Bacterial Enzymes and Multi-enzymatic Systems for Cleaning-up Dyes from the Environment - Microbial Degradation of Synthetic Dyes in Wastewaters - page 30

9 is a dis-
tinctive feature shared with other bacterial laccases from Streptomyces ipomoea
(Molina-Guijarro et al. 2009 ), Bacillus vallismortis (Zhang et al. 2012 )orBacillus
subtilis X1 (Guan et al. 2013 ) which is in contrast with the optimal pH values in the
acidic range shown by laccases of fungal origin (Abadulla et al. 2000 ; Almansa
et al. 2004 ; Maier et al. 2004 ; Camarero et al. 2005 ; Rodriguez Couto et al. 2005 ;
Zille et al. 2005a , b ; Pogni et al. 2007 ). In order to explore the enzymatic mech-
anism of azo dyes degradation, Sudan orange G (SOG) was selected for more
detailed investigations. Two pKa values for SOG were measured using potentio-
metric measurements, 6.90

The optimal pH for dye-decolorization by CotA-laccase around 8

-

0.02, which were attributed to ortho
and para hydroxyl groups of the azo dye (Pereira et al. 2009b ). Based on this data,
the oxidation of SOG is mostly dependent on the protonation-deprotonation equi-
librium of the more acidic hydroxyl group of the substrate molecule, since maximal
rates are found at pH 8, above the pKa value of the ortho hydroxyl group of SOG.
This is in contrast with fungal laccases, which, in agreement with their optimal
pH at acidic ranges, oxidise more easily the protonated form of the dye. The
results obtained with CotA for the oxidation of SOG are consistent with data
obtained using syringyl-type phenolic compounds, where maximal enzymatic
rates were also observed at pH values above the pKa value of the compounds
tested which con

± 0.02 and 11.74 ±

rmed the preference of CotA for deprotonated phenolic groups
(Rosado et al. 2012 ). The differences in the optimal pH, as observed in bacterial
and fungal laccases, are most probably related to the presence of a conserved
negatively charged residue close to the substrate binding cavity of fungal laccases
and absent in CotA or in any bacterial laccase identi ed so far and proposed to

stabilize the formation of the phenoxy radical during the catalytic reaction of
fungal laccases (Bertrand et al. 2002 ; Piontek et al. 2002 ; Madzak et al. 2006 ;
Kallio et al. 2009 ; Rosado et al. 2012 ). Therefore, the oxidation of phenolic
groups by bacterial laccases without any carboxylic acid residue in the substrate
binding site is strictly dependent on the chemical nature of the substrates i.e.
maximal rates are found at pH values above the pKa values, when phenolate
anions, which are more prone to oxidation than the phenol form, are present at
higher concentrations.

2.2 Azo Dyes Biotransformation

The transformation of SOG (Pereira et al. 2009a ) resulted in a decrease in the
intensity of the dye absorption band, at λ max = 430 nm, with concomitant increase in

absorption bands at 325 and 530 nm, indicating the generation of biotransformation
products (Fig. 2 a).
The time course of SOG biotransformation was additionally monitored by HPLC
(Fig. 2 b), where SOG was chromatographically separated from products of the
enzymatic reaction. A major peak with R t of 5 min, corresponding to the substrate
which decreased over the time course of the reaction and disappeared after 7 h