**5. Evolutionary issues and scope for improvement of dye-degrading enzymes**

Enzymes are capable of carrying out a tremendous range of biochemical functions, including dye bioremediation. However, their efficiency, stability, and costs often do not correspond to the needs of industrial operation [127]. In dye bioremediation, the choice of enzymes also depends on the effluent characteristics, operational requirements, and costs. Although some peroxidases and laccases are being employed successfully in industry, there is still no enzyme that combines the desired attributes of being stable and active over a range of temperatures and pH values, with high reduction potential [128].

To overcome this limitation, tailor-made biocatalysts can be created from wild-type enzymes by protein engineering using either rational design via computer-aided molecular modeling and site-directed mutagenesis, or by directed evolution techniques. These techniques can be used to successfully modify protein activity, stability, enantioselectivity, soluble expression, and binding affinity. In this regard, the availability of the structure of the enzyme and knowledge about the relationships between structure and function is requisite to undertake rational design and is consequently very information intensive [129]. Rapid progress in solving protein structures, and the enormously increasing number of sequences stored in public data bases have significantly eased access to data and structures, making rational protein engi‐ neering possible.

To overcome challenges faced by rational design, directed evolution has emerged as a key technology for protein engineering, generating impressive results [129]. Direct evolution involves four key steps: (1) selecting a starting gene sequence, (2) creating a library of variants, (3) selecting variants by high-throughput screening with improved function, and (4) repeating the process until the improvement or function is achieved [130]. The most common mutagen‐ esis method used includes error-prone PCR (ep-PCR), saturation mutagenesis, and DNA shuffling [131]. For both approaches to protein engineering, the gene(s) encoding the en‐ zyme(s) of interest, a suitable expression system, and a sensitive detection system are prereq‐ uisites.

## **5.1. Engineering for specificity**

by the formation of a light reddish-brown product (Figure 4). Similar results have been

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

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.

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

that both laccase and peroxidase utilize similar mechanism for AQ degradation.

reported in other DyP peroxidases [75-78, 82, 83, 114-117].

126 Advances in Bioremediation of Wastewater and Polluted Soil

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

ketone, a metabolic dead-end product [125].

There have been many attempts to use rational approaches to engineer laccases over the last couple of decades. Using site-directed mutagenesis, Xu et al. [132] developed a collection of variants with structural perturbations at the T1 Cu center to determine what parameters define the catalytic activity and the redox potentials of laccase. In the study, F463M mutation resulted in a T1 Cu site with an EPR signal intermediate between that of the wild-type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation also led to a more basic optimal pH as well as an increase in *k*cat and *K*m. Similarly, triple mutations V509L/S510E/G511A and L466V/E467S/ A468G near T1 Cu center of *Myceliophthora* and *Rhizoctonia* laccase, respectively, resulted in a phenol-oxidase activity with an altered *K*m, *k*cat, fluoride inhibition, and pH optimum shifted 1 unit lower and higher, respectively [133]. These observations were attributed to mutationinduced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the T1 Cu center. Modifications in the amino acid composition in the enzyme active site of *Tinea versicolor*laccase also improved enzyme activity and affinity toward larger phenolic substrates [134].

Random mutagenesis experiments on *Pleurotus ostreatus* laccase POXC and POXA1B cDNAs, using ep-PCR, have been reported to result to variant library with altered enzyme properties [135]. In this study, two variants 2L4A and 3L7H showed a higher specific activity than the wild-type enzyme toward typical aromatic substrates and expanded dye degradation specif‐ icities [136]. Several directed evolution studies of bacterial laccase CotA have also been used to successfully improve enzyme substrate specificity and functional expression [137-140]. Gupta and Farinas [138] reported a variant of CotA having 120-fold more specificity for ABTS with unexpectedly enhanced thermal stability with the half-life for the heat inactivation (*t*1/2) at 80°C increased by 62 min. This newly generated laccase variant represents a helpful "evolved form" of the enzyme that is more durable and versatile as a biocatalyst, with respect to both the varieties of xenobiotics degraded and the operative conditions.

