**2.2 Fungi and culture conditions**

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 inoculation.

### **2.3 Enzyme assay**

A schematic representation of the strategies employed to distinguish between individual phenol-oxidizing enzymes by subtractive activity assays (Szklarz et al., 1989) and sequential enzymatic staining of gels using native-PAGE is shown in Fig. 1.

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, respectively.

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes

**2.6 Identification of isozymes by mass spectrometry** 

(FDR) on acquired MS/MS data against decoy database.

bromophenol blue used as a dye.

**2.5 Glycosidase treatment** 

Involved in Lignin-Degradation by *Lentinula edodes* on Native-PAGE 397

MnP-e (57) etc. indicated the relative mobility of each isozyme relative to the mobility of the

To purify the enzymes in the crude enzyme solutions prior to electrophoresis, 1 ml acetone was added to a 100 µl aliquot of the enzyme solution and kept at −20°C for 3 h to precipitate the proteins. The proteins were then resuspended in 100 µl of 10 mM phosphate buffer (pH 6.0). To determine whether the enzymes were glycosylated, the protein suspension in phosphate buffer was incubated with glycosidase (Glycosidases 'Mixed', Seikagaku Biobusiness Corp., Japan) at final concentrations of 0.25−2.0% (w/v) with a protease inhibitor (Complete, Mini, EDTA-free; Roche Diagnostics, Germany) at 37°C overnight. Effects of the glycosidase treatment on activities of each of the isozymes were then examined

Distinguishing between isozymes was performed as described previously (Saeki et al. 2011). Briefly, after native-PAGE had been conducted on the same sample solution in adjacent lanes, each gel was then subjected to enzymatic staining and Coomassie brilliant blue (CBB) staining. Bands of interest, such as those exhibiting the same mobility as bands in the enzymatic staining experiments, were then excised from the CBB-stained gel using a sterile surgical blade and placed in 1.5 ml microcentrifuge tubes. To remove the CBB dye, each polyacrylamide gel section was then repeatedly washed with 50, 30 and 50% v/v acetonitrile containing 25 mM NH4HCO3 under sonication for 20 min with a micromixer (Taitec, Tokyo), before finally being washed with 100% acetonitrile without NH4HCO3 for 5 min. The sections of polyacrylamide gel were then vacuum-dried for 5 min and recovered in 100 µl of 50 mM NH4HCO3 (pH 7.8) containing 10 ng/µl trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, WI) on ice for 30 min. Any extra trypsin solution was then removed and the sections of gel were incubated at 37°C for 16 h. The tryptic fragments in a gel were then extracted by immersing the gel sections in 50 µl of extraction buffer consisting 50% acetonitrile and 5% trifluoroacetic acid under sonication (Ultrasonic cleaner, SU-3T, Shibata, Japan) for 20 min. The extraction buffer was placed into new tube and replaced with 25 µl of fresh extraction buffer. The extraction process was repeated a further three times and the collected buffer containing the tryptic fragments was finally concentrated to approximately 5 µl by drying under vacuum. Analysis of the tryptic peptides by tandem mass spectrometry was performed on a nanoelectrospray ionization quadrupole time-offlight (Q-TOF) hybrid mass spectrometer (Q-TOF Premier, Waters Micromass, MA) coupled with a nano-HPLC (Cap-LC; Waters Micromass). The peptides were separated on a BEH 130-C18 column (1.7 µm, 100 μm × 100 mm, Nano Ease, Waters, MA) at flow rate of 0.2 µl/min according to the manufacturer's instructions. The peptide sequences thus obtained were then either matched automatically to proteins in a non-redundant database (National Center for Biotechnology Information, NCBI, www.ncbi.nlm.nih.gov) using the Mascot MS/MS ions search algorithm (Mascot Server version 2.2, Matrix Science), or BLAST searches were manually performed against the DNA Data Bank of Japan database (DDBJ, www.ddbj.nig.ac.jp). Mascot search was also performed to calculate the false discovery rate

by enzymatic staining after native-PAGE as described in section 2.4 above.

