Bacterial Enzymes and Multi-enzymatic Systems for Cleaning-up Dyes from the Environment - Microbial Degradation of Synthetic Dyes in Wastewaters - page 38

( a) ( b)

120 120

100 100

80

80

PpAzoR + Catalase

PpAzoR

60

60

40

40

Catalase

20

20

0

0

0 100 200 300 400 500 0 1 2 3 4 0 1 2 3 4 5

[O 2 ] (nmol/ml) Time (min) Time (min)

Fig. 9 a Inhibition of dye decolorization by increasing concentrations of dioxygen. b Consump-
tion of oxygen with an equipped with a Clark oxygen electrode. The chamber volume

(1 mL) contained 0.25 mM of NADPH in 20 mM Tris-HCl, pH 7.6 buffer. Reaction was initiated
by the addition of 3.5

“ Oxygraph ”

M PpAzoR and 1,500 Units of catalase (Sigma) was added at different time
periods, as indicated by the arrows

µ

The steady state kinetics of PpAzoR for oxygen reduction (V max =5Umg − 1 , K m
app = 0.1 mM, k cat app = 238 min − 1 , k cat /K m = 7

10 4 M − 1 .s − 1 ) showing higher

×

af ty as compared to dyes reduction

which explains the competitive catalytic behaviour of oxygen and thus, the

nity and one order of magnitude higher speci

“ oxygen

sensitive character of PpAzoR and the need to perform the decolorization of dyes

under anaerobic conditions.

”

3.2 The Catalytic Mechanism of PpAzoR and the Toxicity
of Dye Products

The ping-pong bi bi mechanism is indicative that PpAzoR reduces the substrates in

-

2 distinct steps: rst, a complete reduction of the FMN co-factor; and second,

transfer of these electrons from the

avin to the substrate, resulting in the formation
of the corresponding putative hydrazo derivatives. This reaction cycle proceeds a
second time and delivers the necessary 4 electrons in order to obtain a complete
reduction of the substrates into the nal products. The mechanism of azo reduction

by PpAzoR was supported by the detection of aromatic amines by HPLC (Fig. 10 ).
The reported high toxicity of the azoreductase products relates to the toxic nature
of the aromatic amines formed (Pinheiro et al. 2004 ). Therefore, the toxicity of the
azo dyes and PpAzoR enzymatic products was tested based on the inhibitory effects
on the growth of Saccharomyces cerevisiae and on the reproduction inhibition of
Caenorhabditis elegans (Mendes et al. 2011a ). In general, the toxicity of intact dyes
correlates well between the 2 model eukaryotic organisms tested (Fig. 11 ). Nev-
ertheless, C. elegans seems more susceptible to the presence of intact dyes, since
some dyes show more than 2-fold higher inhibition for the nematodes reproduction
than for S. cerevisiae growth.