p-hydroxycinnamic acids as natural mediators for laccase oxidation of recalcitrant compounds

Article abstract of DOI:10.1021/es8008979

The capabilities of p-coumaric acid (PCA), ferulic acid (FA), and sinapic acid (SA) as laccase mediators are compared in oxidation of industrial dyes and polycyclic aromatic hydrocarbons (PAH). SA behaved as highly efficient mediator in decolorization of dyes, including the recalcitrant Reactive Black 5. This mediating capacity was related to the specificity constant of the enzyme oxidizing this p-hydroxycinnamic acid, which was 16 times higher than for the typical substrate 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). The kinetics of ABTS oxidation by laccase in the presence of p-hydroxycinnamic acids suggested that the stable phenoxyl radical of a SA transformation product acts as laccase mediator. On the other hand, FA and, especially PCA, easily mediated benzo[a]pyrene oxidation, the latter also promoting the oxidation of the more recalcitrant phenanthrene. Phenanthrene transformation by laccase-PCA was enhanced by Tween 80. This fact, together with the detection of TBARS (thiobarbituric acid-reactive-substances) from unsaturated fatty acids, revealed that laccase can also initiate lipid peroxidation reactions in the presence of p-hydroxycinnamic acids enabling oxidation of the most recalcitrant PAH.

Full text of DOI:10.1021/es8008979

Environ. Sci. Technol. 2008, 42, 6703–6709
fact, the so-called laccase-mediator system has been dem-
p-Hydroxycinnamic Acids as Natural
Mediators for Laccase Oxidation of
Recalcitrant Compounds
onstrated to be efficient for degradation of aromatic con-
taminants (5), paper pulp bleaching (6), and pitch control
(7) or dye decolorization (8). Nevertheless, the utilization of
synthetic mediators in industrial processes is hindered by
their high cost and the possible generation of toxic species.
The availability of low cost and environmentally friendly
natural mediators could facilitate the application of laccase-
mediator systems in white-biotechnological processes.
Lignin biodegradation has been described as an “enzy-
matic combustion” (9) wherein enzymatic and radical
reactions result in degradation of nonphenolic aromatic
structures, even by white-rot fungi that produce laccase as
the sole ligninolytic oxidoreductase (10). Contribution of free
radicals from lignin-degradation products as laccase media-
tors might explain this phenomenon. Exploring the mediating
capabilities of naturally occurring phenols is a good starting
point for searching alternative mediators and might also help
to understand the natural process of lignin degradation. The
ability of some lignin-derived phenols, such as acetosyrin-
gone, syringaldehyde, or vanillin, to mediate laccase decol-
orization of dyes (11), oxidation of polycyclic aromatic
hydrocarbons (PAH) (12), and removal of lignin (13) and
recalcitrant lipids (14) from paper pulps has been proved.
p-Hydroxycinnamic acids are precursors of lignin in
vascular plants being also present as extractives or forming
lignin-carbohydrate bridges in grasses (15). p-Coumaric (4-
hydroxycinnamic) acid (PCA) and ferulic (3-methoxy-4-
hydroxycinnamic) acid (FA) are abundant in soil, where they
incorporate to the humic substances (16). Moreover, p-
hydroxycinnamic acids are ubiquitous in plant foods (seeds
and fruits) where they act as antioxidants, e.g., the high
concentration of sinapic (3,5-dimethoxy-4-hydroxycinnamic)
acid (SA) in rapeseed oil has been related to its stability toward
peroxidation (17). Treatment of p-hydroxycinnamic acids
with laccase results in a variety of oxidation and coupling
products depending on their aromatic ring substituents and
the reaction conditions (18, 19).
