Purification, characterization and synergism in autolysis of a group of 1,3-β-glucan hydrolases from the pilei of Coprinopsis cinerea fruiting bodies

Abstract

Using a combined chromatography method, we simultaneously purified three protein fractions (II-2, II-3 and II-4) with 1,3-b-glucanase activity from extraction of pilei of Coprinopsis cinerea fruiting bodies. MALDI-TOF/TOF amino acid sequencing showed that these three fractions matched a putative exo-1,3-b-glucanase, a putative glucan 1,3-b-glucosidase and a putative glycosyl hydrolase family 16 protein annotated in the C. cinerea genome, respectively; however, they were characterized as a 1,3-b-glucosidase, an exo-1,3-b-glucanase and an endo-1, 3-b-glucanase, respectively, by analysis of their substrate specificities and modes of action. This study explored how these three 1,3-b-glucoside hydrolases synergistically acted on laminarin: the endo-1,3-b-glucanase hydrolysed internal glycosidic bonds of laminarin to generate 1,3-b-oligosaccharides of various lengths, the exo-1,3-b-glucanase cleaved the longer-chain laminarioligosaccharides into short-chain disaccharides, laminaribiose and gentiobiose, and the 1,3-b-glucosidase further hydrolysed laminaribiose to glucose. The remaining gentiobiose must be hydrolysed by other 1,6-b-glucosidases. Therefore, the endo-1,3-b-glucanase, exo-1,3-b-glucanase and 1,3-b-glucosidase may act synergistically to completely degrade the 1,3-b-glucan backbone of the C. cinerea cell wall during fruiting body autolysis. These three 1,3-b-glucoside hydrolases share a similar optimum pH and optimum temperature, supporting the speculation that these enzymes work together under the same conditions to degrade 1,3-b-glucan in the C. cinerea cell wall during fruiting body autolysis.

Transcriptomic atlas of mushroom development highlights an independent origin of complex multicellularity

“…A large suite of β-glucanases, chitinases, laccases, endo-β-1,4-mannanases, α-1,3-mannosidases were developmentally regulated, many of which are also expanded in Agaricomycetes (Table 1, Supplementary Fig 7). The expression of glucan-, chitin-and mannose-active enzymes is consistent with active FCW remodeling during fruiting body formation and recent reports of similar genes upregulated in the fruiting bodies of Lentinula 12,13,26 , Flammulina 27 and Coprinopsis 28 . Kre9/Knh1 homologs are developmentally regulated in all species and are overrepresented in mushroom-forming fungi (P = 1.45×10 −5 , FET).…”

Transcriptome data reveal conserved patterns of fruiting body development and response to heat stress in the mushroom-forming fungus Flammulina filiformis

“…Certain CAZymes were shown or assumed to participate in cell wall remodeling during fungal tissue differentiation [13,49,[75][76][77]. We annotated 407 CAZymes genes in F. filiformis, 137 of them were differentially expressed (Fig 2E; S7 Table), which is consistent with previous studies in other mushroom-forming fungi [13,20,49].…”

Transcriptomic atlas of mushroom development reveals conserved genes behind complex multicellularity in fungi

“…S12). The expression of glucan-, chitin-, and mannose-active enzymes is consistent with active FCW remodeling during fruiting body formation and recent reports of similar genes upregulated in the fruiting bodies of Lentinula (16,17,29), Flammulina (30), and Coprinopsis (31). Kre9/Knh1 homologs are developmentally regulated in all species and are overrepresented in mushroomforming fungi (P = 1.45 ×10 −5 , FET; Dataset S3).…”