On the mechanism of biotransformation of the anthraquinonic dye acid blue 62 by laceases

Article abstract of DOI:10.1002/adsc.200900271

We used the recombinant CotA-laccase from the bacterium Bacillus subtilis to investigate the biotransformation of the commercial anthraquinonic dye Acid Blue 62. Kinetics of dye biotransformation at pH6 follow a Michaelis-Menten model. NMR and several MS techniques allowed the identification of intermediates and final products of the enzymatic biotransformation. The main final product obtained, l-[(4-amino-9,10-dioxo-3-sulfo-9,10-dihydroanthracen-l-yl)diazenyl]-4- cyclohexylamino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid, is formed through the creation of an azo link and has been previously identified as an intermediate compound in the biodegradation of Acid Blue 62 by crude fungal preparations. The identification of 1,4diamino-9,10-dioxo-3-sulfo-9,10- dihydroanthracene 2-sulfonic acid and of cyclohexanone, in reaction mixtures with CotA-laccase and also its presence in reactions performed with the LAC3 lacease from the fungus Trametes sp. C30, suggest the occurrence of coupling reactions between the intermediate products of dye oxidation. Based on these results, we propose a mechanistic pathway for the biotransformation of Acid Blue 62 by laceases. A bioassay based on the inhibitory effects of the dye and its enzymatic products on the growth of Saccharomyces cerevisiae shows the importance of laceases in reducing dye toxicity.

Full text of DOI:10.1002/adsc.200900271

FULL PAPERS
DOI: 10.1002/adsc.200900271
On the Mechanism of Biotransformation of the Anthraquinonic
Dye Acid Blue 62 by Laccases
Luciana Pereira,a Ana V. Coelho,a Cristina A. Viegas,b Christelle Ganachaud,c
Gilles Iacazio,c Thierry Tron,c M. Paula Robalo,d and Lꢀgia O. Martinsa,*
a
Instituto de Tecnologia Quꢀmica e Biolꢁgica, Universidade Nova de Lisboa, Av. da Repfflblica, 2780-157 Oeiras, Portugal
Fax : (+35)-12-1441-1277; e-mail: lmartins@itqb.unl.pt
IBB-Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia Biolꢁgica e Quꢀmica, Instituto Superior TØcnico,
b
Av Rovisco Pais, 1049-001 Lisboa, Portugal
Laboratoire BiosCiences, ISM2 UMR6263, case 432, Faculte des Sciences et Techniques de Saint JØrôme, Aix-Marseille
c
UniversitØ, Av. Escadrille Normandie-Niemen. 133397 Marseille Cedex 20, France
Departamento de Engenharia Quꢀmica, Instituto Superior de Engenharia de Lisboa, Rua Cons. Emꢀdio Navarro, 1,
d
1959-007 Lisboa, Portugal and Centro de Quꢀmica Estrutural, Complexo I, Superior TØcnico, Av Rovisco Pais, 1049-001
Lisboa, Portugal
Received: April 17, 2009; Revised: June 3, 2009; Published online: July 21, 2009
Abstract: We used the recombinant CotA-laccase 2-sulfonic acid and of cyclohexanone, in reaction
from the bacterium Bacillus subtilis to investigate mixtures with CotA-laccase and also its presence in
the biotransformation of the commercial anthraqui- reactions performed with the LAC3 laccase from the
nonic dye Acid Blue 62. Kinetics of dye biotransfor- fungus Trametes sp. C30, suggest the occurrence of
mation at pH 6 follow a Michaelis–Menten model. coupling reactions between the intermediate prod-
NMR and several MS techniques allowed the iden- ucts of dye oxidation. Based on these results, we pro-
tification of intermediates and final products of the pose a mechanistic pathway for the biotransforma-
enzymatic biotransformation. The main final product tion of Acid Blue 62 by laccases. A bioassay based
obtained, 1-[(4-amino-9,10-dioxo-3-sulfo-9,10-dihy- on the inhibitory effects of the dye and its enzymatic
droanthracen-1-yl)diazenyl]-4-cyclohexylamino-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonic acid,
products on the growth of Saccharomyces cerevisiae
is shows the importance of laccases in reducing dye
formed through the creation of an azo link and has toxicity.