S U S A N A C A M A R E R O , * , † A N A I . C A Ñ A S , †
P A U L A N O U S I A I N E N , § E R I C R E C O R D , ‡
A N N E L O M A S C O L O , ‡
M A R ´I A J E S Ú S M A R T Í N E Z , † A N D
Á N G E L T . M A R T Í N E Z †
´
Centro de Investigaciones Biologicas, CSIC, Ramiro de Maeztu
9, E-28040 Madrid, Spain, Laboratory of Organic Chemistry,
P.O. Box 55, FI-00014 University of Helsinki, Finland, and
´
Unite de Biotechnologie des Champignons Filamenteux, INRA,
´
Universite Aix Marseille, 163 Avenue de Luminy, CP 925,
F-13288, Marseille 09, France
Received March 31, 2008. Revised manuscript received
May 31, 2008. Accepted June 9, 2008.
The capabilities of p-coumaric acid (PCA), ferulic acid (FA),
and sinapic acid (SA) as laccase mediators are compared in
oxidation of industrialdyes and polycyclic aromatic hydrocarbons
(PAH). SA behaved as highly efficient mediator in decolorization
of dyes, including the recalcitrant Reactive Black 5. This
mediating capacity was related to the specificity constant of
the enzyme oxidizing this p-hydroxycinnamic acid, which was 16
times higher than for the typical substrate 2,2′-azinobis-(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS). The kinetics of ABTS
oxidation by laccase in the presence of p-hydroxycinnamic
acids suggested that the stable phenoxyl radical of a SA
transformation product acts as laccase mediator. On the other
hand, FA and, especially PCA, easily mediated benzo[a]pyrene
oxidation, the latter also promoting the oxidation of the more
recalcitrant phenanthrene. Phenanthrene transformation by
laccase-PCA was enhanced by Tween 80. This fact, together
with the detection of TBARS (thiobarbituric acid-reactive-
substances) from unsaturated fatty acids, revealed that laccase
can also initiate lipid peroxidation reactions in the presence
of p-hydroxycinnamic acids enabling oxidation of the most
recalcitrant PAH.
PCA was included in a preliminary screening of natural
phenols as laccase mediators (11) and yielded promising
results in a recent study on transformation of PAH (12).
Therefore, PCA and their analogues, FA and SA, are compared
here regarding the kinetics of their oxidation by laccase or
other oxidizers (antioxidant activity) and their capabilities
to act as mediators in laccase oxidation of compounds of
environmental interest (such as industrial dyes and PAH)
including initiation of lipid peroxidation reactions for
degradation of the most recalcitrant ones.
Introduction
Production of extracellular peroxidases and laccases confers
white-rot fungi a unique capability to degrade the lignin
polymer in wood, and a variety of recalcitrant contaminants
(1, 2). Laccases are multicopper oxidases produced by most
white-rot basidiomycetes with broad substrate specificity (3).
Moreover, the presence of synthetic redox mediators, like
ABTS (2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid))
or 1-hydroxybenzotriazole (HBT), enhances the oxidative
capabilities of laccases allowing the oxidation of high-redox
potential aromatic compounds (4). This fact together with
the use of oxygen as electron acceptor, make laccases highly
interesting for industrial and environmental applications. In
Materials and Methods
Chemicals. PCA, FA, SA, HBT, 2,6-dimethoxyphenol (DMP),
Acid Blue 74, Azure B, benzo[a]pyrene, phenanthrene, linoleic
acid, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxy-
lic acid (Trolox) were obtained from Sigma-Aldrich. ABTS
was supplied by Roche, and Reactive Black 5 by Dye Star
(Frankfurt, Germany). Syringaresinol (3,6-bis-(4-hydroxy-3,5-
dimethoxyphenyl)-tetrahydrofuro[3,4-c]furan) was synthe-
sized by oxidative ꢀ-ꢀ’coupling of sinapyl alcohol in a “zulauf”
dehydrogenation reaction catalyzed by horseradish peroxi-
dase in the presence of hydrogen peroxide. Isolation of the
dimeric product (with 25% yield) was performed using
Biotage flash-chromatography equipment, and purity was
checked by NMR.
* Corresponding author phone: 34 918373112; fax: 34 915360432;
e-mail: susanacam@cib.csic.es.
