**Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes Involved in Lignin-Degradation by** *Lentinula edodes* **on Native-PAGE**

Eiji Tanesaka, Naomi Saeki, Akinori Kochi and Motonobu Yoshida *Kinki University Japan* 

#### **1. Introduction**

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;

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes

**2. Experimental procedures** 

**2.2 Fungi and culture conditions** 

**2.1 Terminology** 

inoculation.

respectively.

**2.3 Enzyme assay** 

Involved in Lignin-Degradation by *Lentinula edodes* on Native-PAGE 395

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

The Hokken 600 variety of *L. edodes* (Hokken Co., Ltd., Tochigi, Japan; hereafter referred to as H600) and monokaryotic progenies derived from basidiospores were used in this study. To induce the phenol-oxidizing enzymes, mycelia were cultured in MYPG liquid medium (2.5 g malt extract; 1.0 g yeast extract; 1.0 g peptone; 5.0 g glucose in 1,000 ml of distilled water) supplemented with sawdust extract (MYPG-S). This sawdust extract was produced by adding 1 g of *Castanopsis cuspidata* (Thunb. ex Murray) Schottky sawdust to 30 ml of distilled water and then autoclaving the mixture for 15 min before filtering through filter paper (No. 1, Advantec, Tokyo) and collecting the extract. MYPG-S liquid media samples were then prepared from MYPG liquid medium by adding half a volume of sawdust extract instead of distilled water, which gave an MYPG-S extract that contained 500 mg sawdust in 30 ml media. Mycelia were sub-cultured at 25°C on MYPG 1% agar plates. After 14 days, three mycelial disks measuring 3 mm in diameter were harvested from the plates and used to inoculate 30 ml MYPG-S liquid in a 100 ml flask which was then statically cultured at 25°C. MnP activity was induced during culture on MYPG-S. Lcc activity was induced by adding 2 mM CuSO45H2O to the same media seven days after initial

A schematic representation of the strategies employed to distinguish between individual phenol-oxidizing enzymes by subtractive activity assays (Szklarz et al., 1989) and sequential

Fig. 1. Strategy underlying the subtractive activity assay and sequential enzymatic staining of a gel to distinguish between individual phenol-oxidizing enzymes. Grey, open, and solid squares represent the activities of Lcc, Per and MnP, respectively. Broken, dotted, and solid lines represent the sequential enzymatic staining of Lcc, Per and MnP in a gel,

enzymatic staining of gels using native-PAGE is shown in Fig. 1.

and produce the same catalytic products as described in the Introduction.

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* spp. (Orth et al., 1993).

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

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.
