*3.3. Extraction of Total Protein and Western Blot Analysis*

Total protein of *L. edodes* was extracted as previously reported [40]. Briefly, 0.1 g of mycelia or fruiting body powder from each group (three replicates for each group) was mixed with 0.5 mL of extraction buffer (0.5 M Tris-HCl, 50 mM EDTA, 0.1 M NaCl and 40 mM dithiothreitol). The supernatants were collected after extraction for 10 min and centrifugation at 10,000× *g* for 15 min at 4 ◦C to remove the insoluble substance. Next, the same volume of saturated Tris-phenol was added to the supernatants, followed by the addition of five volumes of pre-cooled 0.1 M ammonium acetate in methanol to precipitate the protein. After washing with pre-cooled 80% acetone several times, the precipitated proteins were resolubilized and denatured for 10 min in 40 μL solution buffer (7 M urea, 50 mM Tris-HCl, 25 mM EDTA, 10 mM NaCl and 60 mM dithiothreitol). Finally, the pelleted proteins were diluted to 200 μL for further analysis. The concentration of the total protein was tested by the Coomassie Brilliant Blue G250 method [41], and the quality of protein was checked by 10% SDS-PAGE (Supplementary Figure S2) [42].

Western blot was used to analyze the expression of γ-glutamyl transpeptidase (Leggt, EC 2.3.2.2) and S-alkyl-L-cysteine sulfoxide lyase (Lecsl, EC 4.4.1.4) at different growth stages of *L. edodes.* After 50 μg of each protein sample was run on 10% SDS-PAGE gels (Bio-Rad Mini, Hercules, CA, USA), Western blot was performed by standard protocols using 1:200 anti-Leggt and anti-Lecsl polyclonal antibody sera. The antibodies against Leggt and Lecsl were raised by immunizing rabbits with the mixture of purified recombinant protein, which was expressed in *Escherichia coli* BL21 and purified by Ni-NTA Agarose column (Genscript, Nanjing, China) and Freund's adjuvant [43]. The specificity of polyclonal antibodies was detected by Western blot. The results showed that anti-Leggt polyclonal antibody sera had special bands at 68 kDa, 45 kDa and 23 kDa and anti-Lecsl polyclonal antibody sera had special band at 54 kDa, respectively. 1:50,000 horseradish peroxidase conjugated secondary antibody (BOSTER, Wuhan, China). Meanwhile, the β-actin antibody (BOSTER, Wuhan, China) was treated with the same protocol as an internal control [21].

#### *3.4. Enzyme Activity Assays*

GGT activity was determined by the transfer rate of γ-glutamyl from γ-glutamyl *p*-nitroanilide (GPNA) as reported by Liu et al. [18]. The mixture including 1 mL crude enzyme extract from *L. edodes*, 1 mL GPNA (3.5 mM) and 3 mL Tris-HCl (0.5 M, pH = 7.6) was incubated at 37 ◦C for 20 min and the reaction was stopped by adding 3 mL of 1.5 M cold (4 ◦C) acetic acid. Then, the amount of p-nitroaniline released was measured at 410 nm. The specific activity of GGT was defined as the amount of enzyme that released 1 μmol of p-nitroaniline from the substrate per min per g protein (U/g).

C-S lyase activity was measured as previously described with some modifications [44]. The mixture containing 0.3 mL crude enzyme extract from *L. edodes*, 0.5 mL S-ethyl-L-cysteine sulfoxide and 0.2 mL Tris-HCl (0.5 M, pH = 7.6) was incubated at 37 ◦C for 5 min. The reaction was terminated by adding 1 mL trichloroacetic acid (TCA, 10%). After supplementation with 1 mL 2,4-dinitrophenylhydrazine (DNPH, 0.1%, *m*/*v*) were added to the mixture was incubated for 5 min at 25 ◦C. Finally, 2.5 mL NaOH (2.5 M) was added to the mixture and incubated for 10 min at 25 ◦C. The absorbance of DNPH at 520 nm was measured. The specific activity of C-S lyase was expressed as units of enzyme per g of *L. edodes* protein (U/g).
