ENZYMES IN AZO DYES DECOLORIZATION

The azo bond (N=N) is the most labile region of azo dyes, which may readily undergo cleavage either by enzymatic biodegradation (i.e., metabolic cleavage) or abiotic (thermal and photochemical) degradation (Weber and Adams, 1995; Ollgaard et al., 1998). Biological systems contain enzymes, azoreductases, laccases, peroxidase, and polyphenol oxidase that involved in degradation of azo dyes. The azo bond of water-insoluble azo dyes is usually not available for the intracellular enzymatic breakdown. However, the possible degr adation of azo dyes may occur through specific pathways (Engel et al., 2010). However, in sulfonated azo dyes, it is restricted to the release of aromatic amines. Several oxidoreductases from microorganisms have effectively doing the breakdown process either intracellularly or specific pathways.

8.6.1 AZOREDUCTASES

Azoreductases (EC 1.7.1.6), otherwise known as azobenzene reductase, are a major group of enzymes produced by bacteria and fungi. It can decolorize azo dye by reducing the azo group (-N=N-) into their corresponding amines (Pandey et al., 2007), which involve the breakdown of azo bond leads to degradation of dye. Azoreductases belongs to the family of oxidoreductases, and it catalyzes the reduction reaction only in the presence of coenzymes like NADH, NADPH, and FADH,. The catalytic reaction of this enzyme proceeds via a ping-pong mechanism by using two molecules of NAD(P)H to reduce one molecule of the azo compound, a substrate. Mainly bacterial decolorization of azo dye is attributed by the

Bacteriological Removal of Azo Dyes

239

enzyme azoreductase (Figure 8.7). In bacteria, the reduction reaction takes place either intracellularly or extracellularly at the cell membrane (Ram Laklian Singh et al., 2015). Several azoreductases have been identified from bacteria and are tabulated in Table 8.4.

However, in recent years, intracellular azo dye reduction has been suspected because of high polarities, high molecular weight, and sulfonate group substitution on azo dyes (Joslmi et al., 2011). Hence, a mechanism other than reduced flavin-dependent azoreductases must exist for sulfonated azo dye reduction in bacteria. Moreover, these reductants may link between the outer membrane of bacterial cells with the intracellular electron transport system and a complex dye molecule. The mediator compounds may be either metabolic products of certain substrates utilized by bacteria or added externally. For example, riboflavin significantly increased mordant yellow 10 reductions by anaerobic granular sludge (Field and Brady, 2003); the addition of anthraquinone-2,6-disulfonate, and a synthetic electron carrier could greatly enhance the reduction of many azo dye (Vander Zee et al., 2001b). Cell-free extract of Spingomonas sp. strain BN6 that was grown in the presence of 2-naphthyl sulfonate under aerobic condition showed 10-20 times better decolorization rate of amaranth in anaerobic condition (Keck et al., 1997). Kudlich et al. (1997) suggested that the membrane-bound and the cytoplasmic azoreductases not be the same enzyme systems because the activity of membrane-bound azoreductase is dependent on redox mediator (Figure 8.8).

Decolorization of azo dye by redox mediator assisted method

FIGURE 8.8 Decolorization of azo dye by redox mediator assisted method.

Azoreductases can be classified based on their oxygen requirements, structure, and function. On the basis of oxygen requirement, it is classified either active in the presence or absence of oxygen (Bafana and Chakrabarti,

  • 2008). There are two types of oxygen-insensitive azoreductases identified in bacteria: one is monomeric flavin-free enzymes containing a putative NAD(P)H binding motif, and the other is polymeric flavin-dependent enzymes (Chen, 2006). Monomeric flavin-free azoreductases also show very narrow substrate specificity, but polymeric flavin-dependent azoreductase families can catalyze a variety of substrates that differ in size and complexity. Biochemical characteristics and the protein structures of several bacterial FMN-dependent azoreductases have been determined (Wang et al., 2007; Chen et al., 2010). Flavin dependent reductases are further categorized into three gr oups based on then requirement of NADH, NADPH, or both coenzymes (Purrj and John, 2009) as an electron donor. Two monomeric flavin-free azoreductases (Azo A and Azo B) have been described in Pigmentiphaga kullae K24 (Bhtmel and Stolz, 2003). AzoB is very efficient hr reducing Orange I compared to AzoA from P kullae K24. Functionally AzoB is higher active than AzoA. AzoB requires two molecules of NADPH (4 electron donor) for the complete reductive cleavage of Orange I to sulfanilic acid and l-arnino-4-naphthaol. The active site of the AzoB has two binding sites that allow the binding of the substrate and NADPH that implies an appropriate position for direct hydride transfer rather than a proton-relay catalytic reaction. Also, the enzyme AzoB utilizes either NADH or NADPH as reductants for the reaction. Chen et al. (2010) revealed that there is a clear correlation between structure, cofactor requirement, and substrate specificity in azoreductases.
  • 8.6.2 LACCASES