Similar to laccases, several attempts have been made to engineer peroxidases specificity using rational approaches [68, 141-143]. Using a combination of site-directed mutagenesis and *in vivo* shuffling, Garcia-Ruiz et al. [68] developed VPL2 variants of *P. eryngii* with enhanced VP activity (~129-fold) compared with the parental VPL2. Engineering of the cavity of cytochrome c peroxidase (CCP) via W191G mutation has been shown to alter the specificity of the enzyme toward substrates 2-aminothiazole [144]. Two mutations (A147M and A147Y) in CCP have also been reported to exhibit unique specificities toward oxidation of small substrates [142].

#### **5.2. Engineering for properties of enzymes**

Protein thermostability is a crucial issue in the practical application of enzymes in dye bioremediation applications. Several studies have reported the application of protein engi‐ neering techniques to improve thermal stabilities of peroxidases [68, 145, 146], azoreductase [147], and laccases [148, 149]. *In silico* design and site-directed mutagenesis of thermo-labile residues of *Coprinus cinereus* peroxidase (CiP) resulted in two variants (S323Y and E328D) with increased thermostability over the wild-type enzyme in addition to conserved catalytic activity [145]. Similarly, five rounds of mutagenesis/recombination followed by high-throughput screening yielded a variant 1B6, showing 300-fold higher half-life at 50°C than that exhibited by the homodimeric wild-type PpAzoR azoreductase from *Pseudomonas putida* [147]. In *P. ostreatus* VPL2, directed evolution involving six rounds of DNA shuffling cycle was used to improve enzyme secretion, activity, and stability [68]. The generated variant had a higher *T*<sup>50</sup> of 8°C and increased enzyme stability at alkaline pH. In addition, the *K*<sup>m</sup> for H2O2 was enhanced

15-fold with the catalytic efficiency maintained, accompanied by an improvement in peroxide stability. five rounds of mutagenesis/recombination followed by high-throughput screening yielded a variant 1B6, showing 300-fold higher half-life at 50°C than that exhibited by the homodimeric wild-type

thermostability over the wild-type enzyme in addition to conserved catalytic activity [145]. Similarly,

Protein thermostability is a crucial issue in the practical application of enzymes in dye bioremediation applications. Several studies have reported the application of protein engineering

5.2 Engineering for properties of enzymes

AQ, under conditions of higher peroxide concentrations.

in a T1 Cu site with an EPR signal intermediate between that of the wild-type laccase and plastocyanin, an altered UV-visible spectrum, and a decreased redox potential (by 0.1 V). In oxidizing phenolic substrate, the mutation also led to a more basic optimal pH as well as an increase in *k*cat and *K*m. Similarly, triple mutations V509L/S510E/G511A and L466V/E467S/ A468G near T1 Cu center of *Myceliophthora* and *Rhizoctonia* laccase, respectively, resulted in a phenol-oxidase activity with an altered *K*m, *k*cat, fluoride inhibition, and pH optimum shifted 1 unit lower and higher, respectively [133]. These observations were attributed to mutationinduced structural perturbations on the molecular recognition between the reducing substrate and laccase and on the electron transfer from the substrate to the T1 Cu center. Modifications in the amino acid composition in the enzyme active site of *Tinea versicolor*laccase also improved

Random mutagenesis experiments on *Pleurotus ostreatus* laccase POXC and POXA1B cDNAs, using ep-PCR, have been reported to result to variant library with altered enzyme properties [135]. In this study, two variants 2L4A and 3L7H showed a higher specific activity than the wild-type enzyme toward typical aromatic substrates and expanded dye degradation specif‐ icities [136]. Several directed evolution studies of bacterial laccase CotA have also been used to successfully improve enzyme substrate specificity and functional expression [137-140]. Gupta and Farinas [138] reported a variant of CotA having 120-fold more specificity for ABTS with unexpectedly enhanced thermal stability with the half-life for the heat inactivation (*t*1/2) at 80°C increased by 62 min. This newly generated laccase variant represents a helpful "evolved form" of the enzyme that is more durable and versatile as a biocatalyst, with respect

Similar to laccases, several attempts have been made to engineer peroxidases specificity using rational approaches [68, 141-143]. Using a combination of site-directed mutagenesis and *in vivo* shuffling, Garcia-Ruiz et al. [68] developed VPL2 variants of *P. eryngii* with enhanced VP activity (~129-fold) compared with the parental VPL2. Engineering of the cavity of cytochrome c peroxidase (CCP) via W191G mutation has been shown to alter the specificity of the enzyme toward substrates 2-aminothiazole [144]. Two mutations (A147M and A147Y) in CCP have also been reported to exhibit unique specificities toward oxidation of small substrates [142].

