Lentinula edodes (Berk.) Pegler, the shiitake mushroom, a white-rot basidiomycete, is one of the most valuable, cultured, edible mushrooms in the world (Chang and Miles 1989). It was traditionally cultivated on Fagaceae logs but is now grown on sawdust-based media. The ability of white-rot basidiomycetes to degrade wood components, especially lignin, affects culture time to harvesting and yields (Smith et al. 1988; Kinugawa and Tanesaka 1990; Tanesaka et al. 1993; Ohga and Kitamoto 1997). L. edodes secretes lignin-degrading enzymes, laccase (Lcc) and manganese peroxidase (MnP), on sawdust-based media (Leatham 1985; Tokimoto et al. 1987; Buswell et al. 1995; Makker et al. 2001), but not usually in liquid media. Sakamoto et al. (2009) found the main isozyme produced by L. edodes on sawdust media was manganese peroxidase, lemnp2. This finding suggests these enzymes play an important role in degrading sawdust during culture, and the expression and properties of the enzymes will influence mycelia growth and fruit body development (Wood et al. 1988).

There have been several reports on 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; 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 called “protein activity staining” or “enzymatic staining” are well established in histochemical studies and genetics (Pasteur et al. 1988). The present study reports improved procedures of enzymatic staining following polyacrylamide gel electrophoresis (native-PAGE) to distinguish Lcc and MnP isozymes induced in liquid cultures of L. edodes.

A variety of L. edodes (Hokken 600: Hokken, Tochigi, Japan) was used as a dikaryotic stock. To induce phenoloxidases, mycelia were cultured on MYPG liquid (2.5 g malt extract; 1.0 g yeast extract; 1.0 g peptone; 5.0 g glucose in 1,000 ml distilled water) supplemented with sawdust extract (MYPG-S). One gram of sawdust from Castanopsis cuspidata (Thunb. ex Murray) Schottky in 30 ml distilled water was autoclaved for 15 min and filtered with filter paper (No. 1; ADVANTEC). The filtrate was used as sawdust extract. MYPG-S liquid media was prepared from MYPG liquid with a half-volume of sawdust extract added in place of distilled water. Mycelia were subcultured at 25ºC on MYPG 1% agar plates. After 14 days, three mycelial disks, which were 3 mm in diameter, were taken from the plates and inoculated into 30 ml MYPG-S liquid in a 100-ml flask and statically cultured at 25ºC. MnP activity was induced during culture on MYPG-S. Lcc activity was induced by adding 2 mM CuSO4·5H2O to the same media 7 days after initial inoculation.

To assay activities of extracellular enzymes, 100 μl culture liquid was sampled every few days during culture and centrifuged at 13,000 rpm for 10 min; the supernatant was used as a crude enzyme solution. The crude enzyme solution was assayed for Lcc, Per, and MnP in identical 5-ml test tubes containing the following reaction mixtures: Lcc assay mixture consisted of 0.1 mM o-dianisidine in 0.1 M sodium tartrate buffer (pH 5.0); additional H2O2 (final concentration 0.1 mM) was added to Lcc assay mixture to assess Per activity; and additional MnSO4·5H2O (final concentration 0.1 mM) was added to the Per assay mixture to assess MnP activity. Aliquots (20 μl) of crude enzyme solution were added to test tubes containing 980 μl of each reaction mixture; the mixtures were incubated at 37ºC for 10 min and the reaction stopped by addition of 50 μl 40 mM NaN3. Sodium azide was added to control tubes containing the Lcc assay mixture before incubation to inactivate enzymes. Catalytic products of the reaction were spectrophotometrically assayed with o-dianisidine as a substrate at absorbance 460 nm; enzyme products were estimated by subtraction of assay measurements (Szklarz et al. 1989): i.e., Lcc activity = Lcc assay minus measurements of control; Per activity = Per assay minus Lcc assay; and MnP activity = MnP assay minus Per assay, respectively. One unit (U) of enzyme activity was defined as amount of enzyme that catalyzes 1 μmol o-dianisidine for 1 min (Paszczynski et al. 1988).

Neither MnP nor Lcc was induced under cultures on MYPG liquid without sawdust extracts (data not shown). Under MnP-induced conditions (cultured on MYPG + S) MnP activity increased suddenly after 20 days, reached maximum activity (60 U/ml) on day 35, and then decreased (Fig. 1a). Yoshikawa et al. (2004) reported that addition of wood tips or sawdust of fagaceous trees, C. cuspidata or Fagus crenata Blume, in MYPG liquid, induced MnP activity. The results from the present study show that sawdust extracts in hot water also induce MnP. Lcc activity was detected 14 days after inoculation (7 days after the addition of 2 mM CuSO4·5H2O) and continued beyond day 50, while MnP activity was suppressed (Fig. 1b). Laccase may often be induced by the addition of aromatic compounds (Scheel et al. 2000; Saparrat et al. 2002) or metallic ions including copper (Galhaup and Haltrich 2001; Soden and Dobson 2001; Galhaup et al. 2002; Shutova et al. 2008). In the white-rot basidiomycete Trametes pubescens (Schum.: Fr.) Pilát., 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 we employed, either Lcc or MnP activity was detected, but not both. These results suggest that induction of MnP and Lcc are controlled by an interlock system, i.e., Lcc induction suppresses the MnP production, or more simply, the addition of CuSO4·5H2O suppresses the MnP production.

