**4. Enzyme-linked biotransformation of industrial dyes**

#### **4.1. Enzymatic degradation of azo dyes**

A general mechanism for the peroxidase-catalyzed oxidation mechanism for azo dye degra‐ dation, via either the symmetrical and/or asymmetrical azo bond cleavage, has been proposed. Goszczynski et al. [108] proposed mechanisms in the course of which these enzymes convert dyes to cation radicals and become susceptible to nucleophilic attack by water or hydrogen peroxide. This results in the simultaneous split of the azo linkage, both symmetrically and asymmetrically, to produce intermediates that subsequently undergo several redox reactions, such as depolymerization, repolymerization, and demethylation, finally leading to more stable intermediates. In this reaction scheme, two successive one-electron oxidations of the phenolic ring, by the hydrogen peroxide–oxidized forms of the enzyme, produce a carbonium ion. A water molecule then reacts with the phenolic carbon, bearing the azo linkage, and an unstable hydroxyl intermediate, which breaks down into a quinone, and an amidophenyldiazine, is formed. The latter compound is then oxidized, by oxygen, into the corresponding phenyldia‐ zene radical, which, after elimination of nitrogen, gives a phenyl radical that is reoxidized by oxygen. This mechanism leads to the detoxification of azo dyes because no aromatic amines are formed. LIP, MnP, and VP from basidiomycetous fungi are able to oxidize several azo dyes by following this mechanism [20, 43, 48, 54, 61-63]. Although exhibiting a general preference toward trichromatic AQ dyes than azo dyes, it is possible that DyP-type peroxidases also use the same mechanism. azo dyes because no aromatic amines are formed. LIP, MnP, and VP from basidiomycetous fungi are able to oxidize several azo dyes by following this mechanism [20, 43, 48, 54, 61-63]. Although they exhibit a general preference toward trichromatic AQ dyes than azo dyes, it is possible that DyP-

type peroxidases also use the same mechanism.

*sphaeroides* [93-101]. There are at least two different types of bacterial azoreductases: those that require flavin and those that do not [94]. Flavin-dependent azoreductases can be further classified into two families according to their amino acid sequences. Azoreductases from *E. coli* and *Bacillus* sp. strain OY1-2 are representative of the two flavin-dependent azoreductases, respectively. *Bacillus* sp. strain OY1-2 azoreductase is a 23-kDa protein with the ability to reduce the azo dyes Rocceline, Sumifix Red B, and Methyl red, producing dimethyl pphenylenediamine and o-aminobenzoic acid in the presence of β-NADPH [102]. On the other hand, *Xenophilus azovarans* KF46 and *P. kullae* K24 azoreductases are monomeric flavin-free enzymes that use NADPH as a cofactor to degrade the azo dyes carboxy-Orange II and I. These two enzymes exhibit different substrate specificities and sizes (21 and 30 kDa) and require the

In addition to peroxidases, laccases, and azoreductases, the bioremediation of synthetic dye compounds with other enzymes such as tyrosinases [103], aryl alcohol oxidases [104], and biosulfidogenic hydrogenases [105] have been demonstrated. Similar to laccases, tyrosinases (monophenol monooxygenase, EC1.14.18.1) are oxidoreductases that can catalyze the oxida‐ tion of phenolic and other aromatic compounds, without the use of a cofactor in presence of oxygen. Catalytically, these enzymes possess both cresolase activity (ortho-hydroxylation of monophenols to *o*-diphenols) and catecholase activity (the oxidation of *o*-diphenols to *o*quinones) [103]. Tyrosinases also degrade aromatic amines and *o*-aminophenols via the similar ortho-hydroxylation and oxidation reactions. The resultant products undergo subsequent

Biotechnologically, tyrosinases are used as markers of the oxidative enzymes involved in the degradation of azo dyes. For example, the involvement of tyrosinase in the degradation of Direct Blue-6 by *Pseudomonas desmolyticum* NCIM 2112 [106], disperse dye brown 3REL by a microbial consortium consisting of *Galactomyces geotrichum* MTCC 1360, and sulfonated azo dyes by *Brevibacterium* sp. strain VN-15 [103] and *Bacillus* sp. VUS [62] has been demonstrated. The activity of aryl alcohol oxidase (AAO) has been reported in *B. adusta* Dec 1; it oxidizes VA to veratraldehyde producing H2O2, which is important for supporting the *in vivo* dye-decol‐ orizing activity of fungi [104]. The involvement of a similar enzyme in *Comamonas* sp. UVS during the decolorization of Red HE7B and Direct Blue GL has also been reported [107].

