**3.1 Selective induction of phenol-oxidizing enzyme**

Phenol-oxidizing enzyme activities under different culture conditions are shown in Fig. 3. Neither MnP nor Lcc was induced when mycelia were cultured on MYPG liquid medium without sawdust extract (data not shown). Under MnP-induced conditions (i.e. when mycelia were cultured on MYPG+S), MnP activity increased suddenly on day 21, before reaching a maximum activity (95 U/ml) on day 35 and then decreasing thereafter (Fig. 3a).

We previously found that supplementing the MYPG liquid medium with wood chips or sawdust from members of the Fagacae, *C. cuspidata* or *Fagus crenata* Blume, induced MnP

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes

each of the phenol-oxidizing enzymes (Saeki et al., 2011).

laccase (Lcc1).

induced conditions are shown in Fig. 5.

**3.2 Isozyme detection and identification** 

Involved in Lignin-Degradation by *Lentinula edodes* on Native-PAGE 401

Of CBB staining bands subjected to protein identification, proteins with FDR (q≤0.05) were described below. Number of entry (MS/MS data) was ranged from 68 to 89. In the extracellular enzyme sample (culture liquid) prepared under MnP-induced conditions, two MnP-e isozyme bands, MnP-e (52) and MnP-e (57) in Fig. 4a, were detected. These two MnP isozymes were identified as the manganese peroxidase, LeMnP2, a major MnP isozyme that is secreted into sawdust medium by *L. edodes* (Sakamoto et al., 2009). Other enzyme, exo-β-1,3-glucanase, was also detected under MnP-induced conditions (Fig. 4b). In the extracellular enzyme samples prepared under Lcc-induced conditions, two major Lcc isozyme bands, Lcc-e (61) and Lcc-e (67), were detected together with broad tailing smears (Fig. 4c). These two isozymes were identified as being laccases (Lcc1; Fig. 4d), and are known to be an extracellular laccase produced by *L. edodes* (Nagai et al., 2002; Sakamoto et al., 2008). These results, combined with enzyme assay data and results of isozyme detection using PAGE, indicate that MnP and Lcc isozyme detection using the improved LccS+EDTA, PerS+EDTA and MnPS enzymatic staining methods can be used to successfully distinguished between

Fig. 4. Protein bands detected by (**a, c**) enzymatic staining and corresponding (**b, d**) CBB staining on native-PAGE (reprinted from Saeki et al., 2011). Lanes (**a**) and (**b**) show bands detected under MnP-induced conditions at 22 days; lanes (**c**) and (**d**) show bands detected

spectrometry in (**b**): band 1, exo-β-1,3-glucanase; band 2, manganese peroxidase (LeMnP2); and band 3, manganese peroxidase (LeMnP2) and in (**d**): band 1, laccase (Lcc1); band 2,

Expression patterns of extra- and intracellular Lcc isozymes during culture under Lcc-

Three major extracellular Lcc isozymes were detected: Lcc-e (61), which was expressed from day 12 (5 days after the addition of CuSO45H2O), had a constant intensity with a broad smear tails, an Lcc-e (67) from day 17, and another Lcc-e (74) from day 22. All of these enzymes were expressed until the end of culture on day 47. The observed changes in the total band intensity of the three Lcc extracellular isozymes was generally associated with

under Lcc-induced conditions at 30 days. Protein bands identified by Q-TOF mass

**3.3 Comparisons of intracellular and extracellular Lcc isozymes** 

activity (Yoshikawa et al., 2004). The results of the present study show that sawdust extracts produced by autoclaving sawdust in hot water (section 2.2) induce MnP activity (Fig. 3a). Compared with mycelial growth on the MYPG (without extract) medium, the sawdust extract also had a marked effect on the promotion of mycelial growth (MYPG-S). Although less marked than that observed on MYPG-S (100 mg/30 ml), extracts produced using 100 mg sawdust in 30 ml media also promoted mycelial growth; however, MnP activity was not induced in cultures grown in MYPG-S media with lower extract concentrations for up to 35 days (data not shown). These observations suggested that MnP was induced by specific functional compounds in the sawdust extract, and not only due to mycelial growth.

