below (Sections 2.4
and 2.5).
Laccases (EC 1.10.3.2) are enzymes that are capable of catalyzing
the one-electron oxidation of phenols to produce phenoxy rad-
icals and concomitantly reduce molecular oxygen to water [27].
Oligomerization or polymerisation of the radicals to form oligomers
or polymers with higher antioxidant properties than the start-
ing materials has been reported [28–31]. Consequently, research
interest in enzymatically transforming ferulic acid is now increas-
ing with some researchers having reported the transformation
of ferulic acid by laccase [32–34]. Although possible structures
of the oxidation products have been proposed [33], none of the
researchers has conclusively elucidated the structures nor reported
the antioxidant capacity of the products. This prompted us to inves-
tigate the modification of ferulic acid by laccase produced from
Trametes pubescens (strain CBS 696.94) as a way of enhancing the
antioxidant capacity of ferulic acid. To the best of our knowledge,
our work for the first time conclusively elucidates the structure of
two dimers formed from laccase-mediated oxidation of ferulic acid
in organic media, one of which has higher antioxidant capacity than
the substrate.
2.4. Chromatographic separation of reaction products
TLC analysis was performed on aluminium-backed silica gel 60
F254 (Merck) plates using toluene: dioxane: acetic acid (10:2.5:0.2,
v/v/v) or ethyl acetate: dioxane: acetic acid (6:0.2:0.05, v/v/v) as the
mobile phase. The compounds were then visualized by exposure to
UV light at 254 nm.
2.5. High performance liquid chromatography (HPLC)
Prior to HPLC analysis, the enzyme was precipitated out of the
reaction solution by the addition of an equal volume of ice-cold
methanol (only when miscible solvents were used; in biphasic sys-
tem, the enzyme was readily separated from the product). The
mixture was incubated at 0 ◦C for 20 min and then centrifuged
at 0 ◦C for 15 min at 14,000 × g. The supernatant (1.5 mL aliquots)
was transferred into clean vials and analyzed by HPLC. HPLC anal-
ysis was carried out using a Hitachi LaChrom HPLC system from
Merk (Merck, Hitachi, Germany). Separation of the reaction prod-
ucts was carried out on a reversed phase LUNA 5 ␮ PFP(2) 100A,
250 mm × 4.60 mm column under isocratic conditions using ace-
tonitrile: water: acetic acid (25:75:0.1, v/v/v) as the mobile phase
at a flow rate of 1 mL min−1, with 1 h running time. The products
were detected at 270 nm. Alternatively gradient elution using 0.1%
formic acid (solvent A) and acetonitrile (solvent B) was used in
order to reduce the running time to 23 min. The gradient set up was
as follows: 98% A to 0% A (20 min); 0% A to 98% A (20–21 min); 98%
A (21–23 min). Peaks were analyzed using HPLC Manager, Merck
Hitachi model D 700 data software.
2. Materials and methods
2.1. Chemicals and enzyme
Ferulic acid and other chemicals were purchased from
Sigma–Aldrich, South Africa. An airlift reactor was used to grow
T. pubescens (strain CBS 696.94) for the production of laccase [29]
which was purified according to the method of Ryan et al. [35].
2.2. Enzyme activity
Laccase activity was determined spectrophometrically by
monitoring the oxidation of 2,2ꢀ-Azinobis (3-ethylbenzthiazoline-
6-sulfonic acid) (ABTS, ε420 = 36,000 M−1cm−1) as the substrate
[36]. The reaction mixture contained 0.330 mL ABTS (5 mM), 2.5 mL
0.1 M sodium acetate buffer (pH 5.0) and 0.17 mL laccase. Oxidation
of the ABTS was monitored by measuring the increase in absorbance
at 420 nm. One unit of laccase activity was defined as the amount
of enzyme required to oxidise 1 ␮mol of ABTS min−1 at 25 ◦C.
2.6. Purification of reaction products
2.3. Oxidation of ferulic acid
The reaction products were purified by Flash Chromatography.
The miscible solvents containing products were evaporated using
a rotary evaporator and the product extracted with ethyl acetate
followed by separation using a separation funnel. The aqueous
phase was washed twice and monitored for the absence of product.