Antioxidant Activity of p-Hydroxycinnamic Acids. The
capacity of p-hydroxycinnamic acids for scavenging the ABTS
cation radical (ABTS•+) was measured using the TEAC (Trolox
† Centro de Investigaciones Biolo´gicas, CSIC.
§ Laboratory of Organic Chemistry University of Helsinki.
‡ Unite´ de Biotechnologie des Champignons Filamenteux, INRA.
9
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 2008 American Chemical Society
VOL. 42, NO. 17, 2008
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ENVIRONMENTAL SCIENCE & TECHNOLOGY 6703
Published on Web 07/23/2008

equivalent antioxidant capacity) assay (20). Reactions were
carried out in ethanol, phosphate buffer saline (PBS, pH 7.4)
and sodium citrate (pH 5). ABTS•+ was generated by adding
potassium persulfate to ABTS, diluted till 0.7 absorbance at
734 nm, 10 µL of each compound (1.25 mM) were added to
990 µL, and the decrease of absorbance (734 nm) after 6 min
reaction was referred to that obtained with Trolox.
Laccase Production and Purification. Laccase was pro-
duced by Beldem (Belgium) from hyperproducer P. cinna-
barinus ss3 (21) and purified according to Camarero et al.
(11). Activity was estimated by 5 mM ABTS oxidation (ε436 29
300 M-1 cm-1) in 0.1 M sodium acetate, pH 5, at 24 °C.
Laccase Oxidation of p-Hydroxycinnamic Acids: Kinetic
Constants. p-Hydroxycinnamic acid oxidation was estimated
spectrophotometrically (Shimadzu UV-160) using 100 mU
of enzyme in 50 mM sodium citrate, pH 5, at 24 °C. Oxidation
rates were calculated from the decrease of substrate absor-
bance. The molar absorbances of PCA (ꢀ312 11 100 M-1 cm-1),
FA (ꢀ320 12 500 M-1 cm-1), and SA (ꢀ312 16 100 M-1 cm-1) were
estimated by substrate consumption under reaction condi-
tions. ABTS and DMP (ε469 27 500 M-1 cm-1) were also used
as substrates. Mean values and 95% confidence limits of Km
and kcat values were obtained.
TABLE 1. Antioxidant Activities of p-Hydroxycinnamic Acids
and HBT Measured by the Absorbance Decrease (734 nm) of
Chemically-Generated ABTS•+ After 6-min Reaction (Referred
to Trolox)
ethanol
PBS (pH 7.4)
citrate (pH 5)
PCA
FA
SA
HBT
Trolox
0.47
1.08
1.15
0.33
1.00
2.02
2.20
2.23
0.06
1.00
1.93
1.88
2.08
0.00
1.00
TABLE 2. Kinetic Constants for Oxidation of the Three
p-Hydroxycinnamic Acids by P. cinnabarinus Laccase
Compared with Two Typical Substrates (ABTS and DMP), Acid
Blue 74 Dye and Syringaresinol
specificity
Km (µM)
kcat (s-1
)
constant (µM-1 s-1
)
PCA
FA
SA
ABTS
DMP
Acid Blue 74
Syringaresinol
88.1
7.4
5.4
18.7
3.6
2.8
0.75
13.0
24.6
5.5
8.7
0.05
7.4
0.01
1.77
4.54
0.29
2.43
0.02
1.27
UV-visible spectra during enzymatic oxidation of 50 µM
p-hydroxycinnamic acids by 50 mU/ml laccase in 0.1 M
sodium citrate, pH 5, at 24 °C, were collected (for a total of
8 h) using a Hewlett-Packard 8453 diode-array spectropho-
tometer.
5.8
Dye Decolorization Assays. Decolorization of Acid Blue
74 (indigoid), Reactive Black 5 (diazo), and Azure B (het-
erocyclic) dyes (25 µM) were performed with 100 mU/mL
laccase and 50 or 250 µM PCA, FA, SA, or syringaresinol.
