Bacterial Enzymes and Multi-enzymatic Systems for Cleaning-up Dyes from the Environment - Microbial Degradation of Synthetic Dyes in Wastewaters - page 35

3 Biotransformation by Bacterial Azoreductases

Azoreductases is a generic name given to enzymes involved in the reduction of azo
bonds ( ed in several organisms

including algae, yeast and bacteria (Saratale et al. 2011 ; Sol

- N=N - ) and azoreductase activity was identi

í

s et al. 2012 ;
Chengalroyen and Dabbs 2013 ; Khan et al. 2013 ). These enzymes are avin-

independent or avin-dependent oxidoreductases which utilize NADH and/or

NADPH as an electron donor and catalyze the reductive cleavage of the azo bonds
to produce colorless aromatic amine products under anaerobic or aerobic condi-
tions. Flavin-dependent azoreductases share strong similarities with regard to
sequence, structure, and reaction mechanism with the larger family of

avin-
dependent quinone reductases that include Lot6p from Saccharomyces cerevisiae
and the mammalian NQO1 (Deller et al. 2008 ). These enzymes are involved in the
reduction of quinones, quinoneimines, azo dyes, and nitro groups to protect the
cells against the toxic effects of free radicals and reactive oxygen species arising
from electron reductions. They are assumed to take part in the organism ' s enzy-

matic detoxi cation systems; e.g., the azoreductases from E. coli and B. subtilis

were recently implicated in the cellular response to thiol-speci

c stress (Towe et al.
2007 ; Leelakriangsak et al. 2008 ; Liu et al. 2009 ) and Lot6p, the azoreductase
homologue in S. cerevisiae has been implicated in the response to oxidative stress
(Sollner et al. 2007 , 2009 ). Furthermore, as additional members of this family of
enzymes are discovered, the list of transformed substrates continues to grow.
Evolutionarily, these enzymes may provide a selective advantage to bacteria under
various conditions of environmental stress (Khersonsky and Taw k 2010 ).

3.1 Decolorization of Azo Dyes by PpAzoR
from Pseudomona putida MET94

In an effort to nd bacterial strains with a superior ability to degrade synthetic dyes,

a collection of 48 bacterial strains was screened to select the strain P. putida
MET94 for its superior ability to decolorize a diverse array of azo dyes to higher
extent (Mendes et al. 2011b ) (Fig. 7 ).
A BLAST search of the P. putida genome was performed and a 612-bp ORF
encoding a 203 amino acid residue was identi ed containing all the conserved motif

patterns of avin-dependent azoreductases (Wang et al. 2007 ) and was, therefore,

named PpAzoR (Pseudomonas putida azoreductase). The ppAzoR gene was cloned
and expressed in E. coli. Subsequently, the recombinant FMN-dependent PpAzoR
protein was puri

ed and thoroughly characterized following kinetic, spectroscopic
and biochemical and structural approaches (Correia et al. 2011 ; Mendes et al. 2011b ;
Gon alves et al. 2013 ). It was observed that PpAzoR reduced several quinones

(anthraquinone-2-sulfonic acid (AQS), 1,4-benzoquinone, catechol, 2-hydroxy-1,
4-naphtoquinone (Lawsone), 1,2-naphthoquinone) at rates 10

ç

100 times higher than

-