**4. Conclusions**

406 Gel Electrophoresis – Advanced Techniques

We performed preliminarily examinations of the degradation of a β-*O*-4 lignin model compound under MnP- and Lcc-induced conditions (culture experiment) and the degradation of the model compound by incubation with enzyme solutions (incubation

**3.7 Degradation of β-***O***-4 lignin model compound** 

1: Extracellular enzyme solution at an initial activity adjusted to 17 U/ml

2: Mixture of the extracellular enzyme solutions (MnP+Lcc), each at an initial activity of 17 U/ml 3: Numerals in parentheses represent enzyme activities (U/ml) , nd = not detected

given culture conditions (incubation experiment) (Data from Kochi et al., 2009)

Table 1. Degradation rate (%) of β-*O*-4 lignin model compound under MnP- or Lcc-induced conditions (culture experiment) and after incubation with enzyme solutions prepared from

Under MnP-induced conditions, the β-*O*-4 compound was not degraded at all during the initial stages of the culture experiment. Indeed, effective degradation only occurred after day 21 when MnP activities suddenly increased; by day 42, 20.0% of the β-*O*-4 compound had been degraded. Conversely, no degradation of the β-*O*-4 compound was observed under Lcc-induced conditions until day 42 (4.2%). In the incubation experiment with MnP solution, the β-*O*-4 compound was effectively degraded in the initial 4 days of incubation (16.8%), with degradation increasing very gradually thereafter and then decreasing markedly near the end of the experiment; i.e., 23.8% at day 10 and only 7% of the compound was degraded in the latter 6 days. Conversely, degradation of the β-*O*-4 compound incubated with Lcc solution was detectable, but weak, until 10 days after inoculation (6.9%). The change in the degradation rates of the β-*O*-4 compound incubated with a mixture of the MnP and Lcc enzyme solutions (each at an initial activity of 17 U/ml) were similar to the degradation patterns of the MnP solution alone. This similarity indicated that no additive or multiplier effects could be attributed to the interaction of the two enzymes on the degradation of the β-*O*-4 compound. Compared to the initial period of the incubation experiment, the shallow slope of degradation rate in the latter period of the incubation was partly attributable to decreased enzyme activities over the course of the experiment (Table 1). Unfortunately, because we conducted this experiment without a protease-inhibitor, the decrease in enzyme activities was observed in enzyme solutions containing both MnP and Lcc, as well as the mixed MnP+Lcc solutions. In addition, laccase is also capable of degrading non-phenolic lignin model compounds in systems incorporating naturally

experiment) (Table 1).

When cultivated on sawdust-based media, the white-rot basidiomycete *Lentinula edodes* frequently produces the lignin-degrading enzymes MnP and Lcc. In this study, MnP produced by *L. edodes* was induced in a liquid culture supplemented with a sawdust extract of *Castanopsis cuspidata*. Lcc activity was induced by the addition of 2 mM CuSO45H2O into the same media 7 days after initial inoculation. In addition to employing native-PAGE and sequential enzymatic staining to detect the MnP and Lcc secreted by *L. edodes*, we also compared the expression of intra- and extracellular MnP isozymes. To distinguish between the phenol-oxidizing enzymes after native-PAGE, the gel was sequentially stained using an improved enzymatic staining solution (referred to as LccS+EDTA). In addition to containing 0.1 mM acetate buffer (pH 4.0) for Lcc detection, the staining solution contained 1.8 mM *o*dianisidine as the substrate and 130 mM EDTA to eliminate Mn2+ contamination. Subsequently, 0.1 mM H2O2 was added to the LccS+EDTA for Per detection (PerS+EDTA), and 0.1 mM MnSO45H2O was added to the PerS, without EDTA, for MnP detection (MnPS). The two extracellular isozyme bands, MnP-e (52) and MnP-e (57), detected in culture medium under MnP-induced conditions, were both identified as manganese peroxidase (LeMnP2). Similarity, the bands Lcc-e (61) and Lcc-e (67), which were detected under Lcc-

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes

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induced conditions, were both identified as laccase (Lcc1) by Q-TOF mass spectrometry. Four major, intracellular, MnP isozyme bands were detected in mycelial extracts obtained from *L. edodes* cultured under MnP-induced conditions. Of these isozyme bands, two exhibited the same mobilities as extracellular MnP isozymes, while the other two bands, MnP-i (63) and MnP-i (66), were strictly intracellular. The intracellular MnP isozymes were expressed during the initial stage of culture, either several days before, or coincident with, the expression of the extracellular MnP isozymes. Compared to intracellular MnP isozymes, the extracellular MnP isozymes maintained relatively high activities for up to 40 days of culture. While glycosidase treatment of crude enzyme solutions prior to electrophoresis had no effect on the activities of the extracellular MnP isozymes, such treatment completely inactivated the two strictly intracellular MnP isozymes, implying that the intracellular isozymes were active as glycosylated proteins. Both of the extracellular MnP isozymes detected in the dikaryon were also detected in monokaryotic progeny, suggesting that although these isozymes may be encoded by different loci, they are not under allelic control. Southern blot analysis revealed that the probe *lemnp2* region hybridized with the four of the monokaryotic strains used, all of which exhibited the same two hybridization signals that were observed in the parent dikaryon. These observations suggest that there are two copies of *lemnp2* in the *L. edodes* haploid genome. Moreover, degradation assays involving the addition of the β-*O*-4 lignin model compound in cultures under MnP- and Lcc-induced conditions suggest that, rather than laccase (Lcc1), manganese peroxidase (LeMnP2) is a critical enzyme for lignin degradation in *L. edodes*.

In response to the crucial role played by basidiomycetous fungi in the carbon cycle by degrading lignocelluloses, considerable effort has focused on the functional genomics related to the enzymatic systems and mechanisms involved in lignin degradation, particularly in a few model fungus species. Nevertheless, fungal succession on dead logs and leaf litter in nature show that complete degradation of lignocelluloses is a commensal and competitive process affected by numerous fungi. The assay system presented here would be practical and convenient, not only as a method of screening isozymes of value in mushroom breeding and cultivation, but also for evaluating the lignin-degrading abilities of fungi and assessing the antagonistic interactions of different strains under experimental conditions.