A general mechanism for the peroxidase-catalyzed oxidation mechanism for azo dye degra‐ dation, via either the symmetrical and/or asymmetrical azo bond cleavage, has been proposed. Goszczynski et al. [108] proposed mechanisms in the course of which these enzymes convert dyes to cation radicals and become susceptible to nucleophilic attack by water or hydrogen peroxide. This results in the simultaneous split of the azo linkage, both symmetrically and

presence of hydroxyl groups in the aromatic ring of the substrate [95].

**4. Enzyme-linked biotransformation of industrial dyes**

**3.4. Other dye-degrading enzymes**

122 Advances in Bioremediation of Wastewater and Polluted Soil

polymerization, giving rise to oligomeric products.

**4.1. Enzymatic degradation of azo dyes**

Figure 3. Decolorization of azo dyes in the presence of mediators by AnaPX. (a) Biotransformation of Reactive Black 5 by AnaPX in the presence of various mediators. Inset shows the structures of the mediators and the resultant product upon decolorization. (b) Decolorization profile of several azo dyes by AnaPX in presence of different mediator. **Figure 3.** Decolorization of azo dyes in the presence of mediators by AnaPX. (a) Biotransformation of Reactive Black 5 by AnaPX in the presence of various mediators. Inset shows the structures of the mediators and the resultant product upon decolorization. (b) Decolorization profile of several azo dyes by AnaPX in presence of different mediator. com‐ pounds.

Some azo dyes are oxidized effectively only in the presence of mediator compounds. In previous studies, we have shown that the azo degradation ability of AnaPX was significantly improved in the presence of redox mediators [75]. The decolorization range and oxidation rates of AnaPX, compared with HRP for azo dyes, increased markedly (2- to 5-fold) in the presence of a lignin-derived phenolic redox mediator, syringaldehyde (SA) (Figure 3). The degradation of Reactive Black 5, in presence of SA, was pH dependent, with the AnaPX-mediator reaction exhibiting maximal activity at pH 6.0, although the enzyme optimum pH is 4.5 [75]. Similar results have been reported in the presence of NOH-type (1-hydroxy-1H-benzotriazole (HOBt), *N*-hydroxyphthalimide (NHPI), 1 nitroso-2‐naphthol), and quinine-containing mediators such as 10-methylphenothiazine (10-MP); Some azo dyes are oxidized effectively only in the presence of mediator compounds. In previous studies, we have shown that the azo degradation ability of AnaPX was significantly improved in the presence of redox mediators [75]. The decolorization range and oxidation rates of AnaPX, compared with HRP for azo dyes, increased markedly (2- to 5-fold) in the presence of a lignin-derived phenolic redox mediator, syringaldehyde (SA) (Figure 3). The degradation of Reactive Black 5, in presence of SA, was pH dependent, with the AnaPX-mediator reaction exhibiting maximal activity at pH 6.0, although the enzyme optimum pH is 4.5 [75]. Similar

however, decolorization efficiencies were different, possibly due to differences in the type of dye structure (Figure 3). Differences in the position of the substituents (-CH3, -OCH3) and/or their substitution with -Cl or -NO2 groups on the phenolic ring affect the electronic character of dye compounds and may render the azo dye more or less susceptible to oxidation by enzymes, resulting in the observed dye-structure-related effects on decolorization efficiencies [109]. Our earlier work also showed that the transformation of Reactive Black 5 resulted in a decrease in the intensity of the dye absorption band, at *λ*max = 600, 400, and 310 nm, indicating the degradation of the dye. The complete decolorization of the dye resulted in the formation of light brownish products within 1 min in the presence of SA and 1-nitroso-2-naphthol. In contrast, other mediators showed varied decolorization efficiency for Reactive Black 5, illustrating the importance of mediator specificity toward different functional groups. It has been postulated that phenoxy radicals from SA act similarly to the -NO-

