**6. Ligninolytic enzymes and their characteristics**

So far, the white rot fungi are the main eukaryotic microorganisms reported that produce these enzymes and play a crucial role in the degradation of plant raw materials, as well as numerous phenolic contaminants in soil bioremediation and in industrial waters [7]. The four major lignin modifying enzymes are lignin peroxidase (LiP), manganese peroxidase (MnP), laccase (Lac), and the versatile peroxidase (VP). White rot fungi contain the four enzymes and therefore can decompose and mineralize several environmental contaminants in non-toxic forms [18].

It has been observed that white rot basidiomycetes such as *Coriolus versicolor*, *P. chrysosporium*, and *T. versicolor*, degrade lignin more efficiently. However, electron microscopy studies of the early stages of fungal degradation have shown that oxidative ligninolytic enzymes are too large to penetrate the micropores of the cell wall of wood. Therefore, it has been suggested that before enzymatic attack, low molecular weight reactive oxidant compounds should initiate changes in lignin structure [20].

Manganese peroxidase (MnP) was first isolated from *P. chrysosporium* [28]. This enzyme is produced by almost all basidiomycete fungi that cause white wood rot and contain a heme group as prosthetic group, which is always extracellular [29]. The production of MnP in *P. chrysosporium* is enhanced by the limitation of nutrients such as C, N, and also by Mn2+. It is also shown that the fungus *Irpex lacteus* has a great potential in the pretreatment of lignocellulose as well as in the biodegradation of xenobiotics, producing MnP as the main enzyme in many controlled conditions tested [30]. This peroxidase, requires Mn2+ as its reducing substrate, oxidizing it to Mn3+, which is then chelated by several organic acids as oxalate, which are secreted by fungi [18]. Mn3+ chelating complexes in turn act as low molecular weight diffusible oxidants of the relatively labile phenolic substructures in the lignin. However, Mn3+ chelating complexes act as low molecular weight oxidants of the phenolic substructures of lignin. Although in natural lignin contains other non-phenolic substructures and these are abundant and recalcitrant to degradation by this mechanism. The efficiency of MnP is dependent on the peroxidation of unsaturated fatty acids, generating in the process, species that are capable of oxidizing and excising non-phenolic structures in synthetic lignins. It has also been shown that unsaturated fatty acids increase the ability of MnP to bleach (i.e., delignify) wood pulps [29].

The discovery of the enzyme lignin peroxidase (LiP), was confirmed in 1983 from the fungus *Phanerochaete chrysosporium*. LiP oxidizes aromatic lignin rings and a wide range of dangerous contaminants such as phenols, dyes, xenobiotics, and this enzyme has a redox potential (E0 ~ 1.2 V at pH 3.0–4.5) [28, 31]. LiP is a biotechnologically important enzyme that has broad applications such as the delignification of lignocellulosic materials, conversion of coal into low molecular weight fractions, which could be used for the production of basic chemicals, in bio-pulp and bio-bleaching in industries of paper, and on the elimination of recalcitrant organic pollutants and for enzymatic polymerization in the polymer industries [18]. *P. chrysosporium* can produce LiP since it depends only on the limitation of carbon, nitrogen, or sulfur. It is important to mention that this enzyme uses a monomeric compound, veratryl alcohol as an inducer for the catalysis of the reaction [32].

In case of Laccase (Lac), it was first discovered by Yoshida in the Japanese lacquer tree *Rhus vernicifera* in 1883 [28]. This enzyme is capable of oxidizing phenols and aromatic amines. These are typical of basidiomycetes of white rot [33]. Lac is a multi-copper oxidase in its structure, the availability of copper in the medium could allow the synthesis of the enzyme. In addition, the presence of copper in *Pleurotus ostreatus* cultures decreases the activity of extracellular proteases that can degrade laccase [34]. A study with *P. ostreatus* in solid culture, using sugar cane bagasse as a substrate, showed that laccases are induced by copper sulfate and ferulic acid and two concentration levels of organic nitrogen in the form of yeast extract and its regulation of their expression may be substantially diverse among fungal species [22]. Lac is capable of catalyzing the direct oxidation of ortho and para-diphenols, aminophenols, polyphenols, polyamines, and aryl diamines, as well as some inorganic ions. It couples the four single-electron oxidations of the reducing substrate to the fourelectron reducing division of the dioxygen, using four Cu atoms distributed against three sites, defined according to their spectroscopic properties [23].

The other important ligninolytic enzyme is versatile peroxidase (VP), which was first discovered approximately 20 years ago in the fungus *Pleurotus eryngii*, and a few years later in the *Bjerkandera* species [35]. VP has recently been described as a new ligninolytic peroxidase family, together with LiP and MnP, both reported by *Phenerocheate chrysosporium* [18]. In general, VP enzymes, share the same characteristics to most peroxidases, but the VP enzyme is unique with respect to the substrate that can oxidize. VP oxidizes high potential redox dyes [18]. Therefore, VP is an oxidoreductase that degrades lignin and in natural settings this enzyme cooperates with LiP, MnP, and Lac to break down lignocellulose. VP is considered as a hybrid enzyme because it combines catalytic features of MnP, oxidizing Mn2+ to Mn3+, and can oxidize aromatic compounds when complexed with organic acids [36]. This enzyme is distinguished from LiP and MnP in that it has multiple active sites to directly oxidize Mn2+ and some phenolic and non-phenolic substrates (such as Orange II, Reactive Black 5, and Direct Blue 199) in the absence of Mn2+. In addition, VP contains a conserved manganese binding site from MnP and a conserved tryptophan residue from LiP [37].