Reactions were carried out in 0.1 M sodium citrate, pH 5, for
2 h at 24 °C and 160 rev/min. Absorbance decrease was
followed at the Acid Blue 74 (608 nm), Reactive Black 5 (598
nm), and Azure B (647 nm) maxima (deducting the absor-
bances of the oxidized mediators at these wavelengths).
ABTS Oxidation in the Presence of Laccase and p-
Hydroxycinnamic Acids. First, 50 µM ABTS was oxidized with
50 mU/mL laccase in 0.1 M sodium citrate, pH 5 at 24 °C, for
5 min. Once oxidation to the ABTS+• radical was completed, 50
µM PCA, FA, or SA was added, and changes in the UV-visible
spectra were monitorized in a diode-array spectrophotometer
until maximal reoxidation of ABTS was attained (240, 500, and
650 s for laccase-SA, laccase-FA, and laccase-PCA, respectively).
Spectra of p-hydroxycinnamic acids oxidized by laccase alone
were recorded for comparison.
1.3 mL of 0.85% thiobarbituric acid and 40 µL of 0.8%
butylated hydroxytoluene (in acetic acid) (22). Reactions were
boiled for 30 min and, after adding 3 mL of butanol, clarified
by centrifugation, and absorbance measured at 532 nm.
Results
Oxidation of p-Hydroxycinnamic Acids. The antioxidant
activities of PCA, FA, SA, and HBT were evaluated by
measuring the reduction of chemically generated ABTS•+
(Table 1). Activities were tested as usual in ethanol and PBS,
but also in citrate, pH 5. SA exhibited the highest antioxidant
activity in the three media assayed.
The kinetic constants for oxidation of PCA, FA, and SA by
P. cinnabarinus laccase were estimated under steady-state
conditions, and compared with those for the typical sub-
strates ABTS and DMP (Table 2). SA was promptly oxidized
by laccase, with an outstanding specificity constant (16-fold
ABTS, twice DMP and more than 400-fold PCA).
Oxidation of PAH by Laccase in the Presence of p-
Hydroxycinnamic Acids. Oxidation of benzo[a]pyrene (50
µM) was investigated with 2 U/mL of laccase and 500 µM
PCA, FA, SA, HBT, or ABTS. Reactions were performed in 0.1
M sodium acetate, pH 5, with 0.1% Tween 20 (as surfactant)
in 4 mL Teflon-sealed vials under continuous rolling (6 rev/
min) in darkness at 30 °C, for 24 or 48 h. Oxidation of
phenanthrene by laccase was investigated analogously, but
in the presence of 1% Tween 80 (as source of unsaturated
lipids) or 1% Tween 20 (as control), and incubated up to 9
days. Samples from benzo[a]pyrene and phenanthrene
treatments were extracted with ethyl acetate, evaporated and
dissolved in 100 µL of acetonitrile for HPLC analyses according
to Can˜as et al. (12), using a linear gradient of 20% acetonitrile/
80% water (0.1% acetic acid).
Lipid Peroxidation: TBARS Production. Lipid peroxi-
dation by laccase-p-hydroxycinnamic acids and laccase-HBT
was assayed with 2 U/mL of enzyme in 0.1 M sodium acetate,
pH 5, in 4 mL vials at 24 °C, under continuous rolling and
darkness. Two concentrations of mediator (0.5 or 2 mM) and
0.7 mM linoleic acid or 2 mM Tween 80 were used. TBARS
production was analyzed after different times in 0.5 mL
samples, by adding 1.1 mL of 3.3 M sodium acetate, pH 3.5,
UV-visible spectra in the course of SA oxidation by laccase
are shown in Figure 1. The maximum around 300 nm
decreased (10 s) and was replaced by maxima at 334 nm (30
s) and 512 nm (60 s). At an advanced stage (1 h) a maximum
at 425 nm appeared, which is the only one in the visible
region at the end of the reaction (8 h). These results suggest
the existence of at least three chemical species during SA
oxidation.