To assay the activities of extracellular enzymes, 100 µl of culture liquid was sampled every two to three days during culture and centrifuged at 13,000 rpm for 10 min; this 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), with additional H2O2 (final concentration 0.1 mM) added to the Lcc assay mixture to assess Per activity. Additional MnSO45H2O (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, which were then incubated at 37°C for 10 min. The reactions were stopped by the addition of 50 µl of 40 mM NaN3. To inactivate the enzymes in the control tubes, sodium azide was added to the control tubes containing the Lcc assay mixture before incubation. Catalytic products of the reaction were spectrophotometrically assayed using *o*-dianisidine as a substrate, and the activity of enzyme products was estimated by subtracting the respective absorbance values at 460 nm: i.e., Lcc activity = Lcc assay minus the control assay; Per activity = Per assay minus the Lcc assay; and MnP activity = MnP assay minus the Per assay, respectively. One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyze 1 µmol of *o*dianisidine in 1 min (ε460 = 29,400 M-1cm-1: Paszczynski et al., 1988).

#### **2.4 Native PAGE and enzymatic staining**

Each of the phenol-oxidizing isozymes was detected by native PAGE as described previously (Saeki et al., 2011). Briefly, whole cultures were filtered through a nylon stocking to separate the mycelia from the culture liquid. The collected mycelia were then ground with a ceramic mortar and pestle in two volumes (v/w) of crushing buffer (0.05 M Tris-HCl, pH 7.2, 0.1% β-mercaptoethanol) before being centrifuged at 12,000 rpm for 10 min. The resulting supernatant was considered to represent the intracellular enzyme sample. To prepare the extracellular enzyme sample, the culture liquid was centrifuged at 13,000 rpm for 10 min, and the supernatant was filtered (No.2 filter paper, Advantec) and then concentrated 15-fold by ultrafiltration using the centrifugal filter unit, Centriprep YM-10 (10-kDa cut-off membrane, Millipore, MA). Aliquots containing 15 µl enzyme sample, 1.5 µl glycerol and 1.5 mg bromophenol blue (BPB) as a dye marker were then loaded into the wells of 12.5% (for intracellular) or 17.5% (for extracellular) polyacrylamide gels. Native-PAGE gels were run at 15 mA for 15 min followed by 25 mA for 3−4 h. After electrophoresis, the gel was sequentially incubated at 37°C for 30 min in three different staining solutions. The first staining solution was an improved enzymatic staining solution containing additional ethylenediaminetetraacetic acid (EDTA) (for Lcc and Per) to remove the Mn2+ typically used in conventional staining solutions. Staining for enzymes was performed as follows. To the Lcc staining solution (LccS+EDTA), 1.8 mM *o*-dianisidine, 0.1 mM acetate buffer (pH 4.0) containing 130 mM EDTA (LccS+EDTA), an additional H2O2 (final concentration 1.0 mM) was added to produce the Per staining solution (PerS+EDTA). In the same way, additional MnSO45H2O (final concentration 0.1 mM) was added to the PerS without EDTA to produce the MnP staining solution (MnPS). The gels were rinsed with distilled water between each staining procedure to remove the previous staining solutions, particularly the EDTA from PerS+EDTA used for MnP staining. Isozyme nomenclature employed an (-e) or (-i) in Lcc-e, MnP-e or MnP-i to indicate whether the Lcc and MnP enzymes were extra- or intracellular. Numerals in parenthesis, e.g., MnP-e (52), MnP-e (57) etc. indicated the relative mobility of each isozyme relative to the mobility of the bromophenol blue used as a dye.

#### **2.5 Glycosidase treatment**

396 Gel Electrophoresis – Advanced Techniques

To assay the activities of extracellular enzymes, 100 µl of culture liquid was sampled every two to three days during culture and centrifuged at 13,000 rpm for 10 min; this 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), with additional H2O2 (final concentration 0.1 mM) added to the Lcc assay mixture to assess Per activity. Additional MnSO45H2O (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, which were then incubated at 37°C for 10 min. The reactions were stopped by the addition of 50 µl of 40 mM NaN3. To inactivate the enzymes in the control tubes, sodium azide was added to the control tubes containing the Lcc assay mixture before incubation. Catalytic products of the reaction were spectrophotometrically assayed using *o*-dianisidine as a substrate, and the activity of enzyme products was estimated by subtracting the respective absorbance values at 460 nm: i.e., Lcc activity = Lcc assay minus the control assay; Per activity = Per assay minus the Lcc assay; and MnP activity = MnP assay minus the Per assay, respectively. One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyze 1 µmol of *o*-