results have been reported in the presence of NOH-type (1-hydroxy-1H-benzotriazole (HOBt), *N*-hydroxyphthalimide (NHPI), 1-nitroso-2-naphthol), and quinine-containing mediators such as 10-methylphenothiazine (10-MP); however, decolorization efficiencies were different, possibly due to differences in the type of dye structure (Figure 3). Differences in the position of the substituents (-CH3, -OCH3) and/or their substitution with -Cl or -NO2 groups on the phenolic ring affect the electronic character of dye compounds and may render the azo dye more or less susceptible to oxidation by enzymes, resulting in the observed dye-structurerelated effects on decolorization efficiencies [109]. Our earlier work also showed that the transformation of Reactive Black 5 resulted in a decrease in the intensity of the dye absorption band, at *λ*max = 600, 400, and 310 nm, indicating the degradation of the dye. The complete decolorization of the dye resulted in the formation of light brownish products within 1 min in the presence of SA and 1-nitroso-2-naphthol. In contrast, other mediators showed varied decolorization efficiency for Reactive Black 5, illustrating the importance of mediator specif‐ icity toward different functional groups. It has been postulated that phenoxy radicals from SA act similarly to the -NO- radicals from -NOH- compounds, i.e., they extract a hydrogen atom from the substrate [110, 111]. It is very likely that the varied oxidative ability observed for the different AnaPX mediators is governed by the dissociation energy of the corresponding bond, which consequently affects the azo dye degradation.

The laccase-catalyzed oxidation of azo dyes has also been postulated to follow a similar mechanism, albeit with slight modifications. In this reaction scheme, fungal laccases oxidize azo dyes through a highly nonspecific free radical mechanism to form phenolic type com‐ pounds without the cleavage of the azo bond [89, 109, 112]. A similar mechanism has been reported in the biotransformation of the azo dye, Sudan Orange G by bacterial Cot-laccase from *B. subtilis*, that exhibits an optimal pH of around 8–9 for dye decolorization [89]. Accord‐ ing to this mechanism, lacasses initially catalyze a one-electron transfer reaction of the dye to generate a phenoxyl radical that is sequentially oxidized to various unstable radical molecules, with the concomitant destruction of the chromophoric structure of the dye. The resultant biotransformation radical species undergo coupling reactions to form less toxic oligomeric or polymeric condensation products. This laccase-catalyzed oxidation of phenolic azo dyes is however dependent on the electronic character and position of the substituent on the phenolic ring [89, 109]. For example, azo dyes with electron-donating 2-methyl or 2-methoxy substitu‐ ents are more easily oxidized than compounds containing a methyl group in position 3, or those unsubstituted or substituted with 2-chloro and 2-nitrogroups. Generally, the laccasecatalyzed oxidation of azo dyes excludes the formation of toxic aromatic amines obtained under reductive conditions, making these enzymes important in azo dye bioremediation.

All azoreductases reduces azo compounds via a Ping Pong Bi Bi mechanism [94, 101]. In the proposed mechanism, azo compound reduction undergoes two cycles of NAD(P)H-depend‐ ent reduction; the azo substrate is reduced to a hydrazine in the first cycle, and the hydrazine is further reduced to two amines in the second cycle. In this reaction, FMN serves as a redox center in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate [94, 113]. However, corresponding aromatic amines, formed during anaerobic azo reduction, are generally more toxic, mutagenic, and carcinogenic than azo decolorization of RB5 and Acid Blue 45 by AnaPX results in a decrease in absorbance at 600 nm and

reduction; the azo substrate is reduced to a hydrazine in the first cycle, and the hydrazine is further reduced to two amines in the second cycle. In this reaction, FMN serves as a redox center in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate [94, 113]. However, corresponding aromatic amines, formed during anaerobic azo reduction, are generally more toxic, mutagenic, and carcinogenic than azo substrates [32, 94, 113]. Moreover, the requirement for expensive cofactors is a barrier to the wider utilization of azoreductases in

