## 4 Lignin Biodegradation and Ligninolytic Enzymes
### Lacase and Lacase-Like Multicopper Oxidase  
Lacase (EC 1.10.3.2) is a polyphenol oxidase belonging to the copper-containing oxidase family that is capable of oxidizing aromatic compounds with substrate spectra in diverse organisms including fungi, bacteria, insects, and plants [38; 39]. With coupling reactions of radical formation as well as one-electron oxidation, a lactose catalyzes the cleaving of C-C or C-O bonds in lignin and other aromatic compounds. This enzyme can oxidize a wide range of aromatic compounds, aliphatic amines, hydroxylindoles, polysaccharides, and inorganic or organic metals [17; 19]. Consequently, it is favored by various industries for oxidation reactions [38; 39; 40]. In eukaryotes, lacase activity has been reported from most higher fungi in the orders Ascomycetes and Basidiomycetes [17; 19; 39]. Besides lignin degradation, lacase and lacase-like multicopper oxidase is an essential enzyme playing a role in multiple functions of development and morphogenesis, pathogenesis and detoxification, the biosynthesis of secondary metabolites (e.g., melanin pigments), and polymerization of polyphenic compounds, etc. [19; 38; 39]. Interestingly, mushroom lacases and related enzymes were found to be intracellular or extracellular proteins. These localized enzymes may participate in different cellular and physical functions in mushrooms. The extracellular enzymes are secreted proteins containing signal sequences and protein N-glycosylation for cellular localization, which catalyze high molecular weight complex aromatic compounds such as lignin [39; 41]. The intracellular enzymes are endogenous cytoplasmic proteins that convert the low molecular weight of intracellular compounds for biosynthesis of secondary metabolites as well as pigment chemicals [25; 42]. As summarized in Table 1[43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59], several species of mushroom, including _Agaricus bisporus_, _Lentinula edodes_, _Pleurotus ostreatus_, and _Trantes versicolor_, produce more than one lacase isoenzyme, each with different biochemical features. Although the biological functions of some of these isoenzymes remain unclear, they display differences in terms of their substrate specificities and expression patterns [74; 79; 86]. In higher eukaryote mushrooms, alternative RNA splicing in the transcription process results in multiple transcript variations for isoenzymes of lacase and lacase-like multicopper oxidase with highly homologous sequences in one species [90; 91; 92]. The number of isoforms varies depending on the mushroom species, presence or lack of an inducer, growth and stress condition, and other environmental conditions [92; 93; 94; 95; 96]. However, the regulatory mechanisms of isoenzymes and the cellular functions have not yet been characterized. Although the biological functions of these lacase isoenzymes are still unclear, most mushroom lacases are secreted extracellular enzymes with different substrate specificities toward aromatic compounds. Therefore, it has been suggested that they play an important role in the degradation of lignin in nutrient uptake to support the growth of mushroom fruit bodies. _L. edodes_ H600 harbors extracellular and intracellular lacases, Lec1 and Lec2, within different substrate specificities [59; 60]. The activities of both enzymes for typical lacase substrates such as ABTS, p-phenylenediamine, 2,6-dimethoxyol, ferulic acid, and guaiacol were similar, but those of intracellular lacase for catechol and L-DOPA were noticeably lower than the extracellular enzyme [60]. The expression of these two lacase-coding genes was high in the caps of the fruiting body, and high enzyme activities were also detected when the pigment of the fruit bodies changed during post-harvesting preservation. Thus, the intracellular lacase could play a role in pigment synthesis by oxidation of phenolic compounds, such as dihydroxyphenols, to making the fruiting bodies turn brown [60].