been previously identified as an intermediate com-
pound in the biodegradation of Acid Blue 62 by Keywords: anthraquinonic dyes; azo dye synthesis;
crude fungal preparations. The identification of 1,4- biocatalysis; biotransformations; laccase; radical cou-
diamino-9,10-dioxo-3-sulfo-9,10-dihydroanthracene-
pling
Introduction
radical intermediates.[4] Recently, we have shown that
CotA-laccase from the bacterium Bacillus subtilis is
Laccases are oxidoreductases belonging to the multi- able to decolourise a variety of azo and anthraquinon-
copper oxidase family of enzymes.[1] Their catalytic ic dyes at alkaline pH and in the absence of redox
centres consist of three structurally and functionally mediators.[5] We have additionally reported on the
distinct copper sites; T1 copper (“blue copper”) is a synthesis of 2,2-bis(3’-indolyl)indoxyl, a yellow com-
mononuclear centre involved in the substrate oxida- pound obtained from trimerisation of indole using the
tion, whereas T2 and T3 form a trinuclear centre in- LAC3 laccase from the fungus Trametes sp. C30.[6]
volved in the dioxygen reduction to water.[2] These
Around 106 tons of synthetic dyes are produced an-
enzymes are useful biocatalysts for diverse biotechno- nually, of which 1–1.5”105 tons are released to the
logical applications, owing to their relatively non-spe- environment in wastewaters.[7] Dyes are in general
cific oxidation capacity, the lack of requirement for stable organic pollutants, persisting in the environ-
co-factors and the use of readily available oxygen as ment, and concerns have been raised that such artifi-
an electron acceptor.[3] Laccases also have uses in or- cial compounds may be xenobiotic.[8] Therefore, the
ganic synthesis, where their typical substrates are phe- development of methods for their degradation has
nols or amines, and the reaction products are dimers been increasingly explored.[7] Antraquinonic dyes are
and oligomers derived from the coupling of reactive used either as primary or secondary dyes in commer-
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Luciana Pereira et al.
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cial trichromatic dyeing formulations.[9] However, lim-
ited information exists on their physicochemical or
biological degradation[10–12] and even less is known on
the molecular mechanisms of transformation.[13,14]
Nevertheless, this knowledge is important, not only
for the development of efficient bioremediation pro-
cesses, but also in the search for harmless dyeing com-
pounds to be synthesised by green chemistry process-
es.
In this study, we report on the enzymatic biotrans-
formation of the anthraquinonic dye Acid Blue 62
(AB62, compound 1) into 1-[(4-amino-9,10-dioxo-3-
sulfo-9,10-dihydroanthracen-1-yl)diazenyl]-4-cyclohex-
ylamino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic
acid) (compound 4[14]). The enzymatic reactions were
followed by UV-vis and HPLC. Based on the charac-
terisation of intermediates and final products of the
reaction, a possible mechanistic pathway for the bio-
transformation of AB62 through the formation of an
azo link is discussed. Moreover, the toxicities of
AB62 and its biotransformation products were evalu-
ated, confirming the environmental friendly character
of laccase-mediated processes.
Figure 1. Time course for AB62 biotransformation by CotA-
laccase as monitored by HPLC. Insert: HPLC peak areas at
500 nm versus time of the reaction. [&, AB62 and products:
&
*
, Rt 13 min and, , Rt 50 min].
Results and Discussion
case towards AB62 oxidation (4.7”103 sÀ1·MÀ1) is 2–3
orders of magnitude lower than that of fungal laccas-
Monitoring the Biotransformation of AB62 by UV-vis es.[11] Nevertheless, the lower catalytic efficiency to-
and HPLC
wards AB62 oxidation proved to be advantageous for
the detection and purification of the intermediate 2
Transformation of AB62 by CotA-laccase resulted in (DAAS) in reactions with this enzyme.