Laccases (EC 1.10.3.2) are multicopper phenol oxidases and were first identified from the sap of the Rhusvernicifera, Japanese lacquer tree (Ram Lakhan Singh et al., 2015). Laccases act on phenol containing compounds and similar other molecules and perform one-electron oxidations. It decolorizes azo dyes and produces phenolic compounds rather than toxic aromatic amines while decolorizing azo dyes through absorbing a highly nonspecific free radical mechanism (Chivukula and Renganathan, 1995; Wong and Yu, 1999). It shows less substrate specificity and has the capability to degrade different types of xenobiotic substances, including dyes (De Souza et al., 2006). Therefore, it has been attaining great importance in the bioremediation of colored textile wastewater. Laccases so far reported were mainly from fungi and plant and also from a small number of bacteria (Gianfreda et al., 1999).

Laccases use O, as an electron acceptor to catalyze the oxidation of aromatic amines, phenols, polyphenols, and various nonphenolic compounds (Kiiskinen et al„ 2002; Viswanath et al., 2008). Industrially relevant laccase enzyme is produced by bacteria, insects, higher plants, and fungi (Delanoy et al., 2005). Laccase has been described from a large number of bacteria like Azospirillum lipoferum, Escherichia coli, Bacillus lichenifomiis, Bacillus halodurans, Streptomyces coelicolor and Thermus thermophillus (Sharma et al., 2007; Singh et al., 2007; Koschorreck et al., 2009). Fungal and bacterial laccases have a similar structure, but their amino acid sequences are quite different (Claus, 2004), and bacterial laccases often occurred in the monomeric form, but some fungal laccases are isozymes/isoenzymes that form a multimeric complex by oligomerization. Laccase uses low molecular weight substances like 2,2-azino-bis-

3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a redox mediator in the actual electron transfer steps (Wong and Yu, 1999). However, on the other hand, laccase enzymes that purified from mushroom Hypsizygus ulmarius decolorized methyl orange without using redox mediator (Ravikumar et al., 2013). It has been suggested that in the presence of redox mediators, dye decolorization could be improved considerably (Reyes et al., 1999; Abadulla et al., 2000; Soares et al., 2001).

Laccases are an oxidative enzyme oxidizes phenol compounds by transfer of one electi on to generate a phenoxy radical which is further oxidized by the same enzyme to produce a carbonium ion in which the charge is restricted on the phenolic ring of carbon bearing the azo linkage. Water makes a nucleophilic attack on the carbonium ion and produces 4-sulfophenyldiazene and a benzoquinone. 4-Sulfophenyldiazene is an apparently unstable compound in the presence of oxygen, which oxidizes it to analogous phenyl diazene radical. Then it readily loses molecular nitrogen and to produce a sulfopherryl radical, which is then scavenged by O, to yield 4-sulfophenylliydroperoxide (Figure 8.9). 4-sulfophenylliydroperoxide is uncommon peroxide and is known to be formed only oxidation of sulfonated azo dyes by peroxidases, whereas organic peroxides ar e unstable in presence of metal ions.