Decolorization of Industrial Dyes. Different dyes were
treated with the enzyme in the absence or presence of each
p-hydroxycinnamic acid. Pycnoporus cinnabarinus laccase
was able to directly oxidize Acid Blue 74. The enzyme has
high affinity for this dye as revealed by the Km value that was
the lowest among the different substrates and mediators
investigated (Table 2). However, due to its low catalytic
constant, 2 h incubation was required for high decolorization
(Table 3). p-Hydroxycinnamic acids promoted laccase oxi-
dation of Acid Blue 74 after short incubation times, attaining
90% decolorization after 5 min with SA. However, the
mediating effect of the p-hydroxycinnamic acids was espe-
cially significant for the recalcitrant dyes Reactive Black 5
and Azure B (Table 3) that were not modified by the enzyme
alone due to their high redox potential (both around 1.3 V)
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FIGURE 1. Changes in the UV-visible spectrum of SA (50 µM) during oxidation by laccase. The different scans correspond to 0
(black line), 10 s (gray line), 30 s (orange line), 60 s (blue line), 10 min (red line), 1 h (purple line), and 8 h (green line).
TABLE 3. Dye (25 µM) Decolorization (%) after 5 min and 2 h Treatment with Laccase Alone or in the Presence of
p-Hydroxycinnamic Acids or Syringaresinol Using 2 or 10 Mediator/Dye Ratios (Mean Values with Standard Deviation Values
below 5% Are Shown)
Laccase alone
PCA
FA
SA
Syringaresinol
2
10
2
10
2
10
2
10
Acid Blue 74
Reactive Black 5
Azure B
5 min
2 h
5 min
2 h
5 min
2 h
4.1
96.1
1.8
1.8
0.0
44.1
100
6.9
16.7
1.3
52.0
100
7.8
21.6
11.5
57.6
23.8
96.1
6.5
8.0
10.3
20.6
4.0
100
5.2
7.4
3.2
90.5
100
12.4
56.8
1.6
85.5
92.1
11.7
94.1
3.9
70.9
100
3.0
34.7
0.0
5.1
100
1.5
86.0
4.5
0.0
19.3
38.5
6.1
18.9
2.7
8.3
(23). SA produced almost complete decolorization of Reactive
Black 5, and near 60% decolorization of Azure B was obtained
in the presence of PCA after 2 h.
spectral change observed during ABTS reoxidation, in
agreement with the absence of maxima of interest in the
spectrum of oxidized FA (Figure 2B and inset). The spectral
changes in the PCA reactions were very limited (Figure 2C),
and ABTS reoxidation was extremely low (no changes at 425
nm during the first 240 s, although partial reoxidation was
found later).
Transformation of Benzo[a]pyrene. The capabilities of
the three p-hydroxycinnamic acids as laccase mediators were
also tested in oxidation of PAH, and compared with ABTS
and HBT (Figure 3A). Benzo[a]pyrene was poorly transformed
by the laccase alone (only around 10% in 48 h). However, FA
and PCA significantly promoted benzo[a]pyrene removal by
the enzyme (no effect was caused by SA). The use of PCA
resulted in high transformation rates (over 90%) similar to
those obtained with HBT and higher than with ABTS.
Transformation of Phenanthrene and Lipid Peroxida-
tion. No phenanthrene was transformed by P. cinnabarinus
laccase alone and only 10% was transformed after 9 days
treatment using PCA (or HBT) as mediator (Figure 3B).
However, the presence of Tween 80 produced a significant
enhancement of phenanthrene transformation by laccase-
HBT and also by laccase-PCA, especially after long incubation
times.
Syringaresinol, a phenolic dimer analogue to those formed
by oxidative ꢀ-ꢀ′ coupling of SA catalyzed by laccase, was
also assayed as substrate being easily oxidized by the enzyme
(Table 2). Moreover, its mediating capability was demon-
strated by the increase of decolorization rates compared with
the use of laccase alone, in a similar way to SA (Table 3).