Fig. 1
figure 1

Phenoloxidase activity, laccase (Lcc) (filled circles), peroxidase (Per) (open circles), and manganese peroxidase (MnP) (filled squares), in liquid culture of Lentinula edodes under MnP-induced (a) or Lcc-induced conditions (b). Arrow in b indicates the day of CuSO4 addition. Each value represents mean with standard error (vertical bar) of three replicate cultures

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Each phenoloxidase isozyme was detected by native PAGE. Whole cultures were filtered through a nylon stocking and the filtrate collected and centrifuged at 13,000 rpm for 10 min. The supernatant was filtered (filter paper, No. 2) and concentrated to approximately 1/15 of the original volume by ultrafiltration using Centriprep YM-10 (Millipore). Aliquots containing 15 μl filtrate and 1.5 μl loading dye (50% glycerol and 0.5% bromophenol blue, BPB) were applied to 17.5% polyacrylamide gel. Native-PAGE was run at 15 mA for 15 min followed by 25 mA for 3–4 h. After electrophoresis, the gel was sequentially incubated at 37ºC for 30 min in fundamental enzymatic staining solutions to detect each enzyme in the following order: Lcc staining solution (LccS), 1.8 mM o-dianisidine, and 0.1 M acetate buffer (pH 4.0); additional H2O2 (final concentration, 0.1 mM) to produce Per staining solution (PerS); and additional MnSO4·5H2O added to the PerS (final concentration 0.1 mM) to produce MnP staining solution (MnPS). Nomenclature of the isozyme follows May et al. (1980). Lcc-e and MnP-e refer to extracellular enzymes of Lcc and MnP, respectively, and the following numerals in parentheses represented the relative mobility of each isozyme to the mobility of bromophenol blue used as a dye marker.

We expected Lcc, Per, and MnP bands to be distinguished on the same gel by subtraction of newly appeared bands from sequential staining in the order of LccS, PerS, and MnPS. In practice, however, unexpected bands (two intense bands) often appeared on gels subjected to LccS staining solution, even on the samples prepared from MnP-induced conditions without Lcc activity (Fig. 2a, lane 1). No additional bands appeared in subsequent staining procedures with PerS or MnPS (Fig. 2b, lane 1). At first, we considered these bands developed from MnP isozymes were caused by Mn2+ contamination in the sample solution, gel, or staining solution. However, acetone precipitation of sample solution and pre-run gels at 15 mA for 20 min also gave unexpected results (data not shown).

Fig. 2
figure 2

Effects of the addition of ethylenediaminetetraacetic acid (EDTA) in staining solution on isozyme detection for one gel. a Lcc staining with LccS (lane 1) or with LccS + EDTA (lanes 2–7) conducted on aliquots of the same sample solution prepared from MnP-induced conditions (at 27 days). Lanes 2–7 contain increasing concentrations of EDTA. EDTA concentration: lane 1, 0 mM (LccS); lane 2, 100 mM; lane 3, 110 mM; lane 4, 120 mM; lane 5, 130 mM; lane 6, 140 mM; lane 7, 150 mM. b MnP staining with MnPS in sequential staining after staining with LccS and PerS (lane 1) or with LccS + EDTA and PerS + EDTA (lanes 2–7) on the same gel shown in a. c Lcc staining with LccS (lane 1) or with LccS + EDTA (lanes 2–7) conducted on aliquots of the same sample solution prepared from Lcc-induced conditions (36 days). EDTA concentration of each lane was the same as above

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In subsequent staining procedures, ethylenediaminetetraacetic acid (EDTA) was added at concentrations of 100, 110, 120, 130, 140, and 150 mM into the staining solutions LccS and PerS, named LccS + EDTA and PerS + EDTA, respectively, to eliminate Mn2+ contamination, before the gel was stained with MnPS. The gels were rinsed with distilled water between each stain procedure. When gels were stained with LccS + EDTA, the intensity of the bands weakened with increasing concentration of EDTA and disappeared at 130–150 mM (Fig. 2a, lanes 5–7). These bands reappeared following sequential staining with MnPS. This result indicated that these two bands were MnP isozymes, named MnP-e (52) and MnP-e (57), respectively (Fig. 2b). In sample solutions prepared from Lcc-induced conditions, two bands with broad tailing smears were detected by staining with LccS. Intensity of these bands also weakened as EDTA concentration in the staining solution (LccS + EDTA) increased, but did not completely disappear even at 130–150 mM EDTA (Fig. 2c). As this result is independent of Mn2+ and developed from Lcc isozymes, named Lcc-e (61) and Lcc-e (67), we deduce that treatment with approximately 130 mM EDTA in the staining solution would enable the distinction of Lcc and MnP isozymes from each other on a single gel.