a decrease in the intensity of the dye absorption
bands, at lmax =600 and 630 nm, along with an in-
crease in absorption around 500 nm due to the forma- Identification of Biotransformation Products of AB62
tion of reddish biotransformation products (data not
shown). The time course of the biotransformation of The biotransformation products of AB62 were identi-
AB62 was additionally monitored by HPLC fied in the enzymatic reaction mixtures and after pu-
(Figure 1). In the first minutes of reaction, a peak rification using NMR, MS/MSn, LC-MS and GC-MS
with an Rt of 13 min (formed at a rate of 1.7Æ0.2 analyses. The 1H NMR spectrum of purified inter-
mmol·minÀ1·mgÀ1, Figure 1, insert) was detected in mediate product with an Rt of 13 min is shown in Fig-
chromatograms. However, as the reaction proceed, ure 3A (data summarised in the Experimental Sec-
this peak decreased (at a rate of 0.5Æ0.1 mmol·minÀ1· tion). In the chemical shift region of the aromatic pro-
1
mgÀ1) concomitant with the appearance of a major tons, the H NMR spectrum of compound 2 shows a
peak at 50 min (at a rate of 0.9Æ0.3 mmol·minÀ1· double triplet and a double doublet (two protons
mgÀ1). These observations are consistent with the ini- each) centred at 7.75 and 8.30 ppm, typical for the
tial formation of an intermediate product (compound ring A protons (H6, H7 and H5, H8, respectively) of
2, DAAS, see Scheme 1) before its consumption in an anthraquinone skeleton. Moreover, the singlet at
further reactions. A more detailed kinetic analysis 7.80 ppm, which corresponds to one proton, could be
was performed and as can be seen in Figure 2, we ob- assigned to the ring C proton H3 and proves that the
served an increase in the dye consumption rates with ring C is trisubstituted in positions 1, 2 and 4. No fur-
increasing dye concentration for values up to 1 mM, ther signals are observed in the aliphatic region re-
and above this, the rates become independent of the vealing the absence of the cyclohexyl group initially
dye concentration. Therefore, the enzyme steady-state present in the structure of AB62. The mass spectrum
kinetic constants for AB62 were calculated according of this compound exhibit peaks with m/z=317 and
to the Michaelis–Menten model: Vmax of 1.3Æ0.9 319 in negative and positive ionisation modes, respec-
mmol·minÀ1·mgÀ1, kcat of 1.4Æ0.6 sÀ1 and Km of 296Æ tively (Figure 3B), leading to a molecular mass of
49 mM. The catalytic efficiency (kcat/Km) of CotA lac- 318. Fragmentation of the peak with an m/z of 319
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On the Mechanism of Biotransformation of the Anthraquinonic Dye Acid Blue 62
FULL PAPERS
Scheme 1. Identified products of AB62 biotransformation by CotA-laccase. The ions m/z of 319, 633 and 715 were also iden-
tified in reaction mixtures with LAC3 from Trametes sp. C30.
produced two ions with m/z of 237 and 255 that could
be formed through the loss of SO3H (81 amu) and
SO2 (64 amu) groups. Therefore, this compound, that
results most likely from the loss of the cyclohexyl
moiety of 1 through enzymatic oxidation, can be iden-
tified as 1,4-diamino-9,10- dioxo-3-sulfo-9,10-dihy-
droanthracene-2-sulfonic acid (DAAS, 2). This com-
pound is also an intermediate in the photocatalytic
degradation of Acid Blue 25, a structurally similar an-
thraquinonic dye.[16]
1
The H NMR spectrum obtained for the purified
product with Rt of 50 min contains signals in two dif-
ferent regions (data summarised in the Experimental
Section). The chemical shifts found in the 1–4 ppm
region are within the expected shift range for the cy-
clohexyl moiety, whereas the chemical shifts in the
low-field aromatic region (7–9 ppm) are consistent
with the presence of two slightly different anthraqui-
Figure 2. Non-hyperbolic saturation kinetics of CotA-laccase
with AB62. Insert: Hannes plot.
