520 M.C. Terrón et al. / Biochimie 86 (2004) 519–522
at 436 nm (eo= 29300 M–1 cm–1). The reaction mixture as described above but without the addition of TA. Laccase
usually consists of acetate buffer at around pH 5, ABTS as activity was measured immediately after the addition of TA
the substrate (10 mM ﬁnal concentration), and the fungal solutions to enzymatic crudes using both, ABTS and 2,6-
extracellular medium containing the laccase activity to be DMP as substrates.
In this work, we demonstrate that under these reaction 2.4. Laccase activity determination
conditions but in the absence of any enzyme, tannic acid (TA)
is able to carry out the chemical reduction of ABTS+•. This Laccase activity was measured, using 2,6-DMP  or
results in an underestimation of laccase activity values deter- ABTS  as enzyme substrates. One unit of laccase activity
mined by this method in biological samples containing TA or is deﬁned as the formation of 1 µmol of product per min. All
related aromatic compounds. assays were performed in duplicate using a Shimadzu UV-
2. Materials and methods 2.5. ABTS Spectra
2.1. Chemicals The absorption spectra of an ABTS (10 mM) solution
dissolved in water was carried out in a Jasco V-530 spectro-
TA (Tannic acid powder pure, USP, empirical formula photometer from 400 to 500 nm. Different ABTS spectra
C76H52O46), was obtained from Merck (Darmstadt, Ger- were carried out in the absence and in the presence of TA
many). ABTS was purchased from Boehringer-Mannheim (0.25, 0.50, and 1 µM ﬁnal concentrations) or sodium ascor-
(Germany) and 2,6-DMP from Fluka (Germany). All other bate (25 µM).
chemicals were reagent grade obtained from Merck or
3. Results and discussion
2.2. Organism and maintenance
The interference in the laccase-detection assay using
Basidiomycetes Coriolopsis gallica (A-241) and Tram- ABTS as the substrate (evidenced by the decrease of absor-
etes sp. I-62 (B-24), were obtained from the IJFM (Instituto bance at 436 nm in the presence of different industrial efﬂu-
Jaime Ferrán de Microbiología) collection. The fungal cul- ents), is a frequent observation in our laboratory (data not
tures were maintained on malt agar slants (2% malt extract, published). Given that all these wastewaters come from in-
2% Bacto Agar), grown for 10 days at 28 °C, and stored at dustries that use plants as raw material, we speculated that
4 °C. the common substances which may be responsible for this
interference could be polyphenolic based compounds; which
2.3. Culture conditions in addition are molecules which could be easily oxidized by
the enzyme. Thus, we determined the total phenol and tannin
C. gallica was grown on agar plates with modiﬁed Cza- content in some of these efﬂuents, which indicated that tan-
peck medium  for seven days at 28 °C. Ten plugs (1 cm2) nins represent a signiﬁcant percentage of their phenolic com-
were cut and inoculated under sterile conditions into 500 ml position . For this reason, TA (the most abundant
culture ﬂasks containing 300 ml of Kirk growth medium with polyphenolic molecule in plants after lignin) was selected as
0.4 mM veratryl alcohol . After incubation for 48 h at a model compound in our study.
28 °C in an orbital shaker (200 rpm), an inoculum of 1:10 Experiments were performed initially to elucidate
(v/v) was transferred into 250 ml Erlenmeyer ﬂasks contain- whether the observed interference with laccase activity
ing 100 ml of the same medium. Samples were incubated at might be due to a direct interference with the laccase enzyme
28 °C and 125 rpm for 7 days. Afterwards, a ﬁlter-sterilized itself, or to a chemical interference with ABTS. With this in
(0.22 µm) water-solution of 50 mM TA was added to the mind, we monitored laccase activity in C. gallica over an
culture medium to reach a ﬁnal concentration of 200 µM, and 8-day period; grown in either the presence or absence of
the cultures were incubated under the same conditions for 200 µM of TA. Laccase activity was measured using ABTS
eight more days. During this time laccase activity was mea- or 2,6-DMP (Fig. 1). Signiﬁcant differences were observed
sured daily in the extracellular ﬂuids using ABTS and 2,6- with much higher laccase activity values being detected in
DMP as substrates. Controls without TA were also run and the presence of TA using 2,6-DMP as substrate as opposed to
monitored daily. ABTS. For example, laccase activity determined with 2,6-
The assays to determine laccase activity in the presence of DMP was 3.2-fold higher than in the control without TA, in
different concentrations of TA (0, 0.1, 1, 5, 9, 20, 50, 100 and day 6 samples. On the other hand, laccase activity deter-
200 µM, ﬁnal concentrations) were performed by mixing mined with ABTS was 9-fold lower than that observed on the
them with extracellular ﬂuids from 12-days-old cultures of same day in the control. These results suggested the possible
Trametes sp. I-62, grown under the same culture conditions interference of tannic acid with the ABTS molecule. This is
M.C. Terrón et al. / Biochimie 86 (2004) 519–522 521
Fig. 1. Laccase activity determined as oxidation of (n) 2,6-DMP or (•)
ABTS in the extracellular ﬂuid of Coriolopsis gallica. Each point repre-
sents: [100 × (laccase activity measured in the presence of 200 µM of
TA/laccase activity in the control without TA, measured the same day)].