Although there are many reports on the involvement of peroxidases in the biodegradation of azo dyes as described above, very few studies have reported the degradation of anthraquinoic (AQ) dyes by these peroxidases. Since the first report on the DyP peroxidases' high specificity to AQ dyes [73], several proteins have been isolated and characterized, and their ability to decolorize synthetic dyes was demonstrated. In our study, AnaPX decolorized o v e r 90% of the AQ dyes— Reactive Blue 5 (262 U mg-1), Reactive Blue 4 (167 U mg-1), Reactive Blue 114 (491 U mg-1), and Reactive Blue 19 (401 U mg-1)—within 5 min [75]. These dyes have a vinyl sulfonic reactive moiety in their structure; their aromatic anthracene-9,10-dione structure is highly stabilized by resonance, accounting for their general resistance to both chemical and enzymatic oxidation. The enzyme also decolorized over 70% of Reactive Blue 4 and the triazine dyes, Procion Blue H-ERD and Procion Blue H-EXL, within 2 h. The kinetic parameters determined for AnaPX clearly revealed

> Figure 4. Biotransformation of AQ dye Reactive Blue 5 by AnaPX. The formation of characteristic light reddish brown product (2,2-disulfonyl azobenzene) upon enzymatic degradation of (a) Reactive Blue 5 and (c) other AQ dyes, Reactive Blue 4, Reactive Blue 19, and Reactive Blue 114. (b) UV absorption spectra of the degradation process showing the decrease in absorbance at 600 nm and an increase in absorbance at 400– 500 nm. Each spectrum was taken

after 4-s intervals.

4.2 Enzymatic biotransformation of anthraquinone dyes

an increase in absorbance at 400–500 nm, accompanied by the formation of a light reddish-brown product (Figure 4). Similar results have been reported in other DyP peroxidases [75-78, 82, 83, 114- 117]. **Figure 4.** Biotransformation of AQ dye Reactive Blue 5 by AnaPX. The formation of characteristic light reddish brown product (2,2-disulfonyl azobenzene) upon enzymatic degradation of (a) Reactive Blue 5 and (c) other AQ dyes, Reac‐ tive Blue 4, Reactive Blue 19, and Reactive Blue 114. (b) UV absorption spectra of the degradation process showing the decrease in absorbance at 600 nm and an increase in absorbance at 400–500 nm. Each spectrum was taken after 4-s in‐ tervals.

substrates [32, 94, 113]. Moreover, the requirement for expensive cofactors is a barrier to the wider utilization of azoreductases in bioremediation. The biotransformation of AB62 (an AQ), by B. subtilis Cot-laccase, also results in a decrease in the intensity of the dye absorption bands, at λmax = 600 and 630 nm, along with an increase in absorption around 500 nm, due to the formation of reddish products [118]. Thus, it is probable that

#### **4.2. Enzymatic biotransformation of anthraquinone dyes** both laccase and peroxidase utilize similar mechanism for AQ degradation. In the transformation of Reactive Blue 5 by B. adusta Dec 1 DyP, analysis of the final enzymatic

bioremediation.

results have been reported in the presence of NOH-type (1-hydroxy-1H-benzotriazole (HOBt), *N*-hydroxyphthalimide (NHPI), 1-nitroso-2-naphthol), and quinine-containing mediators such as 10-methylphenothiazine (10-MP); however, decolorization efficiencies were different, possibly due to differences in the type of dye structure (Figure 3). Differences in the position of the substituents (-CH3, -OCH3) and/or their substitution with -Cl or -NO2 groups on the phenolic ring affect the electronic character of dye compounds and may render the azo dye more or less susceptible to oxidation by enzymes, resulting in the observed dye-structurerelated effects on decolorization efficiencies [109]. Our earlier work also showed that the transformation of Reactive Black 5 resulted in a decrease in the intensity of the dye absorption band, at *λ*max = 600, 400, and 310 nm, indicating the degradation of the dye. The complete decolorization of the dye resulted in the formation of light brownish products within 1 min in the presence of SA and 1-nitroso-2-naphthol. In contrast, other mediators showed varied decolorization efficiency for Reactive Black 5, illustrating the importance of mediator specif‐ icity toward different functional groups. It has been postulated that phenoxy radicals from SA act similarly to the -NO- radicals from -NOH- compounds, i.e., they extract a hydrogen atom from the substrate [110, 111]. It is very likely that the varied oxidative ability observed for the different AnaPX mediators is governed by the dissociation energy of the corresponding bond,