Fig. 3. Changes of phenol-oxidizing activities of (●) Lcc, (○) Per and (■) MnP in a liquid culture medium of *L. edodes* under (**a**) MnP-induced (MYPG-S) or (**b**) Lcc-induced conditions (MYPG-S with 2mM CuSO45H2O) (reprinted from Saeki et al., 2011). The arrow in panel (**b**) indicates the day of 2 mM CuSO45H2O addition. Values are means with standard errors (vertical bars) for three replicate cultures.

Fourteen days after inoculation in MYPG-S containing Cu2+, Lcc activity was detected (7 days after the addition of 2 mM CuSO45H2O) (Fig. 3b). This Lcc activity increased gradually after day 52 while MnP activity was completely suppressed. Lcc has been shown to be induced by aromatic compounds and metallic ions such as copper (Collins & Dobson, 1997; Saparrat et al., 2002; Scheel et al., 2000; Shutova et al., 2008; Soden & Dobson, 2001). Indeed, copper has been reported to be a strong laccase inducer in the white-rot fungi *Pleurotus ostreatus* (Palmier et al., 2000), *Trametes pubescens* (Galhaup & Haltrich, 2001; Galhaup et al., 2002), and *T. versicolor* (Collins & Dobson, 1997). In *Trametes pubescens*, the transcription of the laccase gene is induced within 10 h after the addition of 2 mM CuSO4 (Galhaup et al., 2002). Under the two culture conditions employed in this study, either Lcc or MnP activity were detected, but not both (Fig. 3). This finding suggests that the induction of MnP and Lcc are controlled by a negative feedback system, i.e., Lcc-induction suppresses MnP production, or more specifically, the addition of CuSO45H2O suppresses MnP production. Although the addition of several of the aromatic compounds that were tested did not induce Lcc - 2-methoxyphenol (guaiacol), 2,6-dimethoxyphenol (DMP), 4-anisidine, hydroquinone, or 1,2-benzenediol (catechol) - these substances except for DMP were observed to suppress MnP activity (data not shown).

### **3.2 Isozyme detection and identification**

400 Gel Electrophoresis – Advanced Techniques

activity (Yoshikawa et al., 2004). The results of the present study show that sawdust extracts produced by autoclaving sawdust in hot water (section 2.2) induce MnP activity (Fig. 3a). Compared with mycelial growth on the MYPG (without extract) medium, the sawdust extract also had a marked effect on the promotion of mycelial growth (MYPG-S). Although less marked than that observed on MYPG-S (100 mg/30 ml), extracts produced using 100 mg sawdust in 30 ml media also promoted mycelial growth; however, MnP activity was not induced in cultures grown in MYPG-S media with lower extract concentrations for up to 35 days (data not shown). These observations suggested that MnP was induced by specific functional compounds in the sawdust extract, and not only due to mycelial

Fig. 3. Changes of phenol-oxidizing activities of (●) Lcc, (○) Per and (■) MnP in a liquid culture medium of *L. edodes* under (**a**) MnP-induced (MYPG-S) or (**b**) Lcc-induced conditions (MYPG-S with 2mM CuSO45H2O) (reprinted from Saeki et al., 2011). The arrow in panel (**b**) indicates the day of 2 mM CuSO45H2O addition. Values are means with standard errors