supported by a previous observation by Carbajo and cowork-
ers  where increased laccase activity was observed when
TA (100 µM) was added to C. gallica cultures, suggesting
that TA interferes with ABTS molecule rather than inhibit
laccase production in this fungus.
These results prompted us to perform additional analysis
to verify this interference. The absorption spectra of a solu- Fig. 2. (A) Optical absorption spectra of ABTS in the absence (a) and in the
tion of ABTS both in the presence and absence of different presence of different concentrations of TA (0.25 (b), 0.50 (c), and 1 µM (d))
concentrations of TA were then assessed (Fig. 2A). The or sodium ascorbate (25 µM) (e). (B) Laccase activity determined using (n)
absorbance values of ABTS were markedly lower especially 2,6-DMP or (•) ABTS in the presence of different concentrations of TA. For
the experiment, extracellular ﬂuids containing high laccase activity from
in the 400–460 nm range, at increased concentrations of TA. 12-days old cultures of Trametes sp. I-62 were used.
Moreover at TA concentrations of 0.5 µM and higher, the
characteristic shoulder around 420 nm was absent. The spec- the absence of TA, even in the presence of the highest TA
trum of ABTS in the presence of 25 µM ascorbate, a well- concentration assayed (200 µM).
known chemical ABTS reducer , was also performed; Recently, other organic compounds have also been re-
and were shown to be very similar to those of ABTS in the ported to be capable of reducing ABTS. Johannes and Ma-
presence of 0.5 and 1 µM TA (Fig. 2A). This strongly sug- jcherczyk  have demonstrated that several sulfhydryl
gests that TA can chemically reduce the ABTS molecule. It is organic compounds, described as laccase inhibitors, are not
well established that at ABTS concentrations greater than true inhibitors but rather are substances which are able to
1 mM, solutions appear green-blue but become colourless chemically reduce the coloured radical cation ABTS+• to
when reducing agents such as ascorbate or cysteamine are ABTS, resulting in the decolourising of the solution. The
added, even at relatively low concentrations . A similar reduction of ABTS by some physiological organic acids and
change in colour was also observed here when TA was added a phenolic lignin-related compound has also been described
to ABTS. .
In addition we performed assays on a native laccase en- The results presented here indicate that TA is also capable
zyme crude from an 12-day-old culture of Trametes sp. I-62, of chemically reducing ABTS+• in reaction conditions com-
in order to verify whether TA–ABTS interference also af- monly used to detect laccase activity, leading to the possibil-
fected enzymatic activity measurements. Laccase activity ity that much lower laccase activity values will be obtained
was measured both in the presence and absence of different when this compound is present in the reaction mixture. This
concentrations of TA and using either ABTS or 2,6-DMP as interference could probably be extended to include other
substrates (Fig. 2B). The results obtained indicated a dra- easily oxidised structural-related phenolic compounds; and
matic decrease in ABTS mediated laccase activity following may explain at least in part, the decrease in absorbance
addition of 5 µM TA, reaching minimum values (less that 5% observed in the presence of some industrial efﬂuents (data
of initial activity) at TA concentrations of 50 µM TA and not published).
higher. In marked contrast the activity measured using 2,6- Given that this chemical interference may take place in
DMP showed values close to 90% of the activity measured in identical reaction conditions to those used to measure laccase
522 M.C. Terrón et al. / Biochimie 86 (2004) 519–522
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