The laccase-catalyzed oxidation of azo dyes has also been postulated to follow a similar mechanism, albeit with slight modifications. In this reaction scheme, fungal laccases oxidize azo dyes through a highly nonspecific free radical mechanism to form phenolic type com‐ pounds without the cleavage of the azo bond [89, 109, 112]. A similar mechanism has been reported in the biotransformation of the azo dye, Sudan Orange G by bacterial Cot-laccase from *B. subtilis*, that exhibits an optimal pH of around 8–9 for dye decolorization [89]. Accord‐ ing to this mechanism, lacasses initially catalyze a one-electron transfer reaction of the dye to generate a phenoxyl radical that is sequentially oxidized to various unstable radical molecules, with the concomitant destruction of the chromophoric structure of the dye. The resultant biotransformation radical species undergo coupling reactions to form less toxic oligomeric or polymeric condensation products. This laccase-catalyzed oxidation of phenolic azo dyes is however dependent on the electronic character and position of the substituent on the phenolic ring [89, 109]. For example, azo dyes with electron-donating 2-methyl or 2-methoxy substitu‐ ents are more easily oxidized than compounds containing a methyl group in position 3, or those unsubstituted or substituted with 2-chloro and 2-nitrogroups. Generally, the laccasecatalyzed oxidation of azo dyes excludes the formation of toxic aromatic amines obtained under reductive conditions, making these enzymes important in azo dye bioremediation.

All azoreductases reduces azo compounds via a Ping Pong Bi Bi mechanism [94, 101]. In the proposed mechanism, azo compound reduction undergoes two cycles of NAD(P)H-depend‐ ent reduction; the azo substrate is reduced to a hydrazine in the first cycle, and the hydrazine is further reduced to two amines in the second cycle. In this reaction, FMN serves as a redox center in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate [94, 113]. However, corresponding aromatic amines, formed during anaerobic azo reduction, are generally more toxic, mutagenic, and carcinogenic than azo

which consequently affects the azo dye degradation.

124 Advances in Bioremediation of Wastewater and Polluted Soil

Although there are many reports on the involvement of peroxidases in the biodegradation of azo dyes as described above, very few studies have reported the degradation of anthraquinoic (AQ) dyes by these peroxidases. Since the first report on the DyP peroxidases' high specificity to AQ dyes [73], several proteins have been isolated and characterized, and their ability to decolorize synthetic dyes was demonstrated. In our study, AnaPX decolorized over 90% of the AQ dyes—Reactive Blue 5 (262 U mg-1), Reactive Blue 4 (167 U mg-1), Reactive Blue 114 (491 U mg-1), and Reactive Blue 19 (401 U mg-1)—within 5 min [75]. These dyes have a vinyl sulfonic reactive moiety in their structure; their aromatic anthracene-9,10-dione structure is highly stabilized by resonance, accounting for their general resistance to both chemical and enzymatic oxidation. The enzyme also decolorized over 70% of Reactive Blue 4 and the triazine dyes, Procion Blue H-ERD and Procion Blue H-EXL, within 2 h. The kinetic parameters determined for AnaPX clearly revealed that it has a higher affinity and greater redox potential for H2O2 and RB5 than HRP and other peroxidases. This may explain the higher decolorization activity of AnaPX toward RB5. The decolorization of RB5 and Acid Blue 45 by AnaPX results in a decrease in absorbance at 600 nm and an increase in absorbance at 400–500 nm, accompanied reaction mixtures, by NMR and MS techniques, showed that dye degradation results in three reaction by the formation of a light reddish-brown product (Figure 4). Similar results have been reported in other DyP peroxidases [75-78, 82, 83, 114-117].