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Figure 3. 1H NMR (A) and MS/MSn (B) spectra of the purified 1,4-diamino-9,10-dioxo-3-sulfo-9,10-dihydroanthracene-2-sul-
fonic acid (2).
nonic skeletons. The 13C NMR spectrum shows signals 317. The formation of ions with m/z of 384 and 302
1
in agreement with the H NMR data. The mass spec- can be explained by cleavage of the carbon-nitrogen
trum shows peaks at m/z=713 and 715 in negative bond adjacent to the central azo linkage. Minor ions
and positive mode, respectively. Fragmentation of the result from the loss of the cyclohexyl and SO3H
negative ion generated the major ions with m/z of (m/z=633, 525) or from the cleavage of the azo bond
605, 384 and 302, and minor ions with m/z=633, 525, (m/z=317).[15] From these results, we concluded that
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On the Mechanism of Biotransformation of the Anthraquinonic Dye Acid Blue 62
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Table 1. LC-MSn data for AB62 biotransformation products
by CotA-laccase after 2 h of reaction.
mulates approximately in equal amounts (comparable
yields) and at about the same rate (comparable kinet-
ics). The LC-MS analysis of mixtures after 10 min of
AB62 reaction by LAC3 revealed the presence of 4
compounds with m/z of 319, 633, 702 and 715 in posi-
tive mode. The ions m/z of 319, 633 and 715 could be
assigned to structures 2, 5 and 4 respectively, in ac-
cordance with the results obtained for the biotransfor-
mation with CotA-laccase. The ion m/z of 702 could
result from loss of an NH2 group in the hydrazine in-
termediate which results, in turn, from the coupling of
two radicals 2A, just before the final oxidation step
leading to the final product 4 (see Scheme 2).
Rt
[min] (+)
m/z
MSn product ions
MS3
N
(À) MS2
13
16
319
633
317 237, 255
631 317, 441, 469, 523,
551
–
–
19
24
26
28
45
50
248
576
734
604
401
715
–
148, 230
–
–
–
–
574 414, 496
732 306, 388, 416, 653
602 522, 540
399 319
713 302, 317, 384, 525,
605, 633
237, 255
–
54
622,
797
795 603, 335, 399, 715,
779
406, 469, 491,
521, 585
Proposed Mechanism of the Biotransformation of
AB62 by Laccases
the compound is 1-[(4-amino-9,10-dioxo-3-sulfo-9,10- Based on the intermediates and products identified
dihydroanthracen-1-yl)diazenyl]-4-cyclohexylamino- with the two different laccases, we propose a mecha-
9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid) (4) nistic pathway for the biotransformation of AB62
previously identified by Vanhulle et al.[14]
(Scheme 2). Two routes are possible as the enzyme
LC-MS analysis of the total reaction mixtures after can catalyse a single-electron oxidation, either from
2 h revealed the presence of 9 compounds: DAAS the primary or the secondary amines of compound 1,
(2), compound 4 and compounds with m/z of 248, forming the reactive species 1A and 1B, respectively.
576, 604, 622, 633, 715, 734 and 797 (Table 1). The Radical 1B can be sequentially oxidised into an
structures for those are proposed (except for molecu- imine, which would hydrolyse through the breakdown
À
lar ion m/z=622), based in the molecular ion and of the N C bond and the loss of cyclohexanone (3),
mass spectrometric fragmentation ions (compounds followed by a proton abstraction. This produces com-
5–10, Scheme 1). The mass ion m/z=633 (5) should pound 2 (DAAS), the first intermediate in this path-
result from the coupling of two oxidised DAAS mole- way. This route is supported by the identification of
cules (2A) through formation of an azo bond. Indeed, intermediate (2) and product (3) over the course of
the dimer with m/z of 633 was identified by LC-MS as enzymatic AB62 transformations. Subsequently, inter-
the main product of CotA reaction using compound 2 mediate 2 could be further oxidised and the resulting
(1 mM) as substrate (at a slow rate of 0.19Æ0.02 radical form (2A) should lead to the main product of
mmol·minÀ1·mgÀ1). Compounds 8 and 9 should result the biotransformation process, the azo dimer (4). This
from further degradation of this dimer as can be seen dimer should result from the radical coupling of 2A
in Scheme 1. The ion m/z=797 (compound 6), results with one molecule of oxidised dye (compound 1A),
from the coupling of two molecules of 1 and the ion leading to the formation of a hydrazine, followed by
at m/z=734 (compound 7), corresponds to the an oxidative step that could also be catalysed by lac-
sodium adduct of 4 [m/z=715+Na]+. Incubation of 4 cases. To a very low extent, the dimerisation of single
(M=714), the main product of the reaction with radicals 2A and 1A should end in the formation of
CotA-laccase in different assay conditions did not compounds 5 and 6 by a similar process as described
result in any transformation of the compound in con- for 4. In all cases, the formation of an azo bond
trast to that observed in the study of Vanhulle et al.[14] occurs. The final product 4 was identified by NMR
We simultaneously analysed the products of the and MS techniques as described previously. The for-
biotransformation of AB62 by using LAC3 laccase mation (at low concentrations) of the dimeric com-
from the fungus Trametes sp. C30. This enzyme pres- pounds 5 and 6 was supported by the LC-MSn analysis
ents higher rates of oxidation as compared CotA-lac- and by the presence of products 8 and 9 in the reac-
case.[17,18] Etheral extracts of reaction mixtures ana- tion mixtures (Scheme 1).
