Environmental Engineering Reference
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O
NH
2
SO
3
-
O
HN
O
NH
2
O
NH
2
SO
3
-
SO
3
-
(
1B
)
O
HN
O
N
(
1
)
O
NH
+ H
2
O
SO
3
-
O
NH
SO
3
-
O
NH
2
O
SO
3
-
O
HN
+
O
NH
2
(
2A
)
O
NH
2
(
1A
)
(
2
)
(
3
)
i,ii)
i,ii)
i) radical coupling
ii) oxidation
O
NH
2
O
HN
O
NH
2
SO
3
-
O
N
SO
3
-
O
N
SO
3
-
SO
3
-
O
N
O
N
O
N
O
N
SO
3
-
SO
3
-
O
NH
2
(
5
)
O
HN
O
HN
(
6
)
(
4
)
Fig. 6 Proposed mechanism of AB62 biotransformation by laccases. Two oxidative routes are
possible, since laccases are able to catalyze a single-electron oxidation, either from the primary or
the secondary amines of the compound 1 (AB62). The reactive radical species 1A and 1B are
formed, and 1B was sequentially oxidized into an imine, which hydrolyzes leading to
cyclohexanone (3) and the
rst intermediate 2 (DAAS). Compound 2 could be further oxidized
and the resulted radical (2A) should lead to the formation of the main product of the reaction, the
azo dimer (4) by cross-coupling reaction with 1A, followed by an oxidative step that could also be
catalyzed by the enzyme. Similar dimerization processes of radicals 2A or 1A should end in the
formation of compounds 5 and 6, but in a very low extent. The
nal product 4 was identi
ed by
both NMR and MS techniques and the formation of compounds 5 and 6 was supported by LC-MS
n
analysis of the reaction mixtures (adapted from Pereira et al.
2009a
)
The toxicity of synthetic dyes as well as of their bioconversion products presents
a great environmental concern (O
'
Neil et al.
1999
; Robinson et al.
2001
). AB62
causes a signi
cant inhibitory effect on yeast growth and values of LOEC and IC
50
of around 7 and 420
M (3 and 177 mg L
−
1
) respectively, were estimated. The IC
50
is well above the expected dye concentrations in the environment, but is within the
same order of magnitude of the typical dye concentration in spent dye baths
(Robinson et al.
2001
). The mixture, containing AB62 biotransformation products
after 2 h of reaction with CotA-laccase, was signi
ʼ
cantly less toxic to the yeast cell
population. Consistent with the reduced overall toxicity of AB62 solution, com-
pound 4, the reddish azo product that accumulates during the biotransformation
reaction, is signi
cantly less toxic to the yeast (LOEC
45
ʼ
M, IC
50>
750
ʼ
M) than
*
the parent molecule.
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