Kinetics of ABTS Oxidation in the Presence of Laccase
and p-Hydroxycinnamic Acids. When an equimolecular
concentration of p-hydroxycinnamic acid was added to
ABTS•+, previously obtained by 300 s incubation of ABTS
with laccase (gray line in Figure 2), very rapid reduction of
the cation radical was produced (red line), both the 425 nm
peak and the broad maximum around 700 nm disappearing.
However, the intensities of the p-hydroxycinnamic acid
maxima around 300 nm were only slightly modified (a small
displacement to higher wavelengths occurred). After that,
new UV-visible spectra were collected revealing parallel
decreases of the peak around 300 nm and increase of the 425
nm peak (arrows in Figure 2). After 240 s (blue lines)
reoxidation was (i) complete in the presence of SA (Figure
2A) that recovered the previous intensities at 425 and 700
nm; (ii) intermediate in the presence of FA (Figure 2B) that
required 500 s for spectrum stabilization; and (iii) nearly
absent in the case of PCA (Figure 2C).
Production of TBARS from oxidation of linoleic acid by
laccase in the presence of 2 mM PCA or SA was observed,
whereas FA (or lower PCA or SA concentrations) did not
promote TBARS formation (Figure 4A). The amount of TBARS
obtained with PCA was significantly higher than obtained
with SA. When compared with laccase-HBT, the TBARS levels
from oxidation of linoleic acid with laccase-PCA were higher,
although HBT seemed to act more rapidly promoting linoleic
acid peroxidation (Figure 4B). Peroxidation of Tween 80 was
In the case of SA, a maximum around 512 nm appeared
simultaneously to the decrease of the 300 nm peak that was
displaced to 334 nm (Figure 2A and inset). Similar maxima
had been detected during SA oxidation by laccase (orange
and blue lines in Figure 1). In the case of FA, the initial
displacement of the 300 nm peak till 325 nm was the only
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VOL. 42, NO. 17, 2008

FIGURE 2. Changes in the UV-visible spectra during reoxidation of ABTS by laccase in the presence of SA (A), FA (B), or PCA (C).
ABTS (50 µM) was oxidized by laccase during 300 s (gray spectra). Afterward SA, FA, or PCA (50 µM) were added and reduction of
ABTS+• was immediately observed (red spectra). Reoxidation by laccase-mediator was then followed during additional 240 s (scans
each 10 s during first 100 s, and each 20 s until 240 s) (blue line, final spectra). Insets show the spectra of maximal ABTS oxidation
attained by laccase-SA (240 s), laccase-FA (500 s), and laccase-PCA (650 s), compared with the spectrum of completely oxidized
ABTS (gray line) and the spectra of SA, FA, and PCA oxidized by laccase (green line).
also detected with both laccase-mediator systems, but the
TBARS levels attained were significantly lower than obtained
from linoleic acid according to the lower unsaturation degree
of Tween 80.
increased the antioxidant activity of FA and SA with respect
to PCA, as seen by the TEAC values in ethanol, where no
deprotonation interferences take place.
p-Hydroxycinnamic Acids As Laccase Substrates. Laccase
activity on the three p-hydroxycinnamic acids was in
agreement with their antioxidant power (SA > FA > PCA).
SA was the best substrate of laccase among those investigated
here or reported in the literature (11, 24). The higher laccase
activity toward hydroxycinnamic than simple phenols is
related to the more extended conjugated double-bond system
facilitating electron abstraction.
Discussion
Antioxidant Activities of p-Hydroxycinnamic Acids. p-
Hydroxycinnamic acids appeared as strong antioxidants.
Their antioxidant activities were significantly lower in ethanol
than in aqueous solution. The electron-donating effect of
the cinnamate group would be responsible for these differ-
ences since ethanol does not favor deprotonation of cinnamic
acids (pKa 4-5) whereas they are expected to be fully
deprotonated at pH 7.4 (and partially deprotonated at pH 5).
