## 4 Potential Decolorizing Enzymes and Their Characteristics
### Dye-Decolorizing Peroxidases (DyPs)  
Dye-decolorizing peroxididases (EC 1.11.1.19) represent a new family of heme peroxidase with one heme as a cofactor and a role in H\({}_{2}\)O\({}_{2}\)-dependent oxidation of a wide range of molecules [146,147]. Basidiomycete _Thanatephorus cucumeris_ Decl was the first species suggested to be excreting these enzymes due to its ability to decolorize 18 types of reactive, acidic, and dispersive dyes, most of which were xenobiotics [148]. In 1999, the same author succeeded in the attempt to purify DyP for the first time. It is a glycoprotein with a molecular mass of 60 kD [149]. Later, it was reported that several bacteria have a role in lignin degradation, suggesting that these bacteria possess a unique class of dye decolorizing peroxididases [150,151]. Due to the low sequence identity shown between bacterial and fungal DyPs, they are divided into four classes, from A to D. The DyP from class D are the ones from Basidiomycota [146]. The catalytic cycle of the DYPs is composed of the resting state and transient intermediates comprising compound I and compound II (Figure 2) [152]. When the enzyme is in a resting state, it contains Fe\({}^{3+}\). When this resting ferric enzyme is oxidized by H\({}_{2}\)O\({}_{2}\), a high-valence compound I, [Fe\({}^{4+}\) = O\({}^{\uparrow\bullet}\) is produced. This compound I reacts with 1 eq electrons and, from a reducing state, generates a compound II, [Fe\({}^{4+}\) = O]\({}^{+}\). Compound II is then reduced by a second substrate molecule and the resting state enzyme is produced again [152]. [PERSON] and colleagues [153] have shown that DyPs enzymes, _Pleos_-DyP1 and _Pleos_-DyP4, from fungi _Pleurotus ostreatus_ have the ability to oxidize Mn\({}^{2+}\) to Mn\({}^{3+}\), a feature mostly present in MnPs and versatile peroxididases (VPs). To compare the mechanisms of reaction of lignin modifying enzymes, including peroxididases and laccase, a schematic outline is shown (Figure 3) [142]. Figure 3: Comparison of lignin degradation by peroxididases and laccase. MnP—manganese peroxidase, VP—versatile peroxidase, LiP—lignin peroxidase, DyP—dye-decolorizing peroxidase, ox—oxalate, VA—veratryl alcohol. Figure 2: Schematic description of catalytic cycle of _Thermonospora curvata_ DyPs. Many stains present on paper can be caused by past fungal contaminations but also by several types of synthetic dyes composing inks and stamp inks. As we saw in the previous paragraphs, there are some similarities between the structures of synthetic dyes and fungal pigments. Therefore, starting with the known properties of LMEs and DyPs to degrade synthetic dyes, we illustrate here the potential of these enzymes to also decolorize fungal pigments. [PERSON] and colleagues [55] were the first to present the transformation pathway of Remazol Brilliant Blue R (RBBR) dye by immobilizing laccase. Laccase was derived from the white-rot fungus _Trantetes pubescens_ and has shown a degradation activity of 44% in 42 h. The transformation pathway involved steps of deamination and oxidation and resulted in products that were less toxic than RBBR itself [55]. [PERSON] and colleagues have demonstrated that two different strains of _Trantes_ species produce different lacase isoforms capable of degrading Acid green 27 (AG27), another anthraquinone dye. Furthermore, they have shown that besides anthraquinone dyes, the culture fluids of the strain _Trantes_ sp. LA1 also decolorized the azo dye Reactive Black 5 (RB5) and triphenylmethane CV, suggesting that this strain could be a new laccase source with dye-decolorization potential [154]. Another study showed that due to the presence of laccase, MnP, and LiP, the strain _Aspergillus__tizukae_ EAN60 was able to decolorize RBBR and two other azo dyes, methyl red (MR) and methyl orange (MO) [155]. After 9 days of monitoring, the maximum removal of RBBR was close to 95%, while the removal of MO and MR observed after 3 days was 84.90% and 47.65%, respectively.