Protein thermostability is a crucial issue in the practical application of enzymes in dye bioremediation applications. Several studies have reported the application of protein engi‐ neering techniques to improve thermal stabilities of peroxidases [68, 145, 146], azoreductase [147], and laccases [148, 149]. *In silico* design and site-directed mutagenesis of thermo-labile residues of *Coprinus cinereus* peroxidase (CiP) resulted in two variants (S323Y and E328D) with increased thermostability over the wild-type enzyme in addition to conserved catalytic activity [145]. Similarly, five rounds of mutagenesis/recombination followed by high-throughput screening yielded a variant 1B6, showing 300-fold higher half-life at 50°C than that exhibited by the homodimeric wild-type PpAzoR azoreductase from *Pseudomonas putida* [147]. In *P. ostreatus* VPL2, directed evolution involving six rounds of DNA shuffling cycle was used to improve enzyme secretion, activity, and stability [68]. The generated variant had a higher *T*<sup>50</sup> of 8°C and increased enzyme stability at alkaline pH. In addition, the *K*<sup>m</sup> for H2O2 was enhanced

enzyme activity and affinity toward larger phenolic substrates [134].

128 Advances in Bioremediation of Wastewater and Polluted Soil

to both the varieties of xenobiotics degraded and the operative conditions.

**5.2. Engineering for properties of enzymes**

In our study, we have reported the stabilization of bacterial DyP AnaPX against H2O2-induced inactivation by replacing the Met residues in the heme pocket with high redox residues Ile, Leu, and Phe [80]. The heme cavity variants M401L, M401I, M401F, and M451I had significantly increased H2O2 stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, M401F and M451I variants retained 16% and 5% activity at 100 mM H2O2, respectively. In addition, the two mutants maintained high dye decolorization activity toward AQ and azo dyes at 5 mM H2O2 and exhibited a slower rate of heme degradation than the wild-type enzyme (Figure 5). The observed stabilization of AnaPX was attributed to (1) the replacement of potentially oxidizable Met residues, (2) the increased local stability of the heme pocket, or (3) the alteration of the self-inactivation electron transfer pathways due structural perturbations of the heme pocket by the above mutations. The observed increased stabilities and broad substrate specificity can be potentially useful for the further practical application of these AnaPX mutants in bioremediation of wastewater contaminated with recalcitrant AQ, under condi‐ tions of higher peroxide concentrations. PpAzoR azoreductase from Pseudomonas putida [147]. In P. ostreatus VPL2, directed evolution involving six rounds of DNA shuffling cycle was used to improve enzyme secretion, activity, and stability [68]. The generated variant had a higher T50 of 8°C and increased enzyme stability at alkaline pH. In addition, the Km for H2O2 was enhanced 15-fold with the catalytic efficiency maintained, accompanied by an improvement in peroxide stability. In our study, we have reported the stabilization of bacterial DyP AnaPX against H2O2-induced inactivation by replacing the Met residues in the heme pocket with high redox residues Ile, Leu, and Phe [80]. The heme cavity variants M401L, M401I, M401F, and M451I had significantly increased H2O2 stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, M401F and M451I variants retained 16% and 5% activity at 100 mM H2O2, respectively. In addition, the two mutants maintained high dye decolorization activity toward AQ and azo dyes at 5 mM H2O2 and exhibited a slower rate of heme degradation than the wild-type enzyme (Figure 5). The observed stabilization of AnaPX was attributed to (1) the replacement of potentially oxidizable Met residues, (2) the increased local stability of the heme pocket, or (3) the alteration of the self-inactivation electron transfer pathways due structural perturbations of the heme pocket by the above mutations. The observed increased stabilities and broad substrate specificity can be potentially useful for the further practical application of these AnaPX mutants in bioremediation of wastewater contaminated with recalcitrant

**Figure 5.** (a) H2O2 stability of wild-type AnaPX and Met-substituted variants. (b) Dye decolorization activity of AnaPX and two improved variants (M401F and M451I) on AQ and azo dyes at two different H2O2 concentrations (1 and 5 mM) [80].

Strategies for further improvements of laccase through genetic, metabolic, and protein engineering in suitable heterologous hosts for enzyme overproduction and enhanced enzyme kinetics and substrate binding, improving enzyme activity and stability, have been reported [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].