Fourteen days after inoculation in MYPG-S containing Cu2+, Lcc activity was detected (7 days after the addition of 2 mM CuSO45H2O) (Fig. 3b). This Lcc activity increased gradually after day 52 while MnP activity was completely suppressed. Lcc has been shown to be induced by aromatic compounds and metallic ions such as copper (Collins & Dobson, 1997; Saparrat et al., 2002; Scheel et al., 2000; Shutova et al., 2008; Soden & Dobson, 2001). Indeed, copper has been reported to be a strong laccase inducer in the white-rot fungi *Pleurotus ostreatus* (Palmier et al., 2000), *Trametes pubescens* (Galhaup & Haltrich, 2001; Galhaup et al., 2002), and *T. versicolor* (Collins & Dobson, 1997). In *Trametes pubescens*, the transcription of the laccase gene is induced within 10 h after the addition of 2 mM CuSO4 (Galhaup et al., 2002). Under the two culture conditions employed in this study, either Lcc or MnP activity were detected, but not both (Fig. 3). This finding suggests that the induction of MnP and Lcc are controlled by a negative feedback system, i.e., Lcc-induction suppresses MnP production, or more specifically, the addition of CuSO45H2O suppresses MnP production. Although the addition of several of the aromatic compounds that were tested did not induce Lcc - 2-methoxyphenol (guaiacol), 2,6-dimethoxyphenol (DMP), 4-anisidine, hydroquinone, or 1,2-benzenediol (catechol) - these substances except for DMP were observed to suppress

(vertical bars) for three replicate cultures.

MnP activity (data not shown).

growth.

Of CBB staining bands subjected to protein identification, proteins with FDR (q≤0.05) were described below. Number of entry (MS/MS data) was ranged from 68 to 89. In the extracellular enzyme sample (culture liquid) prepared under MnP-induced conditions, two MnP-e isozyme bands, MnP-e (52) and MnP-e (57) in Fig. 4a, were detected. These two MnP isozymes were identified as the manganese peroxidase, LeMnP2, a major MnP isozyme that is secreted into sawdust medium by *L. edodes* (Sakamoto et al., 2009). Other enzyme, exo-β-1,3-glucanase, was also detected under MnP-induced conditions (Fig. 4b). In the extracellular enzyme samples prepared under Lcc-induced conditions, two major Lcc isozyme bands, Lcc-e (61) and Lcc-e (67), were detected together with broad tailing smears (Fig. 4c). These two isozymes were identified as being laccases (Lcc1; Fig. 4d), and are known to be an extracellular laccase produced by *L. edodes* (Nagai et al., 2002; Sakamoto et al., 2008). These results, combined with enzyme assay data and results of isozyme detection using PAGE, indicate that MnP and Lcc isozyme detection using the improved LccS+EDTA, PerS+EDTA and MnPS enzymatic staining methods can be used to successfully distinguished between each of the phenol-oxidizing enzymes (Saeki et al., 2011).

Fig. 4. Protein bands detected by (**a, c**) enzymatic staining and corresponding (**b, d**) CBB staining on native-PAGE (reprinted from Saeki et al., 2011). Lanes (**a**) and (**b**) show bands detected under MnP-induced conditions at 22 days; lanes (**c**) and (**d**) show bands detected under Lcc-induced conditions at 30 days. Protein bands identified by Q-TOF mass spectrometry in (**b**): band 1, exo-β-1,3-glucanase; band 2, manganese peroxidase (LeMnP2); and band 3, manganese peroxidase (LeMnP2) and in (**d**): band 1, laccase (Lcc1); band 2, laccase (Lcc1).

#### **3.3 Comparisons of intracellular and extracellular Lcc isozymes**

Expression patterns of extra- and intracellular Lcc isozymes during culture under Lccinduced conditions are shown in Fig. 5.

Three major extracellular Lcc isozymes were detected: Lcc-e (61), which was expressed from day 12 (5 days after the addition of CuSO45H2O), had a constant intensity with a broad smear tails, an Lcc-e (67) from day 17, and another Lcc-e (74) from day 22. All of these enzymes were expressed until the end of culture on day 47. The observed changes in the total band intensity of the three Lcc extracellular isozymes was generally associated with

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes

Involved in Lignin-Degradation by *Lentinula edodes* on Native-PAGE 403

10−19-days during the initial stage of culture, with transcription increasing from day 22 to day 25 and then decreasing at day 28. These changes in *lemnp2* transcription occurred several days prior to the changes observed in MnP activities in the liquid culture medium.

