**3. Principles of enzymatic bioremediation**

Bioremediation techniques have been gaining increasing prominence worldwide due to high public acceptance, low cost compared to conventional remediation methods, high availability of enzymes, and minimal impact on the environment [31]. The exploration of enzymes for bioremediation has been of great interest due to their ability to function in wider ranges of pH and temperature, in the presence of contaminants and saline concentrations [32]. Enzymatic bioremediation is an ecological, economical, promising, and innovative technique. The process consists of exploring the typical characteristics of microorganisms or genetically modified organisms capable of producing specific enzymes to catalyze or metabolize the pollutant, transforming the toxic form into a nontoxic form and sometimes into new products [33].

Among the enzymes involved in bioremediation processes are laccases, dehalogenases, and hydrolases. Laccases are enzymes capable of catalyzing the oxidation of phenolic compounds, aromatic amines, and their compounds. Dehalogenases degrade a wide range of halogenated compounds by cleaving C – X bonds (X = halogen atom, such as Cl). Hydrolases break chemical bonds using water and convert larger molecules into smaller molecules, decreasing their toxicity. These enzymes facilitate the cleavage of C – C, C – O, C – N, S – S, S – N, S – P, C – P bonds [33].

Enzymes can be used in free or immobilized form, the latter having the following advantages—long-term operational stability, easy recovery, and reuse in industrial applications, which improve process performance and lower overall cost [34].

Immobilization consists of coupling the enzyme with an insoluble support matrix to maintain an adequate geometry, which guarantees greater stability to the enzyme [32]. The bioremediation process using microbial enzymes can be slow and so far, only a few bacterial species have been able to produce enzymes with potent biodegradation capacity. Thus, the use of genetically modified organisms is more common due to their ability to produce large amounts of enzymes under optimized conditions [33].

Enzymes from aerobic bacteria, such as *Pseudomonas*, *Alcaligenes*, *Sphingomonas*, *Rhodococcus,* and *Mycobacterium*, are often used in the bioremediation of pesticides and hydrocarbons, while those produced by anaerobic bacteria are more used in bioremediation of polychlorinated biphenyls (PCBs), trichloroethylene (TCE) decolorization, and chloroform. The main enzymes used in bioremediation processes include those of the cytochrome P450 family, laccases, hydrolases, dehalogenases, dehydrogenases, proteases, and lipases [33]. Fungi can also biodegrade, generally mediated by enzymes, such as azoreductases, lignin peroxidases, manganese peroxidases, and laccases. White rot fungi, for example, are capable of degrading textile dyes through peroxidases and laccases [10].

In the treatment of effluents from the textile industry, enzymes act on the dyes, generating precipitates that can be easily removed or chemically transformed into easy-to-treat compounds [35]. The rate of dye degradation by enzymes will depend on the chemical structure of the dye, salt content, the concentration of metal ions, pH, and temperature of the wastewater [36]. The enzymatic degradation of pollutants in textile effluents has several advantages, such as specificity and selectivity to the substrate, in addition to being an accessible, efficient method that meets the principles of green chemistry [37]. The requirement of large amounts of enzyme, high cost, thermal instability, inhibition of enzymatic activity, attack of certain enzymes by proteases, and the formation of undesirable by-products are the main difficulties or challenges related to the use of enzymatic degradation for wastewater treatment [30].

Some of the problems listed can be solved, at least partially, by immobilizing effective enzymes in low-cost matrices, leading to their separation and reuse, in addition to application in continuous bioreactors [30]. To control the reactions in the biodegradation process, the use of enzymes is often more advantageous than the use of cells [37]. As for the high cost of the enzymes themselves due to the fact of trying to obtain an enzymatic solution as pure as possible, the tendency is that it will decrease as technologies and techniques advance and the exploration of cheaper growth substrates for the reproduction of microorganisms increases.