lysed by TLC upon appropriate staining, revealed the
presence of a single compound of medium polarity. A
comparison with standards led to the conclusion that Assessment of the Environmental Fate of Acid Blue
cyclohexanone (3) could correspond to this com- 62 and its Enzymatic Products
pound, a proposition which was later confirmed by
GC-MS analysis. In the course of the LAC3-catalysed The toxicity of synthetic dyes as well as of their bio-
dimerisation process of AB62, products 3 and 4 accu- conversion products presents a high environmental
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Scheme 2. Proposed mechanism of AB62 biotransformation by laccases.
concern.[7,8] Thus, in this study, a yeast-based bioassay nificantly less toxic to the yeast (LOEC~45 mM,
was used to compare the potential toxicity of AB62 IC50 >750 mM) than the parent molecule (Figure 4).
and products. The use of the eukaryotic model Sac-
charomyces cerevisiae could provide meaningful clues
on the potential cytotoxicity of these compounds for
mammals or aquatic eukaryotic organisms.[19] Our re-
sults show that AB62 causes a significant inhibitory
effect on yeast growth (Figure 4) and values of LOEC
and IC50 of around 7 and 420 mm (3 and 177 mg/L) re-
spectively, were estimated.
The IC50 is well above the expected dye concentra-
tions in the environment but is within the same order
of magnitude as the typical dye concentration in
spent dye baths.[7] The mixture containing AB62 bio-
transformation products after 2 h of reaction with
CotA-laccase was significantly less toxic to the yeast
cell population, resulting in 28.5Æ4.8% of inhibition
of growth as compared with 66.3Æ2.2% inhibition
with the untreated dye solution. Consistent with the
reduced overall toxicity of AB62 solution, we show
that compound 4, the reddish azo product that accu-
&
Figure 4. Toxicity of AB62 (1, &) and compound 4 ( ) as de-
termined by the inhibitory effects to S. cerevisae BY4741
mulates during the biotransformation reaction, is sig- growth.
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On the Mechanism of Biotransformation of the Anthraquinonic Dye Acid Blue 62
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Moreover, compound 4 is at least 5-fold less toxic to Spectroscopic Analysis
yeast than the azo dye SOG (IC50 ~150 mM[5]).
The UV-visible absorption spectra were obtained at room
temperature using a Nicolet Evolution 300 spectrophotome-
ter from Thermo Industries. H and 13C NMR spectra were
1
Conclusions
obtained at room temperature with an Avance Bruker 400
1
spectrometer in MeOD-d4 solvent. The H and 13C chemical
shifts are reported in parts per million (ppm) using the sol-
vent signal as internal reference. Spectral assignments
follow the numbering scheme shown in Figure 3A. Two-di-
mensional HH COSY and HMQC experiments were used to
assign all members of the coupled spin network.