Fig. 6. Northern blot analysis of *lemnp2* gene transcript under (**a**) MnP-induced conditions and (**b**) ribosomal RNA used as a loading control. Days after inoculation are shown above

Expression patterns of extra- and intracellular MnP isozymes during culture under MnPinduced conditions are shown in Fig. 7. The extracellular MnP isozymes, MnP-e (52) and MnP-e (57), were strongly expressed during the initial stage of culture on days 11 to 19, before gradually decreasing until day 43. Four major bands were considered to be intracellular MnP isozymes, and of these, two bands, MnP-i (52) and MnP-i (57), exhibited the same mobility 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 the intracellular MnP isozymes, the extracellular MnP isozymes maintained relatively high activities for up to 43 days of culture. However, changes in the intensities of bands that were neither extracellular nor intracellular MnP isozymes coincided with changes in MnP activity in the liquid culture medium during culture. Although we have no experimental data to explain why this may have occurred, it is worth noting that the intracellular enzyme solution (cell lysate) did not exhibit any phenol-oxidizing activities when assayed spectrophotometrically. In addition, the addition of intracellular enzyme solution to extracellular enzyme solutions caused marked inactivation of the latter (data not shown). Taken together, these observations either imply that the cell lysate contained a specific inhibitor of phenol-oxidizing enzymes when these enzymes and the inhibitor in cell lysate were not separated on a gel, or that there was an error in the manner in which the different experimental culture lots were processed,

While treatment with glycosidase completely inactivated the two strictly intracellular MnP isozymes, glycosidase treatment had no effect on the activities of the extracellular MnP isozymes (Fig. 8). This finding indicates that the intracellular isozymes were active as

each lane. Arrowheads indicate position of 26S and 18S rRNA.

including the replicate flasks.

**3.5 Comparisons between intracellular and extracellular MnP isozymes** 

changes in Lcc activity in the culture liquid (refer Fig. 3). Three major intracellular Lcc isozymes, Lcc-i (61), Lcc-i (67) and Lcc-i (74), were also detected, and all exhibited the same mobilities as their respective extracellular Lcc isozyme counterparts. The intracellular Lcc were coincidentally expressed with the extracellular Lcc isozymes.

Fig. 5. Laccase isozyme banding patterns detected as (**a**) extracellular and (**b**) intracellular isozymes during culture under Lcc-induced conditions. Days after inoculation are shown above each lane.

Although we successfully extracted total RNA from mycelia under Lcc-induced conditions to examine the transcription of Lcc1, extraction of native (undigested) total RNA was unsuccessful. Native total RNA, which was prepared from mycelia under MnP-induced conditions (described in section 3.4 below), was degraded considerably quicker and to a greater extent after the addition of small amounts of cell lysate obtained under Lcc-induced conditions compared to when cell lysate obtained under MnP-induced conditions was added (data not shown). This relatively quicker degradation of native total RNA suggests a relatively high internal RNase activity in the cell lysate of the Lcc-induced condition, which may be attributed to the decrease observed in mycelial growth after the addition of CuSO45H2O and subsequent induction of Lcc, as well as the antagonistic expression of Lcc in the mycelial contact zone of any adjacent and competing basidiomycetes or other fungi (Iakovlev & Stenlid, 2000; Mercer, 1982; White & Boddy, 1992).

#### **3.4 Manganese peroxidase gene transcription**

The sequence of the fragment amplified from cDNA, which was prepared from a mixed pool of total RNAs obtained from the mycelia of 10-, 15- and 18-day-old cultures under MnP-induced conditions, was identical to that of *lemnp2a* but slightly different from *lemnp2b* (data not shown), corroborating the results obtained from the protein identification deduced by Q-TOF mass spectrometry (section 3.2). The finding that these sequences were similar also indicated that hot-water sawdust extracts induced the secretion of the same isozyme, LeMnP2, which is a major MnP isozyme that is secreted into sawdust media (Sakamoto et al. 2009). The results of the Northern blotting experiments with *lemnp2* are shown in Fig. 6. Under MnP-induced conditions, a detectable amount of *lemnp2* mRNA was present at

changes in Lcc activity in the culture liquid (refer Fig. 3). Three major intracellular Lcc isozymes, Lcc-i (61), Lcc-i (67) and Lcc-i (74), were also detected, and all exhibited the same mobilities as their respective extracellular Lcc isozyme counterparts. The intracellular Lcc