The presence of one or two electron-donor methoxy groups
Formation of successive chemical species during p-
hydroxycinnamic acid oxidation by laccase was shown by
the spectral changes observed with SA. The phenoxyl radical
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FIGURE 3. (A) Benzo[a]pyrene (50 µM) transformation (%) by P. cinnabarinus laccase alone (---) or using 500 µM PCA (9), FA (2), SA
(b), HBT (∆) or ABTS (]) as mediators (0.1% Tween 20). B) Phenanthrene (50 µM) transformation (%) by laccase alone (rhombuses)
or using 500 µM HBT (triangles) or PCA (squares) in the presence of 1% Tween 20 (black symbols) or Tween 80 (open symbols).
Mean values and 95% confidence limits are shown.
FIGURE 4. Lipid peroxidation. (A) Production of TBARS by laccase in the presence of 0.5 (---) or 2 mM (s) PCA (9), FA (2) or SA (b)
using 0.7 mM linoleic acid as substrate. (B) Comparison of TBARS production from 0.7 mM linoleic acid (black symbols) and 2 mM
Tween 80 (open symbols) by laccase and 2 mM PCA (squares) or HBT (triangles) as mediators. Mean values and 95% confidence
limits are shown.
would be the first species, followed by ꢀ-ꢀ′ coupling of its
quinone methide resonant form yielding a dihydrosinapic
acid dimer. Side-chain lactonization will restore the phenolic
groups forming the dehydrodisinapic acid dilactone (3,6-
bis-(4-hydroxy-3,5-dimethoxy-phenyl)-tetrahydro-furo[3,4-
c]furan-1,4-dione) (19, 25, 26). This dilactone is also substrate
of laccase and yields one (or various) products absorbing at
512 nm (19). The high molar antioxidant capability of
p-hydroxycinnamic acids is related to this fact, i.e. each
cinnamic acid molecule reduced more than one ABTS•+
radical. The 512 nm peak shift to 425 nm, initiated after 1 h
of reaction, was in agreement with nonenzymatic breakdown
of the dimeric product yielding p-benzoquinone as an end-
product from SA oxidation by laccase (19).
A wider range of condensation products is expected during
oxidation of FA and PCA due to formation of 5-5′, ꢀ-5′, and
4-O-5′ linkages that are not being formed during SA oxidation
due to the presence of the two methoxy groups (27).
Efficient Decolorization of Dyes by Laccase and Natural
Mediators. The best substrate of P. cinnabarinus, laccase,
SA, was the most rapid mediator in dye decolorization. Its
strong mediating capacity might be explained by its fast
oxidation by the enzyme producing a high concentration of
free radicals.
Other dimethoxy-substituted phenols, acetosyringone and
syringaldehyde, had been demonstrated to be good laccase
substrates and efficient mediators for dye decolorization (11).
The two methoxy substituents increase the lifetime of their
phenoxyl free radicals by preventing 5-5′ coupling reactions,
among others. In the case of the SA phenoxyl radicals, their
high tendency for ꢀ-ꢀ′ coupling (19) immediately rendered
the above-mentioned lactones that would be oxidized by
laccase yielding stable phenoxyl radicals since additional
coupling reactions are not possible. Easy oxidation of these
lactones is in agreement with their strong antioxidant activity
(26). Thus, these phenolic dimeric products would act as
laccase mediators as demonstrated here by the significant
increase of dye decolorization by laccase in the presence of
the homologous phenolic dimer syringaresinol.
Kinetics of p-Hydroxycinnamic Acid-Mediated ABTS
Oxidation. ABTS was also used to investigate the behavior
of p-hydroxycinnamic acid radicals as laccase mediators.
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Reduced ABTS was obtained by adding p-hydroxycinnamic
acid to enzymatically generated ABTS•+. After its immediate
reduction, ABTS reoxidation started and the spectral changes
produced were analyzed.