The enzymatic biotransformation process of AB62
was examined by kinetic studies and HPLC, NMR
and MS analyses, leading to the identification of reac-
tion intermediates and final products. In a previous
study on the biodegradation process of AB62 by
crude fungal enzyme preparations, compound 4 is de-
scribed to be an intermediate product transiently
formed before its degradation.[14] In this work we
have unambiguously identified the two main final re-
action products {1-[4-amino-9,10-dioxo-3-sulfo-9,10-
dihydroanthracen-1-yl)diazenyl]-4-cyclohexylamino-
9,10-dioxo-9,10- dihydroanthracene-2-sulfonic acid (4)
and cyclohexanone (3)} and a major intermediate
[1,4-diamino-9,10-dioxo-3-sulfo-9,10-dihydroanthra-
cene-2-sulfonic acid (2)]. The mechanistic scheme
that can be drawn corresponds to a two-step sequence
of oxidation reactions dominated by the loss of the
cyclohexyl substituent of the amino group on AB62
and the further coupling of AB62 and DAAS radicals.
We show that the enzymatic biotransformation pro-
cess is effective in reducing the toxicity of solutions
containing the dye AB62. This study demonstrates
the potential use of laccases in the oxidative degrada-
tion of anthraquinonic dyes in the environment. It
also gives insights over the biosynthesis of azo dye
compounds with low toxicological properties.
HPLC Analysis
HPLC analyses were performed in a HPLC Merck Hitachi
with a diode-array system, using a reverse phase C-18
column (250”4 mm length, 5 mm particle size and pore of
100 Š, from LiChrospher 100 RP-18, Merck). Samples were
diluted 1:1 in acetonitrile (ACN, 99.9%, Lab-SCAN, Dublin,
Ireland) and centrifuged before injection (50 mL). Com-
pounds were eluted with a linear gradient of a mobile phase
from 20 to 40% of ACN plus 0.1% (v/v) of trifluoroacetic
acid (TFA), over 50 min, and maintained isocratic at 40%
for 10 min. The flow rate was 1 mLminÀ1 and temperature
408C.
Purification and Identification of Reaction Products
Two products of AB62 biotransformation by CotA-laccase
as monitored by HPLC with Rt, at 13 and 50 min were puri-
fied using a disposable solid phase extraction RP18 tubes
(Supelclean LC-18, SUPELCO, Bellefonte, PA, USA). The
compound with Rt =13 min was purified after 30 min of re-
action at pH 8, and the compound with Rt =50 min after 2 h
of reaction at pH 6. The reactions were stopped with ACN
(1:1) and samples liophilised and ressuspended in 0.1% TFA
(v/v). Elution of the compounds was performed by a gradi-
ent of increasing ACN concentrations in 0.1% TFA. The re-
sulting purified fractions were analysed by MS and NMR as
stated below.
1,4-Diamino-9,10-dioxo-3-sulfo-9,10-dihydroanthracene-2-
sulfonic acid (DAAS, 2): HPLC: Rt =13 min; violet colour
(lmax =560 nm, e560 =92MÀ1·cmÀ1); 1H NMR (400 MHz,
MeOD): d=7.75 (dt, 2H, J=7.2, 2.4 Hz), 7.80 (s,1H), 8.30
(dd, 2H, J=6.8, 1.2 Hz); 13C NMR (400 MHz, MeOD): d=
127.9, 128.2, 129.5, 133.0, 134.4, 134.5, 135.0, 147.5, 152.6,
181.7, 185.1.
1-[(4-Amino-9,10-dioxo-3-sulfo-9,10-dihydroanthracen-1-
yl)diazenyl]-4-cyclohexylamino-9,10-dioxo-9,10-dihydroan-
thracene-2-sulfonic acid (4): HPLC: Rt =50 min; red colour
(lmax =500 nm, e560 =769MÀ1·cmÀ1); 1H NMR (400 MHz,
MeOD): d=1.44 (m, 1H), 1.55 (q, 4H), 1.71 (m, 1H), 1.84
(m, 2H), 2.14 (m, 2H), 3.76 (m, 1H), 7.71 (t, 2H, J=
7.6 Hz), 7.80 (m, 2H, J=7.2 Hz), 7.88 (s, 1H), 7.98 (dt, 2H,
J=6.4, 0.8 Hz), 8.24 (t, 2H, J=8.4 Hz), 8.62 (s, 1H);
13C NMR (400 MHz, MeOD): d=25.4, 25.5, 26.9, 33.9, 34.0,
52.3, 114.4, 118.3, 127.3, 127.6, 127.8, 127.9, 132.1, 133.2,
134.5, 134.7, 134.8, 135.3, 135.5, 135.8, 136.2, 143.5, 145.4,
150.9, 152.0, 185.7, 185.9, 186.4, 186.5; MS (ESI): m/z (nega-
tive mode)=713 ([MÀH]À), 633, 605, 525, 384, 317, 302.