Fig. 5. Laccase isozyme banding patterns detected as (**a**) extracellular and (**b**) intracellular isozymes during culture under Lcc-induced conditions. Days after inoculation are shown

Although we successfully extracted total RNA from mycelia under Lcc-induced conditions to examine the transcription of Lcc1, extraction of native (undigested) total RNA was unsuccessful. Native total RNA, which was prepared from mycelia under MnP-induced conditions (described in section 3.4 below), was degraded considerably quicker and to a greater extent after the addition of small amounts of cell lysate obtained under Lcc-induced conditions compared to when cell lysate obtained under MnP-induced conditions was added (data not shown). This relatively quicker degradation of native total RNA suggests a relatively high internal RNase activity in the cell lysate of the Lcc-induced condition, which may be attributed to the decrease observed in mycelial growth after the addition of CuSO45H2O and subsequent induction of Lcc, as well as the antagonistic expression of Lcc in the mycelial contact zone of any adjacent and competing basidiomycetes or other fungi

The sequence of the fragment amplified from cDNA, which was prepared from a mixed pool of total RNAs obtained from the mycelia of 10-, 15- and 18-day-old cultures under MnP-induced conditions, was identical to that of *lemnp2a* but slightly different from *lemnp2b* (data not shown), corroborating the results obtained from the protein identification deduced by Q-TOF mass spectrometry (section 3.2). The finding that these sequences were similar also indicated that hot-water sawdust extracts induced the secretion of the same isozyme, LeMnP2, which is a major MnP isozyme that is secreted into sawdust media (Sakamoto et al. 2009). The results of the Northern blotting experiments with *lemnp2* are shown in Fig. 6. Under MnP-induced conditions, a detectable amount of *lemnp2* mRNA was present at

were coincidentally expressed with the extracellular Lcc isozymes.

(Iakovlev & Stenlid, 2000; Mercer, 1982; White & Boddy, 1992).

**3.4 Manganese peroxidase gene transcription** 

above each lane.

10−19-days during the initial stage of culture, with transcription increasing from day 22 to day 25 and then decreasing at day 28. These changes in *lemnp2* transcription occurred several days prior to the changes observed in MnP activities in the liquid culture medium.

Fig. 6. Northern blot analysis of *lemnp2* gene transcript under (**a**) MnP-induced conditions and (**b**) ribosomal RNA used as a loading control. Days after inoculation are shown above each lane. Arrowheads indicate position of 26S and 18S rRNA.

#### **3.5 Comparisons between intracellular and extracellular MnP isozymes**

Expression patterns of extra- and intracellular MnP isozymes during culture under MnPinduced conditions are shown in Fig. 7. The extracellular MnP isozymes, MnP-e (52) and MnP-e (57), were strongly expressed during the initial stage of culture on days 11 to 19, before gradually decreasing until day 43. Four major bands were considered to be intracellular MnP isozymes, and of these, two bands, MnP-i (52) and MnP-i (57), exhibited the same mobility 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 the intracellular MnP isozymes, the extracellular MnP isozymes maintained relatively high activities for up to 43 days of culture. However, changes in the intensities of bands that were neither extracellular nor intracellular MnP isozymes coincided with changes in MnP activity in the liquid culture medium during culture. Although we have no experimental data to explain why this may have occurred, it is worth noting that the intracellular enzyme solution (cell lysate) did not exhibit any phenol-oxidizing activities when assayed spectrophotometrically. In addition, the addition of intracellular enzyme solution to extracellular enzyme solutions caused marked inactivation of the latter (data not shown). Taken together, these observations either imply that the cell lysate contained a specific inhibitor of phenol-oxidizing enzymes when these enzymes and the inhibitor in cell lysate were not separated on a gel, or that there was an error in the manner in which the different experimental culture lots were processed, including the replicate flasks.

