**7. Future prospects and conclusion**

[128]. The substitution of the aromatic amino acids residues with nonaromatic residues of *T. versicolor* laccase resulted in increased resistance to inactivation by free radicals [150]. Directed evolution has been used to increase laccase activity by 170- to 32,000-fold, pH, and temperature stability [151, 152]. The resultant mutants also exhibited increased tolerance to organic solvents such as ethanol and acetonitrile by 30% and 20%, respectively [153]. In addition, the directed evolution of high redox potential laccases has been used to overcome the obstacles associated with their functional expression in host suitable for *in vitro* evolution experiments [151, 153].

**6. Current and potential synthetic dye biodegradation applications**

target effluent in different ways in either cell-free or immobilized form.

catalyst at the end of the reaction from the reaction mixture [155].

**6.2. Immobilized enzyme bioremediation of synthetic dyes**

Enzymes, as potent biocatalysts, have been employed in numerous fields primarily for their immense catalytic potential [36]. In dye bioremediation, cell-free or isolated enzymes are preferred over the intact organism, especially when the effluent to be treated contains pollutants that cannot support growth. The key to successful application of enzymes for dye decolorization is the selection of appropriate enzyme cocktail that will exhibit versatility and efficiency, even under mild reaction conditions. The delivery system selected must be well suited to the purpose, simple, efficient, and cost-effective. Enzymes may be delivered to the

Currently, there are commercial preparation of peroxidases and laccases used for different applications. DeniLite II S®, a commercial laccase formulation containing laccase, a mediator, and a nonionic surfactant, is used in the finishing process for indigo stained clothes [154]. A laccase from ascomycete fungus *Myceliophthora thermophila*, sold commercially by Novozymes as Suberase®, is used in the removal of astringency of cork stoppers for wine bottles. Ligno‐ Zym® system (GmbH), a mixture containing *T. versicolor* laccase and a group of mediators (N-OH-, N-oxide-, oxime-, or hydroxamic acid–functional groups), is used to remove 50% to 70% of lignin from pulp within 1 to 4 h [154]. However, the potential of these commercial laccase

In the industrial scale of operation, the use of pure enzymes in effluent treatment is not economically feasible due to high start-up and running costs. The use of free enzymes as compared with their immobilized forms also show some significant drawbacks such as (1) thermal instability, (2) susceptibility to attack by proteases, (3) activity inhibition, (4) high sensitivity to several denaturing agents, and (5) difficulty of separating or reusing the free

In recent years, it has been shown that many industrial dyes can be decolorized by laccases and peroxidases immobilized on different supports. In contrast to soluble enzymes, immobi‐ lization offers higher enzyme stability, reusability, and capability to work in aqueous as well as in organic solvents due to protection against denaturants and proteolysis and reduced susceptibility to microbial contamination. This may be partly due to enzyme stabilization effect

preparations for treatment of effluents containing dyes has yet to be demonstrated.

**6.1. Free enzyme biodegradation of industrial dyes**

130 Advances in Bioremediation of Wastewater and Polluted Soil

In this chapter, we have discussed the descriptive information on the oxidoreductases from various microorganisms, including their discovery, biochemistry, current biodegradation applications, and limitations to their large scale and efficient use. An ideal enzyme for dye bioremediation application should have the following properties: (i) broad substrate specific‐ ity; (ii) high redox potential; (iii) high tolerance to inactivation by radicals, organic solvents, and shearing forces; (iv) ability to work with a large number of mediators; (v) broad pH and temperature optima; (vi) high enzyme activity and stability; and (vii) low production costs. The current oxidoreductases such as peroxidases and laccases are not well suited for industrial applications that require particular substrate specificities and application conditions (pH and temperatures) in addition to high expression levels. Consequently, effluent treatment using enzymes on a large scale is still not economically viable.

Within the last decade, there is increased research interest in the application of genetic engineering techniques to develop "designer" enzyme cocktails for large-scale dye bioreme‐ diation applications in different industrial sectors. The availability of high-throughput screening assays and functional expression systems plus the crystal structures of the enzymes has helped these efforts enormously. Breakthroughs through protein engineering involving combination of directed evolution with both hybrid and rational approaches, including computational studies, will permit the conversion of the current array of oxidoreductases into versatile biotechnological products for dye bioremediation. In addition, an interdisciplinary approach to wastewater treatment involving nanotechnology and enzyme technology will enable the utilization of peroxidases, laccases, and azoreductases to their full potential. These studies need to be conducted in the context of dye mixtures simulating real dyeing baths, as real bioremediation process is affected by all the factors involved in the dyeing processes, such as components and auxiliaries present in the wastewater, that markedly affects dye decolori‐ zation and/or the enzyme.

In conclusion, the promise of the concerted research efforts evident thus far and the potential of modern microbial and enzyme technologies to make radical improvements in the oxidore‐ ductases give confidence that the development of successful technologies for industrial synthetic dye bioremediation will be possible in the near future.
