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

### **6.1. Free enzyme biodegradation of industrial 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 target effluent in different ways in either cell-free or immobilized form.

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

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

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

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 on immobilization leading to restricting the protein unfolding process, as a result of the introduction of random intra- and intermolecular cross-links [155]. The development of immobilization methods has consequently caused a significant increase in the application of oxidoreductases in various technological processes [156].

Laccases have been immobilized on various supports such as glass-ceramic materials, imidazole-modified silica, montmorillonite, alginate-gelatin mixed gel, hydrophobic sol-gel, and green coconut fiber and applied in decolorization/degradation of various textile and nontextile dyes and phenolic compounds [27, 157-160]. For example, *Trametes modesta* laccase immobilized on alumina decolorized 41 commercial azo, triphenylmethane, indigoid, and heterocyclic dyes. The immobilization of laccase on alginate mixed gels or hydrophobic solgel also led to it improved pH stability, thermostability, and reusability of the enzymes, although a slight decrease in enzyme activity and dye affinity was observed. The entrapment of laccase in alginate-gelatin, alginate-chitosan mixed gels, or hydrophobic sol-gel matrix of trimethoxysilane and proplytetramethoxysilane has been reported to lead to significant lacasse stability toward heat denaturation [161]. The reported improved decolorization of wastewaters by immobilized laccases is attributed to both enzymatic catalysis and support adsorption [112].

There are comparatively fewer investigations on dye decolorization by immobilized peroxi‐ dases, probably due to their requirement of H2O2 for activity. For peroxidase catalysis, H2O2 must be added or generated *in situ* to avoid enzyme deactivation and to achieve a stable decolorization process [158, 162]. For example, the half-life of *Saccharum spontaneum* peroxidase immobilized on polyethylene was favored by careful addition of H2O2 to the reactor to decolorized 15 batches of Procion green HE-4BD [158]. Higher loading rates of H2O2 resulted in 50% loss in decolorization activity of Orange II within 2 h by *Bjerkandera* sp MnP in a membrane reactor; however, the enzyme maintained 96% efficiency under optimized H2O2 and enzyme feeding rates [163]. Similar to laccases, the immobilization of peroxidases into a sol-gel matrix of tetramethoxysilane and propyltrimethoxysilane or in alginate gel and mixed alginate-pectin gel improves their storage stability, pH stability, and thermostability, in addition increased enzymes reusability and decolorization efficiency. The above-mentioned examples illustrates importance of immobilization as a powerful technique in expanding the application of oxidoreductases in bioremediation, particularly in those circumstances where the enzyme can be reused in the application many times to reduce operation costs.