While treatment with glycosidase completely inactivated the two strictly intracellular MnP isozymes, glycosidase treatment had no effect on the activities of the extracellular MnP isozymes (Fig. 8). This finding indicates that the intracellular isozymes were active as

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes

lane 2, #208 (A1B2); lane 3, #305 (A2B1) and lane 4, #105 (A2B2).

they are not allozymes).

Involved in Lignin-Degradation by *Lentinula edodes* on Native-PAGE 405

isozymes may be encoded by different loci, the isozymes are not under allelic control (i.e.

Fig. 9. Extracellular manganese peroxidase isozymes expressed by monokaryotic progenies of H600. Lanes represent strains with mating type factor in parentheses: lane 1, #317 (A1B1);

The results of the Southern blotting experiment of *lemnp2* on *Hin*d III-digested monokaryon genomes are shown in Fig. 10. There was no *Hin*d III restriction site in the amplified region (*lemnp2*S) of the H600 genomic DNA, as expected from the database analysis of different *L. edodes* stock SR-1 (DDBJ Acc. No. AB306944, Sakamoto et al. 2009). Two *lemnp2* hybridization signals appeared at positions between 564-2322 bp in all four of the monokaryotic strains (lanes 1-4 in Fig. 10), and all of the strains exhibited the same two hybridization signals observed in the H600 parent dikaryon (lane 5 in Fig. 10). However, single intense and weak hybridization signals of *lemnp1* were observed using another probe, *lemnp1*S, at different positions between 2322-6557 bp in H600 (lane 6 in Fig. 10), indicating that the two probes did not cross-hybridize with each other. Conversely, it is likely that the weak hybridization signals that appeared between 2322-4631 bp (lane 2 in Fig. 10) were cross-hybridization products between the two probes (see lane 6 in Fig. 10). These observations, combined with the observation of two isozymes being expressed by all of the monokaryons assayed in this study, suggest that there are two copies of *lemnp2* in the haploid genome of *L. edodes*. Nevertheless, to confirm whether the lemnp2 gene is indeed duplicated as proposed here, further analysis will need to be undertaken to assign lemnp2 to a genetic linkage map or on chromosomal DNA which separated by contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. Indeed, such attempts at combining assignments of quantitative trait loci (QTL) related to wood and lignin degradation in fungi would facilitate the identification of new genes involved in another ligninolytic system.

Fig. 10. Southern blot analysis with probe *lemnp2*S on genomic DNAs (digested with *Hin*d III). Lanes represent strains: lane M, size marker (*λ/Hin*d III); lane 1, #317; lane 2, #208; lane

3, #305; lane 4, #105, lane 5, H600; and lane 6, H600 (probed with *lemnp1*S).

glycosylated proteins, and implies that a relationship exists between the secretion of MnP and the simultaneous expression of β-glucanase detected by Q-TOF mass spectrometry.

Fig. 7. Manganese peroxidase isozyme banding patterns detected as (**a**) extracellular and (**b**) intracellular isozymes during culture under MnP-induced conditions. Days after inoculation are shown above each lane.

Fig. 8. Effects of glycosidase treatment on enzymatic staining of (**a**) extracellular and (**b**) intracellular MnP isozymes expressed under MnP-induced conditions (at 20 days). Lanes differ according to concentration of glycosidase: lane 1, control (0%); lane 2, 0.25%; lane 3, 0.5%; lane 4, 1.0%; and lane 5, 2.0%, respectively. MnP-i (57) was not detected.

#### **3.6 MnP isozymes in monokaryons**

Four monokaryotic strains (#317, #208, #305 and #105), each carrying the mating type factor A1B1, A1B2, A2B1 and A2B2, respectively, were derived from basidiospores from dikaryon H600. Although MnP activities of the monokaryons were very weak compared to the MnP activities of H600 (refer Fig. 7), both of the extracellular MnP isozymes (MnP-e (52) and MnP-e (57)) that were detected in the parent dikaryon were also detected in monokaryons, irrespective of their mating-type factors (Fig. 9). This finding suggests that, although these

glycosylated proteins, and implies that a relationship exists between the secretion of MnP and the simultaneous expression of β-glucanase detected by Q-TOF mass spectrometry.