8.6.3 PEROXIDASES

Peroxidases (EC 1.11.1.7) are oxidoreductases that catalyze reactions like reduction of peroxides such as hydrogen peroxide (H,02) and oxidation of a wide variety of organic and inorganic compounds. It is a hernoprotein that catalyzes the oxidations of lignin and other phenolic compounds under acidic pH (pH 3.0 to pH 4.5) and in the presence of hydrogen peroxide (electron acceptor) (Duran et al., 2002). It contains iron (III) protoporphyrin IX as the prosthetic group. It has the potential to reduce environmental pollution by bioremediation of wastewater containing phenols, cresols, chlorinated phenols, and synthetic textile azo dyes (Bansal and Kanwar, 2013). It is well known in fungi to mineralize a wide variety of recalcitrant toxic azo dyes. This character is attributed to their ability to produce exo-enzymes such as lignin peroxidases, manganese peroxidases, and polyphenol oxidases (PPOs). Predominantly, white-rot fungus secretes lignin peroxidases (LiP) under aerobic conditions as a secondary metabolite in the stationary phase. Lignin and phenolic compounds are degraded by (LiP) in the presence of H,0, (cosubstrate) and veratryl alcohol (mediator). In this degradation, H,6, is reduced to H,0 by accepting an electron from LiP (which can oxidize itself). The oxidized LiP returns to its native form (reduced) by gaming an electron front veratryl alcohol, thereby veratryl aldehyde is formed. Veratryl aldehyde gets reduced back to veratryl alcohol by accepting an electron front the substrate. Lignin peroxidase enzyme production has been reported front organisms such as Candida krusei, Phanerochaete chrsosporium, Pleurotus streatus, Citrobacter freundii, and Pseudomaonas desmolyticum (Bansal and Kanwar, 2013).

Lignin-degrading Basidiomycetes fungus produces manganese peroxidases (MnP) extracellularly. MnP oxidizes Mn:+ to Mn3+ that act as a mediator for the oxidation of many phenolic compounds (ten Have and Teunissen, 2001). Phanerochaete crysosporium, P. sordid, C. subvermis- pora, P. radiate, D. squalens, and P rivuJosu can produce MnP extra- cellularly (Bansal and Kanwar, 2013). Microbial production of MnP is dependent on nutrient on which it grows. For example, in the presence of glutamic acid Trametes trogii showed higher laccase and MnP activities, causing decolorization of several azo dyes effectively (Levin et al., 2010). For example, Brevibacterium casei removes azo dye Acid Orange7 (A07) and chromate Cr(VI) under nutrient-limiting conditions. Cr(VI) is reduced by the reduction enzyme of B. casei with the help of A07 that acts as an electron donor. The reduced chromate Cr(III) form complex with the oxidized A07 produced a purple intermediate (Ng et al., 2010). Kalyani et al. (2011) purified 86 kDa peroxidase enzymes from Pseudomonas sp. SUK1, which oxidized various lignin-related phenols and decolorized many textile dyes under the optimum pH (3.0) and temperature (40°C). Compared to fungal peroxidases, the bacterial peroxidases are advantageous, because of bacterial species are more suitable for protein engineering to improve their catalytic properties success- folly (Bugg et al., 2011).

Laccase activity on 2,6-dialkyl-4-(4’ sulfophenyl azo) phenol

FIGURE 8.9 Laccase activity on 2,6-dialkyl-4-(4’ sulfophenyl azo) phenol.

8.6.4 POL YPHENOL OXIDASES (PPOS)

PPOs (EC 1.14.18.1) is referred to as tyrosinase or monophenol monooxygenase. It is a tetrameric protein, contains four copper atoms and has binding sites for oxygen and two aromatic compounds. It uses molecular oxygen as an oxidant, thereby reduces the cost of applying the technology (Wu et al., 2001). It has been reported that tyrosinase enzyme from Bacillus thuringiensis has to be used for decontamination of waste- water having phenols (EL-Shora and Metwally, 2008). It catalyzes phenol oxidation in two phases. In the first step, it catalyzes the hydroxylation of monophenols to o-diphenols. In the second step, o-diphenols is further oxidized to o-quinones. This process has been demonstrated with the aid of tyrosinase enzyme of Pseudomonas desmolyticum NCIM 2112 to degrade Direct blue-6 (Kalme et al., 2007) and mixed culture of Galacto- myces geotrichum and Bacillus sp. YUS to degrade Disperse dye brown 3REL (Jadhav et al., 2008b). Husain and Jan (2000) stated that PPOs are a nonspecific enzyme that can act on a broad range of substrate and have the ability to remove the pollutant from contaminated sites at veiy low concentrations.

 
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