## 4 Potential Decolorizing Enzymes and Their Characteristics
### Dye-Decolorizing Peroxidases (DyPs)
[PERSON] and colleagues [122] investigated the production of ligninolytic enzymes by fungi _Marsmius__clodophyllus_ during its application in the decolorization of RBBR, as well as two azo dyes, such as Orange G and Congo red (CR). By monitoring specific reactions characteristic of ligninolytic enzymes, it has been found that in the presence and absence of RBBR dye, this strain produced both laccase and LiP. After in vitro RBBR dye decolorization in the culture medium containing unpurified enzymes, it has been shown that laccase activity was in agreement with the dye decolorization. Moreover, the fresh dye has been successively added to the already decolorized culture medium. This step actually sped decolorization up in the subsequent round. After reconcentration of unpurified enzymes from decolorized culture medium, the protein precipitates contained laccase that decolorized 76% of RBBR dye in less than one day. The protein precipitates were also tested for in vitro decolorization of a mono-azo dye, Orange G, and a di-azo dye, CR. It has been shown that laccase decolorized 54% of Orange G and 33% of CR at the same time. On the other hand, the protein precipitates did not show the presence of LiP [122]. Another synthetic dye, Crystal Violet (CV), has been widely utilized in medicine and by the textile industry [44]. Several studies have shown that microorganisms can successfully degrade this dye and have proposed the hypothetical pathway of CV degradation [156; 157]. In the first step of degradation, it has been suggested that laccase catalyzed reduction to its leuco derivative before reductively degrading into the next intermediates to the final phenol (Figure 4) [156]. An additional enzyme that appeared to be involved in the process was aminopyrine N-demethylase. Laccase catalytic activity in the degradation of Bisphenol A and decolorization of synthetic dyes was observed by [PERSON] et al. (2013) [40]. The purified enzyme from fungi _Trantes__polyzona_ WRF710-1-induced oxidation of Bisphenol A within 3 h with redox mediator 1-hydroxybenzotriazole (HBT) present. Triphenylmethane dye (Bromophenol Blue; BRB), anthraquinone dye (RBBR), and azo dye (MO) were decolorized without the redox mediator present at a rate of 80%. Another ascomycete, _Leptosphaerulina_ sp., has been described as a producer of high amounts of laccase and MnP. These enzymes have shown the activity of decolorization of Remazol Black, Novacron Red, and Turquoise Blue. To improve the production and decolorizing activity of _Leptosphaerulina_ sp., [PERSON] et al. [158] co-cultivated this species with other fungal species. Based on response surface methodology, they inferred that co-cultivation with _Trichoderma viride_ and _Aspergillus terreus_ was the best treatment for the production of ligninolytic enzymes, specifically lacase, MnP, and versatile peroxidase, as well as the degradation of RB5 [158]. [PERSON] et al. [159] cloned a novel MnP gene (_mnp3_) from the fungal strain _Cerrena unicolor_ BBP6 and expressed it in the yeast _Pichia pastoris_. They have demonstrated that this recombinant enzyme, rMnP3-BBP6, is an effective decolorizing agent against different dyes. Additionally, it has been shown that rMnP3-BBP6 has great potential in denim bleaching. The decolorization potential of dyes such as RBBR, MO, BPB, and CV by recombinant MnP was higher than 50%. Besides lacase and MnPs, LiP was confirmed to decolorize synthetic dyes. This enzyme showed degradative activity in a variety of substrates such as Mg\({}^{2+}\), tryptophan, L-Dopa, hydroquinone, and synthetic dyes. Specifically, LiP isolated and purified from bacteria, _Acinetobacter calcoaceticus_ decolorized azo-dyes such as MR and MO at a very high rate, 98% and 96% in one day, respectively. Other azo dyes were also decolorized by this enzyme, up to approximately 90% within two days [140]. The reaction mechanism of LiP in the decolorization of azo dyes was demonstrated by [PERSON] and colleagues [160,161].