The oxidation reactions were carried out either in a biphasic
system comprising buffer with ethyl acetate as co-solvent or in
a monophasic system with miscible solvents (dioxane, methanol,
ethanol or acetone) as co-solvents. For the biphasic system the

O.E. Adelakun et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 29–35
31
The organic phase was dried using a rotary evaporator (Heidolph,
Germany). For the biphasic system, the organic phase was sep-
arated using a separation funnel and the aqueous phase washed
twice with ethyl acetate. The organic phase was evaporated under
reduced pressure with a rotary evaporator and the crude residue
purified by silica Flash Chromatography using ethyl acetate: diox-
ane: acetic acid (6:0.2:0.05,v/v/v) as mobile phase for purifying
product 1 (P1) or toluene: dioxane: acetic acid (10:2.5:0.2, v/v/v)
as mobile phase for purifying product 2 (P2). The pure fractions
were dried using a rotary evaporator and the products sequentially
washed with acetone, methanol and then acetone again to remove
the acetic acid.
2.8.2. TEAC (Trolox equivalent antioxidant capacity) assay
The ABTS radical scavenging activity of ferulic acid and the two
products were determined according to the method described by
Re et al. [38]. The trolox equivalent antioxidant capacity (TEAC)
method is based on the ability of antioxidant molecules to quench
ABTS•+, a blue–green chromophore with characteristic absorption
at 734 nm, compared with that of Trolox, a water soluble vitamin E
analog. The addition of antioxidants to the preformed radical cation
decolourizes the ABTS•+ as it is reduced to ABTS. ABTS•+ solution
was prepared 12–16 h before use by mixing ABTS salt (7 mM) with
potassium persulfate (2.45 mM) and then stored in the dark until
the assay was performed. The ABTS•+ solution was diluted with
methanol to give an absorbance of 0.70 0.002 at 734 nm. Each
sample (100 ␮L) prepared at different concentration was mixed
with 1100 ␮L ABTS•+ solution and the absorbance was read after
30 min incubation at 25 ◦C.
2.7. Characterization of products
The purified products (P1 and P2) were characterized by mass
spectrometry and nuclear magnetic resonance (NMR) analysis.
3. Results and discussion
2.7.1. Liquid chromatography–mass spectrometry (LC–MS)
LC–MS was performed on a Dionex HPLC system equipped with
a binary solvent manager and autosampler coupled to a Brucker
ESI Q-TOF mass spectrometer. The products were separated using
the same linear gradient of acetonitrile (solvent B) and 0.1% formic
acid (solvent A) as described in Section 2.5 above, at a flow rate of
1 mL min−1, using an injection volume of 10 ␮L and an oven tem-
perature of 30 ◦C. MS spectra were acquired in negative mode and
electrospray voltage was set to +3500 V. Dry gas flow was set to
9 L min−1 with a temperature of 350 ◦C and nebulizer gas pressure
was set to 35 psi.
3.1. Oxidation of ferulic acid in organic solvents
Biocatalysis is conducted in organic media for a number of rea-
sons, mainly poor solubility of some compounds in aqueous media;
ability to carry out new reactions which are impossible in water
because of kinetic or thermodynamic restrictions; relative ease of
product recovery from organic solvents as compared to water; and
the insolubility of enzymes in organic media, which permits their
easy recovery and reuse (thus eliminating the need for immobi-
lization) [39,40]. Ferulic acid is not soluble in aqueous media, thus
organic solvents were employed for this study in either a monopha-
sic or biphasic system. Both the biphasic system and monophasic
systems produced three main oxidation products which were des-
ignated as P1 (tR = 10.535); P2 (tR = 12.202); P3 (tR = 13.446) (Fig. 2).
LC–MS analysis of the oxidation products in negative mode showed
dominant signals at m/z 385.1143 (P1) and m/z 385.1077 (P2)
(Fig. 3) which suggested that these oxidation products were dimers
of ferulic acid (exact mass [M] 386.1). When the mass spectrom-
eter was set to run in positive mode [M+H]+ ion signals were
observed at m/z 387.1, while signals at m/z 409.1 indicating Na
adducts, confirmed that the molecules were indeed dimers. LC–MS
results of the product P3 (m/z 579.2154) suggest oligomerization to
form a trimer. In addition, a tetramer (m/z 769.1948; tR = 13.7) was
observed with the more sensitive MS detector (not observed with
HPLC). After purification, the yields of P1 and P2 were 11.190% and
38.189%, respectively. Work is currently underway to purify prod-
uct P3. Significant amounts of insoluble polymeric products were
also observed.