Each of the phenol-oxidizing isozymes was detected by native PAGE as described previously (Saeki et al., 2011). Briefly, whole cultures were filtered through a nylon stocking to separate the mycelia from the culture liquid. The collected mycelia were then ground with a ceramic mortar and pestle in two volumes (v/w) of crushing buffer (0.05 M Tris-HCl, pH 7.2, 0.1% β-mercaptoethanol) before being centrifuged at 12,000 rpm for 10 min. The resulting supernatant was considered to represent the intracellular enzyme sample. To prepare the extracellular enzyme sample, the culture liquid was centrifuged at 13,000 rpm for 10 min, and the supernatant was filtered (No.2 filter paper, Advantec) and then concentrated 15-fold by ultrafiltration using the centrifugal filter unit, Centriprep YM-10 (10-kDa cut-off membrane, Millipore, MA). Aliquots containing 15 µl enzyme sample, 1.5 µl glycerol and 1.5 mg bromophenol blue (BPB) as a dye marker were then loaded into the wells of 12.5% (for intracellular) or 17.5% (for extracellular) polyacrylamide gels. Native-PAGE gels were run at 15 mA for 15 min followed by 25 mA for 3−4 h. After electrophoresis, the gel was sequentially incubated at 37°C for 30 min in three different staining solutions. The first staining solution was an improved enzymatic staining solution containing additional ethylenediaminetetraacetic acid (EDTA) (for Lcc and Per) to remove the Mn2+ typically used in conventional staining solutions. Staining for enzymes was performed as follows. To the Lcc staining solution (LccS+EDTA), 1.8 mM *o*-dianisidine, 0.1 mM acetate buffer (pH 4.0) containing 130 mM EDTA (LccS+EDTA), an additional H2O2 (final concentration 1.0 mM) was added to produce the Per staining solution (PerS+EDTA). In the same way, additional MnSO45H2O (final concentration 0.1 mM) was added to the PerS without EDTA to produce the MnP staining solution (MnPS). The gels were rinsed with distilled water between each staining procedure to remove the previous staining solutions, particularly the EDTA from PerS+EDTA used for MnP staining. Isozyme nomenclature employed an (-e) or (-i) in Lcc-e, MnP-e or MnP-i to indicate whether the Lcc and MnP enzymes were extra- or intracellular. Numerals in parenthesis, e.g., MnP-e (52),

dianisidine in 1 min (ε460 = 29,400 M-1cm-1: Paszczynski et al., 1988).

**2.4 Native PAGE and enzymatic staining** 

To purify the enzymes in the crude enzyme solutions prior to electrophoresis, 1 ml acetone was added to a 100 µl aliquot of the enzyme solution and kept at −20°C for 3 h to precipitate the proteins. The proteins were then resuspended in 100 µl of 10 mM phosphate buffer (pH 6.0). To determine whether the enzymes were glycosylated, the protein suspension in phosphate buffer was incubated with glycosidase (Glycosidases 'Mixed', Seikagaku Biobusiness Corp., Japan) at final concentrations of 0.25−2.0% (w/v) with a protease inhibitor (Complete, Mini, EDTA-free; Roche Diagnostics, Germany) at 37°C overnight. Effects of the glycosidase treatment on activities of each of the isozymes were then examined by enzymatic staining after native-PAGE as described in section 2.4 above.