The biotransformation of AB62 (an AQ), by *B. subtilis* Cot-laccase, also results in a decrease in the intensity of the dye absorption bands, at *λ*max = 600 and 630 nm, along with an increase in absorption around 500 nm, due to the formation of reddish products [118]. Thus, it is probable that both laccase and peroxidase utilize similar mechanism for AQ degradation.

In the transformation of Reactive Blue 5 by *B. adusta* Dec 1 DyP, analysis of the final enzymatic reaction mixtures, by NMR and MS techniques, showed that dye degradation results in three reaction products: (1) phthalic acid, (2) product 2 (*m*/*z* = 472) lacking the anthraquinone frame, and (3) product 3 (*m*/*z* = 305), formed from the loss a 2,5-diaminobenzene sulfonic acid (ABS) molecule from product 2 [119]. In the proposed reaction mechanism, the anthraquinone frame undergoes initial oxidative ring opening due to attack of the carbonyl group by the H2O molecule. This oxygenase/hydrolase-like activity leads to the production of phthalic acid. In contrast, the formation of products 2 and 3 proceeds via cationic radical catalysis, typical of peroxidases, followed by the subsequent dimerization and polymerization of the intermedi‐ ates to form final products with high molecular weights, such as 2,2-disulfonyl azobenzene. The formation of 2,2-disulfonyl azobenzene resulted in the characteristic reddish-brown product observed during the DyP-catalyzed degradation of Reactive Blue 5 (Figure 4) [75, 119]. Further treatment of the final product with TcVP1, a VP from *B. adusta* Dec 1, decolorized these colored intermediates to colorless by products [74]. The concerted action of these two enzymes, for the complete decolorization of Reactive Blue 5, illustrates the potential utility of DyPs in dual-enzyme systems as a novel strategy in the treatment of dye wastewater.

#### **4.3. Biodegradation of other synthetic dyes by enzymes**

Dye-degrading enzymes can also be applied in the degradation of other synthetic dyes such as indigoid, triarylmethane, and phthalocyanine dyes. Similar to the laccase-catalyzed degradation mechanism for azo dyes, the initial hydrolytic attack by the water molecule, coupled with laccase-catalyzed electron transfer, causes the cleavage of the indigoid frame, forming an intermediate isatin. The subsequent decarboxylation of isatin leads to the formation of anthranilic acid as a final stable oxidation product [120]. This process is used industrially to achieve the stonewashed effect of indigo-dyed denim fabric via mild enzymatic decolorization. This process can be used to treat textile wastewaters containing indigoid dyes. *P. chrysospori‐ um* extracellular lignolytic enzymes such as MnP and LiP have been demonstrated to success‐ fully decolorize indigoid dyes [121, 122]. In the peroxidase-catalyzed decolorization of indigo carmine, isatin sulfonic acid is formed as a final yellowish product when LiP is used. In MnPcatalyzed oxidation of indigo carmine, a stable reddish product, probably a dimeric conden‐ sation product, is formed instead. *P. chrysosporium* cultures, extracellular fluid, and purified peroxidases have been reported to degrade generally recalcitrant crystal violet and six other triphenylmethane dyes [123, 124]. The degradation of these dyes follows N-demethylation reactions. For example, the decolorization of crystal violet has been shown to form Michler's ketone, a metabolic dead-end product [125].

The removal of phthalocyanine dyes in aqueous solution by peroxidase has been widely reported, especially by white rot fungi [51, 67]. Phthalocyanine dyes are reactive dyes con‐ taining metallic complexes, mostly Cu, used to produce blue and green shades in textile dyes. The peroxidase-catalyzed degradation of these dyes involves cleavage of the nitrogen bonds in the inner ring of the phthalocyanine molecule and release of Cu2+ from the metal complex [126]. However, the resultant products tend to be more toxic in the environment [126].