The NMR results indicated that P1 is a dimer of two ferulic
acid monomers covalently bound through a ˇ-5 linkage while the
results for P2 suggest dimerization through a ˇ–ˇ linkage (Fig. 4).
The symmetry of the molecule P2 made assignment easy due
to the signals being clear and separate from each other. In the 1H
NMR spectrum 2-H appeared as a single peak at ı 3.56 while the
methoxy group appeared as a three proton singlet at ı 3.91. The
phenolic OH appeared as a single peak at ı 5.86 in close proximity
to 3-H which appeared as a single peak at ı 5.69. Coupling between
2-H and 3-H was not observed due to the dihedral angle being in
the vicinity of ∼90◦. The aromatic region displayed a typical pattern
of a 1,3,4-trisubstituted system viz., a 2-proton multiplet at ı 6.79
assigned to 2ꢀ-H and 6ꢀ-H while 5ꢀ-H appeared as an ortho coupled
doublet at ı 6.93 with J = 8.8 Hz. In the 13C NMR spectrum all signals
could be assigned according to the structure with C2 at ı 48.5, the
methoxy group at ı 56.3 and C3 at ı 82.0. Three strong signals at ı
107.6, 115.1 and 117.5 were assigned to C2ꢀ, C5ꢀ and C6ꢀ respectively
while C1ꢀ appeared at ı 130.0. The more deshielded carbon signals
2.7.2. Nuclear magnetic resonance (NMR) analysis
Nuclear magnetic resonance (NMR) spectra were recorded using
a VARIAN 200 spectrometer (1H, 200 MHz; 13C, 50 MHz). The
spectra were determined at ambient temperature in deuterated
chloroform (CDCl3) and methanol solutions, with CHCl3 at ı 7.26
for 1H NMR spectra and chloroform (ı 77.00) for 13C NMR spectra
as internal standards. In the NMR spectra, assignments of signals
with the same superscripts are interchangeable. Splitting patterns
are designated as “s”, “d”, “t”, “q”, “m” and “bs”. These symbols indi-
cate “singlet”, “doublet”, “triplet”, “quartet”, “multiplet” and “broad
singlet”.
2.8. Antioxidant activity determination
2.8.1. DPPH (2,2ꢀ-diphenyl-1-picrylhydrazyl) scavenging effect
Antioxidant capacity was determined by measuring DPPH
radical-scavenging activity [37]. Briefly, 3.9 mL of DPPH dissolved in
methanol (0.025 mg/mL) was added to 0.1 mL sample (dissolved in
methanol) at various concentrations. The mixture was shaken vig-
orously and incubated at room temperature in the dark for 60 min,
and the decrease in absorbance at 517 nm determined using a spec-
trometer. The remaining concentration of DPPH in the reaction
medium was then calculated from a calibration curve obtained with
DPPH at 517 nm.
The percentage of remaining DPPH (DPPHR) was calculated as
follows:
ꢀ
ꢁ
(DPPH)T
(DPPH)T=0
% DPPHR
=
× 100
where (DPPH)T is the concentration of DPPH at time 60 min and
(DPPH)T = 0 is the concentration of DPPH at time zero (initial
concentration). The percentage of remaining DPPH against the sam-
ple/standard concentration was plotted to obtain the amount of
antioxidant (mM) necessary to decrease the initial concentration
of DPPH by 50% (EC50).

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O.E. Adelakun et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 29–35
Fig. 2. HPLC chromatogram of products formed during laccase-catalyzed oxidation of ferulic acid. P1, P2 and P3 – oxidation products.
Fig. 3. Mass spectrum of Product P1 (A) and P2 (B) formed during laccase-mediated oxidation of ferulic acid.
Fig. 4. Dimers (P1 and P2) formed during laccase-catalyzed oxidation of ferulic acid.

