**2.1 Terminology**

394 Gel Electrophoresis – Advanced Techniques

Gold & Alic, 1993), Mn2+ is considered to be an important physiological substrate for MnP. Further, while LiP expression has been observed in certain white-rot fungi (e.g. *Phanerochaete chrysosporium* and *Phlebia radiata*) under specific culture conditions (e.g., temperature, agitation, and nutritional constraints), MnP expression has been observed in a wide range of white-rot fungi (Gold & Alic, 1993), including cultivated edible fungi, such as *Agaricus bisporus* (Bonnen et al., 1994), *Ganoderma lucidum, Lentinula edodes*, and *Pleurotus*

The shiitake mushroom, *Lentinula edodes* (Berk.) Pegler, a white-rot basidiomycete, is one of the most valuable, cultured, edible mushrooms in the world (Chang & Miles 1989). Shiitake mushrooms were traditionally cultivated on Fagaceae logs, but they are now grown on sawdust-based media. The ability of white-rot basidiomycetes to degrade wood components, especially lignin, therefore affects both culture-time to harvesting and yields (Kinugawa & Tanesaka, 1990; Ohga & Kitamoto, 1997; Smith et al., 1988; Tanesaka et al., 1993). Although *L. edodes* secretes the lignin-degrading enzymes laccase (Lcc) and MnP when cultivated on sawdust-based media (Buswell et al., 1995; Leatham, 1985; Makker et al., 2001), it does not usually secrete these enzymes in liquid media. It was previously reported that the main isozyme produced by *L. edodes* cultured on sawdust was the manganese peroxidase, LeMnP2 (Sakamoto et al., 2009). In addition, we previously reported that a β-*O*-4 lignin model compound, 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (Umezawa & Higuchi, 1985) was effectively degraded by *L. edodes* under MnP-induced conditions, but not under Lcc-induced conditions (Kochi et al., 2009). These observations supported the hypothesis that these enzymes, particularly MnP, play an important role in degrading sawdust during cultivation, and corroborating reports that the expression and properties of these enzymes is likely to influence mycelial growth and fruit body development (Smith et al., 1988; Wood et al., 1988). Several reports have been published on the purification and characterization of the lignin-degrading enzymes secreted by *L. edodes* using sophisticated biochemical procedures (Forrester et al., 1990; Nagai et al., 2002, 2003, 2007; Sakamoto et al., 2008, 2009). However, these methods are impracticable for routine isozyme analysis during breeding trials. Methods for isozyme detection by electrophoresis using enzyme catalytic properties - referred to as "protein activity staining" or "enzymatic staining" - are well established in histochemical studies and genetics (Pasteur et al., 1988). It was expected that Lcc, peroxidases (Per, EC 1.11.1.7), and MnP bands could be distinguished on the same gel by subtraction of newly appeared bands produced by sequential enzymatic staining. In practice, however, unexpected bands frequently appeared on gels exposed to conventional Lcc staining solutions. Indeed, in samples exhibiting strong MnP activity without Lcc activity, no additional bands appeared in subsequent staining procedures for either Per or MnP. We recently reported improved methods for enzymatic staining using native-PAGE to distinguish between Lcc and MnP isozymes induced in

In this chapter, we describe an assay system for the induction and identification of phenoloxidizing enzymes produced by *L. edodes* grown under liquid culture conditions. In addition, the assay system was used to compare the glycosylation characteristics of these extra- and intracellular isozymes, as well as their modes of inheritance within monokaryotic progenies and β-*O*-4 lignin model compound degradation characteristics under Lcc- and MnP-induced conditions. Based these findings, the potential application of this assay system

to elucidate the ligninolytic mechanisms employed by this fungus is also discussed.

spp. (Orth et al., 1993).

liquid cultures of *L. edodes* (Saeki et al., 2011)*.*

We use the term "phenol-oxidizing enzymes" to describe all phenoloxidases and peroxidases. We do so because of the ability of these enzymes to utilize the same substrates and produce the same catalytic products as described in the Introduction.
