Bacterial Enzymes and Multi-enzymatic Systems for Cleaning-up Dyes from the Environment - Microbial Degradation of Synthetic Dyes in Wastewaters - page 40

Fig. 12 Inhibitory effects of
intact dyes on Saccharomyces
cerevisiae (dark bars) and of
24 h-reaction mixtures treated
with PpAzoR (dashed bars)
(adapted from Mendes et al.
2011a )

120

100

80

60

40

20

0

AR266 DB1 AB194 AR299 DB38 RR4 RB5 RY145 DR80

Azo dyes

correlates with longer life-times of enzymes and frequently relates also to higher
tolerance to the presence of organic co-solvents, extreme pH values and high salt
concentration or pressures, harsh conditions frequently found in industrial pro-
cesses. Therefore, thermostable PpAzoR variants were generated by directed evo-
lution (Brissos et al. 2014 ). Directed evolution is considered to be the most
powerful approach for improving the thermostability of proteins. Different prop-
erties in various target enzymes have been successfully improved using directed
evolution approaches (Kaur and Sharma 2006 ;B

ö ttcher and Bornscheuer 2010 ;

Wang et al. 2012 ). After ve rounds of random mutagenesis, recombination and

high-throughput screening, a thermostable 1B6 variant was identi

ed. Noteworthy

puri ed 1B6 variant enzyme maintains its full activity after incubation for 1 h at

temperatures between 40 and 85 C in clear contrast with the wild type enzyme that

totally looses activity after 1 h at 50

°

C (Fig. 13 a). The kinetic or, the so called,
long term stability was measured, showing that 1B6 is remarkably more stable than
wild type with nearly a 300-fold higher half-life, i.e. retaining 50 % of activity after
58 h at 50

55 °

-

C, while wild type enzyme takes 13 min to lose half of its initial activity
(Fig. 13 b). Therefore, a hit variant of PpAzoR was identi

°

ed with increased
resistance to inactivation, showing full reversibility of the unfolded state upon
thermal inactivation i.e. it could be maintained at high temperatures for prolonged
periods of time without losing its ability to be active at lower temperatures with an
encouraging potential for biotechnological applications.

4 Biotransformation of Dyes Using Bacterial
Dye-Decolorizing Peroxidases

Heme peroxidases catalyse the H 2 O 2 -dependent oxidation of a variety of substrates,
most commonly small organic substrates, playing multiple physiological roles in a
wide range of living organisms. Considering their broad speci city, these enzymes