### **2.6 Identification of isozymes by mass spectrometry**

Distinguishing between isozymes was performed as described previously (Saeki et al. 2011). Briefly, after native-PAGE had been conducted on the same sample solution in adjacent lanes, each gel was then subjected to enzymatic staining and Coomassie brilliant blue (CBB) staining. Bands of interest, such as those exhibiting the same mobility as bands in the enzymatic staining experiments, were then excised from the CBB-stained gel using a sterile surgical blade and placed in 1.5 ml microcentrifuge tubes. To remove the CBB dye, each polyacrylamide gel section was then repeatedly washed with 50, 30 and 50% v/v acetonitrile containing 25 mM NH4HCO3 under sonication for 20 min with a micromixer (Taitec, Tokyo), before finally being washed with 100% acetonitrile without NH4HCO3 for 5 min. The sections of polyacrylamide gel were then vacuum-dried for 5 min and recovered in 100 µl of 50 mM NH4HCO3 (pH 7.8) containing 10 ng/µl trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, WI) on ice for 30 min. Any extra trypsin solution was then removed and the sections of gel were incubated at 37°C for 16 h. The tryptic fragments in a gel were then extracted by immersing the gel sections in 50 µl of extraction buffer consisting 50% acetonitrile and 5% trifluoroacetic acid under sonication (Ultrasonic cleaner, SU-3T, Shibata, Japan) for 20 min. The extraction buffer was placed into new tube and replaced with 25 µl of fresh extraction buffer. The extraction process was repeated a further three times and the collected buffer containing the tryptic fragments was finally concentrated to approximately 5 µl by drying under vacuum. Analysis of the tryptic peptides by tandem mass spectrometry was performed on a nanoelectrospray ionization quadrupole time-offlight (Q-TOF) hybrid mass spectrometer (Q-TOF Premier, Waters Micromass, MA) coupled with a nano-HPLC (Cap-LC; Waters Micromass). The peptides were separated on a BEH 130-C18 column (1.7 µm, 100 μm × 100 mm, Nano Ease, Waters, MA) at flow rate of 0.2 µl/min according to the manufacturer's instructions. The peptide sequences thus obtained were then either matched automatically to proteins in a non-redundant database (National Center for Biotechnology Information, NCBI, www.ncbi.nlm.nih.gov) using the Mascot MS/MS ions search algorithm (Mascot Server version 2.2, Matrix Science), or BLAST searches were manually performed against the DNA Data Bank of Japan database (DDBJ, www.ddbj.nig.ac.jp). Mascot search was also performed to calculate the false discovery rate (FDR) on acquired MS/MS data against decoy database.

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes

(anisoin), which was used as an internal standard.

β-guaiacyl ether (from Umezawa & Higuchi, 1985).

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

**2.9.2 Incubation experiment** 

**3. Results and discussion** 

(Fig. 3a).

Involved in Lignin-Degradation by *Lentinula edodes* on Native-PAGE 399

TMS derivative was then subjected to gas chromatography-mass spectrometry (GC-MS) analysis (6890N, Agilent Technologies, CA), which was fitted with an capillary column (HP-5 MS, 30 m × 0.25 mm i.d., 0.25-µm; J&W Scientific, CA) coupled to an MS (JMS-K9, JEOL, Japan) according to the manufacturer's instructions. Helium was used as the carrier gas at 1.5 ml/min. GC oven conditions consisted of 150°C for 1 min, initially ramped at 10°C/min to 200°C and then at 5°C/min to 250°C. The electron impact mass spectra were obtained at an acceleration energy of 70 eV. The degradation rate (%) of the β-*O*-4 compound was calculated using the rate of quantities of the TMS derivative before and after culture, compensating for the recovery of the β-*O*-4 compound with 4,4'-dimethoxybenzoin

Fig. 2. β-*O*-4 lignin model compound used in this study; 4-ethoxy-3-methoxyphenylglycerol-

Degradation of the β-*O*-4 compound was also examined by incubation with the extracellular enzyme solution, which was prepared using the same procedures used for electrophoresis (section 2.4) with the following slight modifications. The extracellular enzyme solutions were diluted with 0.2 M sodium tartrate buffer (pH 5.0, final concentration of 0.1 M) and distilled water to bring the volume to 10 ml and keep the activity of MnP, Lcc and the mixed solution (MnP+Lcc) at 17 U/ml. For the MnP and MnP+Lcc reactions, additional H2O2 (final concentration 0.1 mM) and MnSO45H2O (final concentration 0.1 mM) were added to the reaction for Lcc. Then, 500 µg of the β-*O*-4 compound in 50 µl of acetone was added to the 10 ml enzyme solution and incubated at 37°C with agitation at 100 rpm for up to 10 days. The

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

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

rate of degradation of the β-*O*-4 compound was evaluated using GC-MS as above.