Experimental Section
Enzymatic Biotransformation of AB62
CotA-laccase activity towards Acid Blue 62 dye (AB62,
Yorkshire Europe, Belgium, 97% purity) was measured
using 1 mM of dye and 1 U·mLÀ1 of CotA in 20 mM sodium
phosphate buffer, pH 6.[18] Reactions were conducted at
378C, with shaking (180 rpm) and time-point samples were
withdrawn, diluted and analysed by spectrophotometry and
HPLC. The decrease in the dye concentration was moni-
tored at 630 nm (lmax of AB62). The effect of pH on the en-
zymatic activity (pH 4–10) was determined in Britton–Rob-
inson buffer (100 mM phosphoric acid, 100 mM boric acid
and 100 mM acetic acid mixture titrated to the desired pH
with 0.5M NaOH). Kinetic parameters were determined
from Lineweaver–Burk plots. The enzymatic transformation
of AB62 (6.6 mM) by LAC3 from Trametes sp. C30
(2 U·mLÀ1)[17] was performed under dioxygen over-pressure
at 2 bar, in Schlenk tubes, with a final volume of 10 mL in
0.1M acetate buffer, pH 5, and at 308C. All enzymatic
assays were performed at least in triplicate.
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LC-ESI-Ion Trap Analysis
The ESI-ion trap MS system was a LCQ ion trap mass spec-
trometer (Thermofinnigan) equipped with electrospray
source and run by Xcalibur (Thermofinnigan) version 1.3
software. The HPLC separation was performed using the
conditions described above. The following conditions were
used in experiments with the ESI source in positive and neg-
ative modes: temperature of the heated capillary, 3508C;
source voltage 4.5 kV. Nitrogen was used as sheath gas and
auxiliary gas. The sheath and auxiliary gas flow rates were
80 and 20 arbitrary units, respectively. HPLC-MS was per-
formed in the full scan mode from m/z 50 to 2,000. All the
fragmentation experiments were done with 50% collision
energy.
GC-MS Analysis
The LAC3 reaction was stopped at times 0, 20, 40 and
60 min and the mixture extracted with diethyl ether (v/v)
containing 5 mg of 2-cyclohexenone as an internal standard.
The organic phase was first dried over sodium sulfate and
then analysed by GC-MS using an HP-6890 GC apparatus
coupled to an HP-5973 mass spectrometer. The column was
a DBWAX (30 m”0.25 mm, 0.25 mm) and He was the carri-
er gas delivered at a 1 mL·minÀ1 flow rate. The following
programme was used for the elution: 408C for 2 min then
rising to 1508C at 108CminÀ1 and hold for 10 min. GC-MS
analysis of CotA reactions were performed at the Analytical
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Toxicity Analysis
A previously described microplate toxicity assay based on
the inhibitory effects on the growth of a cell population of
S. cerevisiae BY4741 (at pH 6.5) was used with minor adap-
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(LOEC) is defined as the lowest concentration of the dye
that affected yeast growth by around 5% and IC50 is the
concentration at which toxic effects produce 50% inhibition
of growth. Data reported are average values with standard
deviations of results from at least three independent toxicity
tests (with three replicas).
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This work was supported by FP6-NMP2-CT-2004-505899
and PTDC/BIO/72108/2006 grants. L. Pereira holds a Post-
Doc fellowship (SFRH/BPD/20744/2004) from Fundażo
para a CiÞncia e Tecnologia (FCT), Portugal. We acknowl-
edge CERMAX at ITQB and Rede Nacional de RMN
(FCT) for access to the NMR facilities, and Helena Matias
for help in data acquisition. We thank Ana Rita Ferreira and
Catarina Costa for help in the toxicity assays.
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