Environmental Engineering Reference
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( a)
( b)
2.0
160
140
1.6
120
100
1.2
80
7h
0.8
60
5h
40
2h
0.4
1h
20
30min
t0
0.0
0
300
400
500
600
700
800
0
5
15
25
35
45
55
Wavelength [nm]
Retention time [min]
Fig. 2 Time course for Sudan orange G (SOG) biotransformation as monitored by absorbance
(a) and by HPLC (b). [(b) black circle SOG and products: black square R t 2.2 min and, black
triangle R t 15 min] (Pereira et al. 2009b )
(Fig. 2 b). Two major products emerged at R t of 2.2 and 15 min. The CotA ef
-
10 3 M 1 s 1 , is in the same order of magnitude to those calculated for
fungal laccases (Pereira et al. 2009b ). The assay mixtures became browner in color
over the course of reaction, presumably due to formation of products (Fig. 3 ). After
centrifugation the
ciency of 8
×
nal reaction mixture, the supernatant contained the compounds
corresponding to the major peak with R t of 2.2 min and the pellet contained the
major product with R t of 15 min. The full identi
cation of this latter fraction was
impaired by its low solubility in several solvents: acetone, ethanol, methanol,
chloroform, dichloromethane, ethyl ether, toluene, hexane and tetrahydrofuran.
A partial solubility (25 %) was found on acetonitrile and thus, the identi
cation of
products was performed only in the soluble part of acetonitrile-dissolved fraction.
The structural identi
cation of twelve SOG biotransformation products (Fig. 4 b)
was based on ESI-MS and MALDI-TOF MS data in combination with a putative
degradation pathway (Fig. 4 a) based on the accepted model for azo dye degradation
by laccases (Chivukula and Renganathan 1995 ; Zille et al. 2005a , b ).
Our results indicate that the enzymatic electron transfer occurs upon oxidation of
SOG deprotonated hydroxyl group. The one-electron oxidation of SOG molecule
by the enzyme results, therefore, in the formation of unstable radical molecules and
in the concomitant destruction of dye chromophoric structure in accordance with
previous reports (Chivukula and Renganathan 1995 ; Zille et al. 2005a , b ). In
addition, the presence of these products can undergo coupling reactions between
themselves or with unreacted dye molecules, producing a large array of oligomeric
products (Fig. 4 b). The presence of these compounds is in accordance with the
darkening of the enzymatic reactions, the high insolubility of products formed, and
also with the reduced toxicity of the
nal reaction mixture as compared to solutions
of intact SOG which was tested using a yeast-based bioassay (Pereira et al. 2009b ).
 
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