Fig. 7. Manganese peroxidase isozyme banding patterns detected as (**a**) extracellular and (**b**) intracellular isozymes during culture under MnP-induced conditions. Days after inoculation

Fig. 8. Effects of glycosidase treatment on enzymatic staining of (**a**) extracellular and (**b**) intracellular MnP isozymes expressed under MnP-induced conditions (at 20 days). Lanes differ according to concentration of glycosidase: lane 1, control (0%); lane 2, 0.25%; lane 3,

Four monokaryotic strains (#317, #208, #305 and #105), each carrying the mating type factor A1B1, A1B2, A2B1 and A2B2, respectively, were derived from basidiospores from dikaryon H600. Although MnP activities of the monokaryons were very weak compared to the MnP activities of H600 (refer Fig. 7), both of the extracellular MnP isozymes (MnP-e (52) and MnP-e (57)) that were detected in the parent dikaryon were also detected in monokaryons, irrespective of their mating-type factors (Fig. 9). This finding suggests that, although these

0.5%; lane 4, 1.0%; and lane 5, 2.0%, respectively. MnP-i (57) was not detected.

are shown above each lane.

**3.6 MnP isozymes in monokaryons** 

isozymes may be encoded by different loci, the isozymes are not under allelic control (i.e. they are not allozymes).

Fig. 9. Extracellular manganese peroxidase isozymes expressed by monokaryotic progenies of H600. Lanes represent strains with mating type factor in parentheses: lane 1, #317 (A1B1); lane 2, #208 (A1B2); lane 3, #305 (A2B1) and lane 4, #105 (A2B2).

The results of the Southern blotting experiment of *lemnp2* on *Hin*d III-digested monokaryon genomes are shown in Fig. 10. There was no *Hin*d III restriction site in the amplified region (*lemnp2*S) of the H600 genomic DNA, as expected from the database analysis of different *L. edodes* stock SR-1 (DDBJ Acc. No. AB306944, Sakamoto et al. 2009). Two *lemnp2* hybridization signals appeared at positions between 564-2322 bp in all four of the monokaryotic strains (lanes 1-4 in Fig. 10), and all of the strains exhibited the same two hybridization signals observed in the H600 parent dikaryon (lane 5 in Fig. 10). However, single intense and weak hybridization signals of *lemnp1* were observed using another probe, *lemnp1*S, at different positions between 2322-6557 bp in H600 (lane 6 in Fig. 10), indicating that the two probes did not cross-hybridize with each other. Conversely, it is likely that the weak hybridization signals that appeared between 2322-4631 bp (lane 2 in Fig. 10) were cross-hybridization products between the two probes (see lane 6 in Fig. 10). These observations, combined with the observation of two isozymes being expressed by all of the monokaryons assayed in this study, suggest that there are two copies of *lemnp2* in the haploid genome of *L. edodes*. Nevertheless, to confirm whether the lemnp2 gene is indeed duplicated as proposed here, further analysis will need to be undertaken to assign lemnp2 to a genetic linkage map or on chromosomal DNA which separated by contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. Indeed, such attempts at combining assignments of quantitative trait loci (QTL) related to wood and lignin degradation in fungi would facilitate the identification of new genes involved in another ligninolytic system.

Fig. 10. Southern blot analysis with probe *lemnp2*S on genomic DNAs (digested with *Hin*d III). Lanes represent strains: lane M, size marker (*λ/Hin*d III); lane 1, #317; lane 2, #208; lane 3, #305; lane 4, #105, lane 5, H600; and lane 6, H600 (probed with *lemnp1*S).

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes

likely to extend into the future (Cullen & Kersten, 2004).

lignin-degradation.