O.E. Adelakun et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 29–35
33
Fig. 5. Laccase-catalyzed oxidation of ferulic acid to produce ˇ–ˇ and ˇ-5 dimers.
of C3ꢀ and C4ꢀ appeared at ı 146.5 and 147.2. Finally the lactone
carbonyl appeared as expected at ı 175.0.
of an unpaired electron at the ˇ position with another at position
C5 of the next radical yields a ˇ-5 linked dimer. Similarly covalent
coupling of radicals both with unpaired electrons at the ˇ position
produces the ˇ–ˇ linked dimer. While there were several possibil-
ities for coupling due to the large number of mesomeric forms, it
appears that our reaction conditions favored the formation of ˇ-5
and ˇ–ˇ linkages due to the stability of C–C bonds [41] and the
lower heat of formation of ˇ-5 linkages [42]. We have observed
similar findings in which C–C bonds predominated in the coupling
of functional molecules to lignin models, though mostly 5–5 link-
ages were formed due to the absence of unsaturated side chains
[43,44]. In addition, recent reviews agree that in dimerization of
a similar molecule, coniferyl alcohol, the radicals favor coupling
at their ˇ positions, resulting essentially in only the ˇ–ˇ, ˇ-O-4,
and ˇ-5 dimers [45,46] although the ˇ-O-4 are the predominant
linkages in polymerization reactions during lignin synthesis [47].
As expected the spectra were more complicated for P1 due to
a lack of symmetry in the compound and the spectrum was run in
deuteromethanol. The two methoxy groups appeared as 3-proton
singlets at ı 3.83 and 3.91 while 3-H appeared as a sharp doublet at
ı 4.01 (J = 2.6 Hz). A clear doublet at ı 5.61 (J = 2.6 Hz) was assigned
to 2-H. A rather broad multiplet centred around ı 6.70 integrated
for 5 protons and assigned to 6-H, 5ꢀ-H, 1ꢀꢀ-H, 2ꢀꢀ-H and 4ꢀ-OH. A
double doublet at ı 7.01 (J = 8.0 and 2.0 Hz) was assigned to 6ꢀ-H
while the corresponding doublet at ı 7.07 (J = 2.0 Hz) was assigned
to 2ꢀ-H. A downfield doublet at ı 7.57 (J = 1.8 Hz) was assigned to
4-H. The two COOH hydrogen were not observed due to exchange
with protons in the deuteromethanol solvent. The 13C spectrum
was assigned as follows: A broadish signal at ı 55.7 is due to the
two methoxy carbons as well as C3. The signal at ı 81.2 is assigned
to C2. Strong signals in the aromatic region have been assigned as
follows: ı 108.3(C2ꢀ), 112.7(C8), 115.1(C5ꢀ), 118.0(C4), 118.7(C6ꢀ).
The sp2 carbons of the ethylene side chain were assigned as follows:
ı 115.2(C2ꢀꢀ) and 146.3(C1ꢀꢀ). The ring junction carbons C3a and C7a
were assigned as follows: ı 125.5 and 148.9 respectively. C5 and C1ꢀ
were assigned to the signals at ı 125.9 and 131.2 respectively. The
deshielded carbon atoms attached to the oxygens were assigned as:
C7(ı 140.8), C4ꢀ(ı 147.5) and C3ꢀ (ı 148.9). Finally the two carbonyl
signals at ı 169.2 and 172.6 were assigned to the two carboxylic
acid groups.
3.2. Effect of organic solvents
The effect of the nature of the organic solvent and the concentra-
tion of the solvent was studied in order to obtain the best reaction
conditions for product formation.
In the biphasic system, there was an increase in the formation of
P2 (ˇ–ˇ dimer) as the concentration of ethyl acetate was increased
from 80% to 90% after which a decline in product formation was
observed (Fig. 6). A similar pattern was observed during the forma-
tion of P1 (ˇ-5 dimer) except that a decline was observed after the
composition of organic solvent was increased beyond 95% (Fig. 6).
In the monophasic system, four co-solvents namely acetone,
dioxane, ethanol and methanol were investigated. As shown in
Fig. 7 most of the substrate was converted to products in ethyl
acetate: a biphasic system. In the monophasic system, the order of
substrate conversion was ethanol > acetone > dioxane > methanol.
However, for product formation, ˇ–ˇ dimer formation was
Based on the results from LC–MS and NMR, the scheme shown
in Fig. 5 is proposed as the possible reaction pathway for the
dimerization of ferulic acid to form a ˇ-5 and ˇ–ˇ linkage [33].
As depicted in Fig. 5, laccase initiates the reaction by oxidizing the
para-hydroxyl group resulting in a radical. Through resonance sta-
bilization, the unpaired electron can occupy different positions on
the radicalized molecule [27]. The radicals are stabilized through
coupling with other radicals to form dimers. Therefore coupling

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O.E. Adelakun et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 29–35
Table 1
Antioxidant activity of ferulic acid and its dimers.
