Hydrolytic Enzymes in the Secretome of the Mushrooms P. eryngii and P. ostreatus: A Comparison Between the Two Species

Abstract

The fungi belonging to the genus Pleurotus can be cultivated in different substrates and represent excellent producers of several extracellular enzymes. In this study, we analyzed eleven hydrolytic enzymes of the P. eryngii and P. ostreatus secretomes, which were collected at three different growth stages after 23 days (mycelial colonization of about 50% of the substrate), 34 days (100% colonization of the substrate) and 50 days (after the first flush). Mushrooms were axenically cultivated on the same substrate. The results demonstrate that proteases, lipases, amylases, α-glucosidase, cellulases (endoglucanase, β-cellobiohydrolase and β-glucosidase) and hemicellulase (xylosidase, glucuronidase, arabinosidase and mannosidase) activities were higher in the secretomes from P. eryngii than those from P. ostreatus. Time course analysis revealed for both species a similar enzymatic activity profile, in which in the early stages of mycelium development, both species use starch as the main carbon source. Protease and lipase activities increased and remained constant during the subsequent formation of fruiting bodies, whereas cellulase and hemicellulase activities decreased after the complete mycelial colonization of the substrate. The zymographic analysis suggested the presence in the secretomes of proteolytic activities belonging to different classes. In conclusion, both mushroom species released into the secretomes a broad spectrum of hydrolytic enzymes potentially useful in various biotechnological fields.

Keywords: cellulases; hemicellulases; hydrolytic enzymes; lipases; mushroom cultivation; mushroom substrate; proteases; secretome.

Figure 1

The mushroom substrate derived from the axenically cultivated P. eryngii and P. ostreatus used for the preparation of secretomes. Substrates were collected at three growth stages: 23, 34 and 50 days.

Figure 2

Analysis of caseinolytic (gel (A)) and gelatinolytic (gel (B)) activities by zymography. Zymograms (10% polyacrylamide/0.1% casein or gelatin) of the secretomes after 23, 34 and 50 days of incubation. For each sample, 1 μg of protein was applied onto the gels.

Figure 3

Analysis of caseinolytic (upper panel) and gelatinolytic (lower panel) activity by zymography. Zymograms (10% polyacrylamide/0.1% casein or gelatin) of secretomes after 34 days of incubation. Samples were analyzed in the absence (control) and in the presence of specific inhibitors: phenylmethylsulfonyl fluoride (PMSF), 1,10 phenanthroline (PA), iodoacetamide (IA) and pepstatin (PEP). Per: Pleurotus eryngii; Pos: Pleurotus ostreatus.

Figure 4

Enzymatic activity of the P. eryngii (A,C) and P. ostreatus (B,D) secretomes recovered from the substrate at three growth stages: after 23, 34 and 50 days of incubations. The enzymatic activities were evaluated spectrophotometrically as described in the Materials and Methods Section. Values of cellulolytic, amylolytic (A,B) and hemicellulolytic activity (C,D) are reported as the mean ± SD of two extracts analyzed in triplicate (n = 6). Statistically significant differences (one-way ANOVA followed by Tukey’s post hoc test, p < 0.05) are reported in Table 2.

Figure 5

Lipolityc activity of secretomes recovered at three growth stages: after 23, 34 and 50 days of incubations. Lipolytic activity was evaluated spectrophotometrically using p-nitrophenyl labelled substrates: p-nitrophenyl acetate (C2), p-nitrophenyl butyrate (C4), p-nitrophenyl decanoate (C10) and p-nitrophenyl palmitate (C16). Values are reported as the mean ± SD of two extracts analyzed in triplicate (n = 6). For each species, different letters indicate a statistically significant difference. Asterisks indicate a statistically significant difference between the two species at each time point (one-way ANOVA followed by Tukey’s post hoc test, p < 0.05).