**4. Conclusions** 

Involved in Lignin-Degradation by *Lentinula edodes* on Native-PAGE 407

occurring or synthetic redox mediators (Johannes & Majcherczyk, 2000; Srebotnik & Hamel 2000; Tanaka et al., 2009). Nevertheless, based on the above results, manganese peroxidase (LeMnP2) appears to be more important than laccase (Lcc1) in lignin degradation by *L. edodes*. Prior to the discovery of Lip and MnP, one of the major catabolites formed by the degradation of the β-*O*-4 dimer by *P. chrysosporium*, 2-guaiacoxyethanol (II), was identified (Enoki et al., 1980). The results described above suggest that the assay system developed in this study is well suited for identifying phenol-oxidizing isozymes involved in the degradation of lignin model compounds. Further, these phenol-oxidizing isozymes have been effective for elucidating the mechanisms involved lignin degradation, and this role is

Although we attempted to identify other MnP isozymes using another commercial Japanese Shiitake variety, "Bridge 32" (The General Environmental Technos Co. Ltd., Osaka, Japan), which is also used in sawdust cultivation, the variety exhibited the same extracellular MnP isozyme patterns as H600 (data not shown). The estimated heritability (*h*2), which is the ratio of the additive genetic components of variance to the phenotypic components of variance, of the variety's wood-degrading ability was relatively low (32.2%) compared to the heritabilities estimated for other traits in crosses of H600 and Bridge 32 (Tanesaka et al., 2007). This low heritability may be attributable to the low allelic variation that exists between the MnP isozymes of the two varieties. The MnP that is produced by *L. edodes* when it is cultured on sawdust media (Buswell et al., 1995; Leatham 1985; Makker et al., 2001; Sakamoto et al., 2009), and which degrades the β-*O*-4 lignin model compound (Kochi et al., 2009), is likely to be critical for mycelial growth and fruit-body development during sawdust cultivation. The system presented here for assaying phenol-oxidizing enzymes under liquid culture conditions could therefore provide a practical screening method for examining isozymes of value in mushroom cultivation, particularly since the assay system targets the wood-degrading ability and the genomic characteristics of the genes involved in

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-

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

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 experiment) (Table 1).


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

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

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 occurring or synthetic redox mediators (Johannes & Majcherczyk, 2000; Srebotnik & Hamel 2000; Tanaka et al., 2009). Nevertheless, based on the above results, manganese peroxidase (LeMnP2) appears to be more important than laccase (Lcc1) in lignin degradation by *L. edodes*. Prior to the discovery of Lip and MnP, one of the major catabolites formed by the degradation of the β-*O*-4 dimer by *P. chrysosporium*, 2-guaiacoxyethanol (II), was identified (Enoki et al., 1980). The results described above suggest that the assay system developed in this study is well suited for identifying phenol-oxidizing isozymes involved in the degradation of lignin model compounds. Further, these phenol-oxidizing isozymes have been effective for elucidating the mechanisms involved lignin degradation, and this role is likely to extend into the future (Cullen & Kersten, 2004).

Although we attempted to identify other MnP isozymes using another commercial Japanese Shiitake variety, "Bridge 32" (The General Environmental Technos Co. Ltd., Osaka, Japan), which is also used in sawdust cultivation, the variety exhibited the same extracellular MnP isozyme patterns as H600 (data not shown). The estimated heritability (*h*2), which is the ratio of the additive genetic components of variance to the phenotypic components of variance, of the variety's wood-degrading ability was relatively low (32.2%) compared to the heritabilities estimated for other traits in crosses of H600 and Bridge 32 (Tanesaka et al., 2007). This low heritability may be attributable to the low allelic variation that exists between the MnP isozymes of the two varieties. The MnP that is produced by *L. edodes* when it is cultured on sawdust media (Buswell et al., 1995; Leatham 1985; Makker et al., 2001; Sakamoto et al., 2009), and which degrades the β-*O*-4 lignin model compound (Kochi et al., 2009), is likely to be critical for mycelial growth and fruit-body development during sawdust cultivation. The system presented here for assaying phenol-oxidizing enzymes under liquid culture conditions could therefore provide a practical screening method for examining isozymes of value in mushroom cultivation, particularly since the assay system targets the wood-degrading ability and the genomic characteristics of the genes involved in lignin-degradation.
