## 4 Potential Decolorizing Enzymes and Their Characteristics
### Dye-Decolorizing Peroxidases (DyPs)
They have proposed that the cleavage of the dye is done asymmetrically, with an active site  
Figure 4: Mechanism of Crystal Violet degradation by lacase. available for a LiP to excite the molecule. This asymmetric cleavage by LiP occurs between the nitrogen of the azo group (N=N) and the carbon of the aromatic ring and results in the production of different types of naphthalene-2-sulfonic acid, depending on the type of the dye that was subjected to degradation (Figure 5) [160; 161]. The anthraquinone dye degradation pathway has been reported by [PERSON] et al. (2009) [162]. The DyP from the basidiomycete _Thanatephorus cucumeris_ was utilized in the process of degradation of RB5 dye. The pathway has the following intermediates: phthalic acid, Product 2, and Product 3 (Figure 6) [162]. These Product 2 and Product 3 were generated by the peroxidase reaction, while in the case of phthalic acid, it has been suggested that DyP acted as a hydrolase or oxygenase rather than a peroxidase [162]. Only a few attempts have been published, describing the degradation of uncharacterized fungal and melanin stains from various surfaces. Two different approaches were applied, a lacase-based method able to decolorize the fungal stains on paper and parchment [163] and a biomimetic copper-pyridine oxidation system able to remove fungal melanin from paper [164] and decolorize fungal stains from textile [165]. However, it has been shown that many quinone colorants are also synthesized by fungi are isolated from paper [2]. Quinones are a class of organic quinoid compounds widely distributed in nature [65]. A common basic structural pattern is an ortho or a para substituted alone. If this dione is conjugated to an aromatic nucleus, it is called benzene quinone, or to a condensed polycyclic aromatic system, then it can be naphthoquinones, anthraquinones, and anthracyclinones [65]. As was shown above, anthraquinone dyes used in textiles and in other industries can be degraded by LMEs synthesized by microorganisms, thus, we hypothesize that similar action can be applied to the quinoid colorants synthesized by different fungal species on historical paper. When one fungal pigment from the quinone class is compared with RBBR (Figure 7) [158; 159] we can see this common cyclic dione with different functional groups. In the degradation of RBBR dye, lacase appears to be the first enzyme that cleaves hydrogen bonds between N-H groups due to the reduction of Cu\({}^{2+}\) to Cu\({}^{+}\), generating two compounds. In the case of fungal colorants, this cleavage would take place between the OH groups. Figure 5: Reaction mechanism of LiP in a degradation pathway of azo dyes. If we take a look at the structures of pigments produced by fungi and synthetic dyes, we can see that most of them contain a phenolic ring bound to the hydroxy group (for a closer look, refer to [2]). For example, simple flavipin, a yellow pigment produced by _Epicocoumpurpurescens_ and _Chaematonium globosum_[2,166], has a phenyl group bonded to three hydroxyl groups (Figure 8b). Under the catalysis of MnPs, one of those hydroxyl groups can be oxidized, and Mn\({}^{3+}\) is reduced to Mn\({}^{2+}\). As the reaction goes further, with the series of oxidoreductase reactions, the phenolic ring would break down, causing the degradation of the pigment. [PERSON] et al. [39] have demonstrated a hypothetical degradation pathway of a phenol containing an organic compound (Figure 8a, [39]) as many fungal pigments and synthetic dyes are [39]. Based on the reactions shown by [PERSON] and colleagues [39], we proposed a similar degradation pathway of the fungal pigment flavipin (Figure 8b). Figure 6: Degradation pathway of RB5 by DyP. Figure 7: Fungal pigment cyclohexumclone and anthraquinone dye RBBR. Due to the characteristic of DyP to act as a hydrolase/oxygenase, this enzyme can also be employed to degrade quinone structures that have been found abundantly in filamentous fungi, leaving different color hues on the paper.