Despite the fact that SA was totally oxidized due to the
equimolecular amount added, ABTS reoxidation proceeded
slowly suggesting that additional reactions were occurring,
probably involving formation of the phenolic lactones
mentioned above (512 nm absorbance maximum appeared)
acting as laccase mediators. Finally, both ABTS and SA were
completely oxidized.
ABTS reoxidation was produced through the laccase-
mediator systems and not by laccase alone since different
oxidation rates were observed in the presence of each
p-hydroxycinnamic acid. Preferred oxidation of SA and
FA by the P. cinnabarinus laccase was in agreement with
the lower Km and higher kcat values for these compounds
compared with ABTS, and the affinity for their phenolic
dimers should be in the same order, as found for sy-
ringaresinol.
Differences in the nature and yield of the coupling
products from oxidation of p-hydroxycinnamic acids will
affect the final yield and speed of the laccase-mediator
reaction. Fast oxidation of SA results in high concentration
of phenoxyl radicals and fast formation of phenolic dilactones
acting as mediators. PCA is much more slowly oxidized by
laccase, and its phenoxyl radicals could form a wider variety
of dimers and oligomers with a lower phenolic content due
to formation of 4-O-5′ and phenylcoumaran-type structures
(19, 25, 28).
Environmentally Friendly Degradation of Recalcitrant
Xenobiotics. In contrast with that observed for decolorization
of dyes, SA did not mediate PAH oxidation, whereas FA and
especially PCA significantly promoted anthracene and ben-
zo[a]pyrene removal by laccase (12). SA is easily oxidized by
the enzyme and their phenoxyl radicals are more reactive
than PCA radicals, but the redox potential of the latter is
significantly higher (0.8 V of PCA, compared with 0.7 and 0.6
V of FA and SA, respectively) (29). Therefore, besides the
concentration and the reactivity/stability equilibrium of the
mediator radicals, their oxidation power toward the target
substrate will control the yield of the laccase-mediator
reactions.
The higher redox potential of phenanthrene (1.50 V)
compared with anthracene (1.09 V) or benzo[a]pyrene (1.27
V) (30) explains the inability of laccase to oxidize this PAH.
The use of mediators allows laccase removal of anthracene
and benzo[a]pyrene, but not of phenanthrene. However,
laccase-PCA efficiently transformed this compound in the
presence of Tween 80 due to lipid peroxidation reactions
forming peroxyl radicals that act as strong oxidizers. The
involvement of these lipid radicals was confirmed here by
the TBARS assay.
The analysis of the different reaction products obtained
by laccase-PCA and laccase-HBT vs laccase-ABTS during
oxidation of PAH (12) suggested that p-hydroxycinnamic acid
phenoxyl radicals follow an hydrogen atom transfer (HAT)
oxidation mechanism, similar to that described for HBT
nitroxyl radicals, and different from the electron transfer (ET)
mechanism followed by ABTS•+ radicals (31). Peroxidation
of lipids by laccase-PCA, or other phenols (32), supports this
HAT oxidation mechanism that generates substrate free
radicals. Moreover, the absence of TBARS with laccase-ABTS
corroborates the ET mechanism and explains why no
phenanthrene oxidation could be attained in the presence
of Tween 80.
(or other recalcitrant PAH) via lipid peroxidation reactions
can be attained using some laccase natural mediators, such
as PCA. It its noteworthy that in this case both the level of
phenanthrene transformation and the release of TBARS by
laccase-PCA were closely similar than those obtained with
HBT, one of the strongest laccase mediators described up to
date.
Acknowledgments
This work has been supported by the Spanish Projects
CTQ2005-08925-CO2-02 and BIO 2005-3569, and the BIORE-
NEW EU-project (NMP2-CT-2006-026456). The authors thank
Prof. J. Sipilä (University of Helsinki) for advice in syrin-
garesinol synthesis. S.C. and A.I.C. thank R&C Contract and
FPI Fellowship of the Spanish MEC, respectively.
Supporting Information Available
Chemical structures of mediators, dyes and PAH. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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