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**Other Applications of Gel Electrophoresis Technique** 

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**20** 

**Enzymatic Staining for Detection of Phenol-**

Eiji Tanesaka, Naomi Saeki, Akinori Kochi and Motonobu Yoshida

Lignocellulose is the most abundant organic compound in the terrestrial environment. Nonetheless, with the exception of basidiomycetous fungi, most organisms are either unable to degrade lignocellulose, or if they can, they do so with difficulty (Kirk & Fenn, 1982). Wood-decomposing basidiomycetes can be grouped into two categories: white-rot and brown-rot fungi. White-rot fungi have cellulases and lignin-degrading enzymes that decompose most cell wall components, whereas brown-rot fungi have enzymatic systems that selectively degrade cellulose and hemicelluloses, leaving brown shrunken lumps of tissue composed mainly of a loose lignin matrix (Enoki et al., 1988; Highley et al., 1985; Highley & Murmanis, 1987; Kirk & Highley, 1973). The name 'white-rot' is derived from the bleaching effect that this fungus has when degrading wood; the lignin-degrading enzymes that they secrete have the effect of promoting lignin loss and exposing the white cellulose fibrils. White-rot fungi are known to produce polyphenol oxidases (phenoloxidases), which, when the fungi are plated on agar media containing gallic or tannic acids, change the color of the agar to a dark reddish-brown in what is referred to as Bavendamm's polyphenol oxidase test or Bavendamm reaction (Bavendamm, 1928, as cited in JØrgensen & Vejlby, 1953). Based on this reaction, phenoloxidases are considered to be one of putative lignindegrading enzymes (Higuchi 1990). Laccase (Lcc, EC 1.10.3.2), catechol oxidase (EC 1.10.3.1) and tyrosinase (monophenol monooxygenase, EC 1.14.18.1) are phenoloxidases with considerable overlap in their substrate affinities (Burke & Cairney, 2002). Lcc catalyze the reduction of O2 to H2O using a range of phenolics, aromatic amines, and other electron-rich substances as hydrogen donors (Thurston, 1994). Similar phenol-oxidizing activities are also observed in peroxidases (EC 1.11.1.x), which use H2O2 as an electron donor. Lignin peroxidase (ligninase, LiP, EC 1.11.1.14) was first discovered in *Phanerochaete chrysosporium*  in which the H2O2-dependent Cα-Cβ cleavage of non-phenolic lignin model compounds was first described (Tien & Kirk, 1983, 1984). Manganese peroxidase (MnP, EC 1.11.1.13) also strongly degrades lignin model compounds and the reaction is mediated by H2O2 and Mn2+ (Glenn et al., 1983; Glenn & Gold, 1985; Kuwahara et al., 1984). Whereas lignin can effectively be oxidized by LiP directly, as reviewed previously (Cullen & Kersten, 2004;

**1. Introduction** 

**Oxidizing Isozymes Involved in Lignin-**

**Degradation by** *Lentinula edodes*

**on Native-PAGE** 

*Kinki University* 

*Japan* 