By using the improved staining method described above, two MnP isozymes, MnP-e (52) and MnP-e (57), were detected during culture under MnP-induced conditions (Fig. 3a). Lcc isozymes, Lcc-e (61) and Lcc-e (67), were also detected as major isozymes during culture under Lcc-induced conditions with an additional isozyme Lcc-e (74), which was weakly detected between the 22th day and 47th day of culture (Fig. 3b).

Fig. 3
figure 3

Phenoloxidase isozymes detected by the improved staining method (see text) during cultures expressed under MnP-induced (a) and Lcc-induced (b) conditions. Days after inoculation are represented on each lane

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Isozyme distinction was confirmed by identifying peptides of each isozyme detected on gels. After native-PAGE conducted on the same sample solution on adjacent lanes, each lane of a gel was divided and subjected to enzymatic staining and Coomasie brilliant blue (CBB) staining (Fig. 4). From the CBB staining gel, bands of interest including those showing the same mobility as that of the enzymatic staining were excised using a sterile surgical blade and placed in 1.5-ml tubes. Each polyacrylamide piece was repeatedly rinsed with 50%, 30%, and 50% v/v acetonitrile containing 25 mM NH4HCO3 under sonication for 20 min with a micromixer (Taitec, Tokyo, Japan) and finally 100% acetonitrile without NH4HCO3 for 5 min. The pieces were air-dried for 5 min and recovered in 100 μl 50 mM NH4HCO3 with 10 μg/μl trypsin (sequencing grade, Roche Diagnostics) on ice for 30 min. The solution was drained and the pieces were incubated at 37ºC for 16 h. The tryptic fragments were extracted in 50 μl extract buffer consisting 50% acetonitrile and 5% formic acid under sonication for 20 min. The extract buffer was placed into a new tube and replaced with 25 μl fresh extract buffer; this extraction was repeated three or more times. The collected buffer containing the tryptic fragments was concentrated to approximately to 5 μl by drying under vacuum. Analysis of tryptic peptides by tandem mass spectrometry was performed on a nanoelectrospray ionization quadrupole time-of-flight hybrid mass spectrometer (Q-TOF Premier; Waters Micromass) coupled with a nano-HPLC (Cap-LC; Waters Micromass). The peptides were separated on a BEH130C18 column (1.7 μm, 100 μm × 100 mm; Nano Ease, Waters) according to the manufacturer’s instruction manual. The peptide sequences obtained were either matched automatically to proteins in a nonredundant database (National Center for Biotechnology Information, NCBI, http://www.ncbi.nlm.nih.gov) using the Mascot MS/MS ions search algorithm (http://www.matrixscience.com) or manual BLAST searches were performed against the current databases DNA Data Bank Japan (DDBJ, http://www.ddbj.nig.ac.jp).

Fig. 4
figure 4

Protein bands detected by enzymatic staining (a, c) and corresponding Coomassie brilliant blue (CBB) staining (b, d) for quadrupole time-of-flight (Q-TOF) mass spectrometry analysis. a, b Bands detected on MnP-induced conditions (at 22 days). c, d Bands detected on Lcc-induced conditions (at 30 days)

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Table 1 shows protein identifications by Q-TOF mass spectrometry analysis. Two MnP isozymes, MnP-e (52) and MnP-e (57), were both identified as manganese peroxidase, lemnp2, which is known to be a major MnP isozyme secreted into sawdust medium by L. edodes (Sakamoto et al. 2009). Other enzymes, β-glucosidase and exo-β-1,3-glucanase, were also detected under MnP-induced conditions. Makker et al. (2001) reported that β-glucosidase is secreted into sawdust culture media of L. edodes as a multicomponent protein complex with MnP and Lcc. Two Lcc isozymes, Lcc-e (61) and Lcc-e (67), detected under Lcc-induced conditions were both identified as laccase produced by L. edodes (lcc1: Sakamoto et al. 2008). We conclude that MnP and Lcc isozyme detection by the improved enzymatic staining solutions LccS + EDTA, PerS + EDTA, and MnPS correctly distinguishes each phenoloxidase.

Table 1 Extracellular enzymes produced by Lentinula edodes under manganese peroxidase (MnP)- and laccase (Lcc)-induced liquid culture conditions
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The separation and identification of each isozyme and enzyme activity will provide information for the evaluation and screening of breeding varieties of L. edodes. Induction of MnP and Lcc under liquid cultures and the improved method for enzymatic staining presented here are convenient and effective method of screening for isozymes of value in mushroom breeding.