Molecule
Molecular
EC50 DPPHa
TEACb value
weight
Ferulic acid
P1 (ˇ-5 dimer)
P2 (ˇ–ˇ dimer)
194.18
386.11
386.11
1.837 0.012
1.310 0.016
2.320 0.057
0.682 0.005
0.653 0.006
0.700 0.013
a
EC50 parameter is defined as the concentration (mM) of substrate that brings
about 50% loss of the DPPH• [37]. Values are means SD of three replicate determi-
nations.
b
The Trolox equivalent antioxidant activity (TEAC) of the antioxidant is defined
as the concentration of antioxidant giving the same percentage inhibition of
absorbance of the radical cation (ABTS+) at 734 nm, as 1 mM of Trolox, at specific
points [38].
formation of this product decreased in the order of ethyl
acetate > dioxane > methanol > ethanol > acetone. For the water
miscible organic solvents employed in this study, solvents
with higher polarity index favored the formation of the ˇ–ˇ
dimer: ethanol (5.2) > methanol (5.1) > dioxane (4.8). However, the
reverse was observed for the ˇ-5 dimer: dioxane (4.8) > methanol
(5.1) > ethanol (5.2); only acetone did not follow a particular order
for both dimers. The effect of different solvents on enzyme cataly-
sis allows ‘medium engineering’ in order to modulate and possibly
improve, the regioselective outcome of reactions [48]. However,
some miscible solvents tended to inactivate the enzyme over time.
For example, only 0.12% or 2.8% residual activity was observed
after 3 h when methanol or dioxane was used, respectively, which
explains the poor substrate conversion in these solvents. Therefore,
meticulous optimisation is required in order to convert the sub-
strate quickly within the first few hours; the second phase of the
reaction (resonance stabilisation and coupling) is non-enzymatic.
In contrast, the enzyme was more stable in ethyl acetate which
probably explains the higher conversion of substrate to products. In
related work, Ma et al. [49] reported the enhancement of catechol-
laccase catalysis in less polar organic solvents as compared with
highly polar media, due to the hydrophobicity of the end product.
It should be noted that both dimers are less polar than the substrate
(ferulic acid) as demonstrated by the retention times which were
observed to be longer than that of ferulic acid (Fig. 2).
Fig. 6. Effect of ethyl acetate content on the formation of ˇ–ˇ and ˇ-5 dimers in
a biphasic system with sodium acetate buffer pH 5.0 as co-solvent. All results are
means standard deviation (SD) of three replicate determinations.
facilitated by the use of ethanol as co-solvent followed by
methanol, ethyl acetate, dioxane and acetone. Ethyl acetate,
which was the best solvent for combined product forma-
tion, promoted the formation of the ˇ-5 dimer and product
3.3. Antioxidant activity determination
Antioxidant activities of the synthesised dimers were evaluated
in relation to that of ferulic acid. Interestingly, the DPPH scaveng-
ing effect and Trolox equivalent antioxidant capacity (TEAC) of the
products showed that the ˇ-5 dimer exhibited higher antioxidant
activity than ferulic acid (Table 1). However, the ˇ–ˇ dimer showed
lower antioxidant activity than ferulic acid. The antioxidant activity
of phenolic compounds depends on various structural features such
as the O–H bond dissociation energy, resonance delocalisation of
the phenoxyl radical and the steric hindrance due to bulky groups
substituting hydrogen in the aromatic ring [50]. The increase in
antioxidant capacity of the ˇ-5 dimer could be attributed to an
increase in electron donating groups after dimerization [51] and
the carboxylic acid group with an adjacent unsaturated C–C double
bond which can provide additional attack sites for free radicals [20].
In the ˇ–ˇ dimer, the unsaturated C–C bonds and carboxyl groups
are lost during the dimerization process which could explain the
reduction in antioxidant capacity.
4. Conclusions
This study has, for the first time, resulted in the elucidation of the
structures of two isolated dimers formed during laccase-catalyzed
oxidation of ferulic acid in organic media. By altering the nature of
Fig. 7. Effect of organic co-solvent on laccase-catalyzed oxidation of ferulic acid
to form ˇ-5 and ˇ–ˇ dimers. All results are means SD of three replicate
determinations.

O.E. Adelakun et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 29–35
35
the organic solvent used, it was possible to facilitate the formation
of the ˇ-5 dimer which is more potent than ferulic acid in terms of
antioxidant capacity. This new product may find useful application
in the health and cosmetic industries.
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