Isolation, Purification, Identification And Physiological Function Of Polysaccharide Hydrolytic Enzyme From

Fungal polysaccharide hydrolase there are many reports, many of which are involved in the autolysis of mycelium or fruiting body. For autolysis have in-depth understanding of the original thought is the result of cell death under harsh environmental conditions, it has now generally considered sophisticated regulatory mechanisms have important physiological functions in the process. It also has important significance for the survival of the fungus, sporulation and dissemination. But the mechanism of fungal autolysis on further more detailed studies are needed. Basidiomycete fungi especially agaricales, after the mature fruit body has rapid autolysis phenomenon, so we study the polysaccharide hydrolysis enzyme hydrolysis of the cell wall structure of Coprinopsis cinerea, a model fungus, and thus try to explain the mechanism of fungal autolysis.Using a combined chromatography method, we simultaneously purified four protein fractions with 1,3-β-glucanase activity and a chitinase from extraction of pilei of Coprinopsis cinerea fruiting bodies. MALDI-TOF/TOF amino acid sequencing showed that these four fractions matched a putative extracellular β-glucosidase, a putative exo-1,3-β-glucanase, a putative glucan 1,3-β-glucosidase and a putative glycosyl hydrolase family 16 protein annotated in the C. cinerea genome, respectively; however, they were characterized as a β-glucosidase, a 1,3-β-glucosidase, an exo-1,3-β-glucanase and an endo-1,3-β-glucanase, respectively, by analysis of their substrate specificities and modes of action. Substrate specificity showed that extracellular β-glucosidase not single-minded 1,3-β-glucosidase, but hydrolysis the β-1,3/1,4/1,6 bond. TLC (thin-layer chromatography) analysis explored how these three 1,3-β-glucoside hydrolases synergistically acted on laminarin:the endo-1,3-β-glucanase hydrolysed internal glycosidic bonds of laminarin to generate 1,3-β-oligosaccharides of various lengths, the exo-1,3-β-glucanase cleaved the longer-chain laminarioligosaccharides into short-chain disaccharides, laminaribiose and gentiobiose, and the 1,3-β-glucosidase further hydrolysed laminaribiose to glucose.The remaining gentiobiose must be hydrolysed by other 1,6-β-glucosidases. The extracellular P-glucosidase may play a role in this. Therefore, the endo-1,3-β-glucanase, exo-1,3-β-glucanase and 1,3-β-glucosidase may act synergistically to completely degrade the 1,3-β-glucan backbone of the C. cinerea cell wall during fruiting body autolysis. These three 1,3-β-glucoside hydrolases share a similar optimum pH and optimum temperature,5.0 and 60 ℃, supporting the speculation that these enzymes work together under the same conditions to degrade 1,3-β-glucan in the C. cinerea cell wall during fruiting body autolysis.MALDI-TOF/TOF MS analysis characterized this purified chitinase as a putative class V chitinase, ChiB1. Substrate specificity and mode of action shows that ChiB1 is an exochitinase. ChiBl has an optimum temperature range of 35 to 40℃ and an optimum pH of 5.0. ChiB1 exhibited Km and Vmax values of 2.63 mg ml-1 and 2.31 μmol min-1 mg protein-1 for colloidal chitin, respectively. ChiBl incubation released a large amount of soluble β-glucan fractions from alkali-insoluble cell wall fractions of C. cinerea fruiting bodies. We presume that chitinase removal of the chitin chains could expose a large amount of free non-reducing termini of β-(1,3)/1,6-glucan or release β-(1,3)/1,6-glucan molecules, thereby promoting the degradation of cell walls in synergy with β-1,3-glucanases for pileus autolysis.By C. cinerea fruiting bodies glucanase and chitinase physiological functions of research, we believe glucanase and chitinase are involved in fruiting body autolysis. Exo-chitinase ChiBl hydrolysis chitin structure of cell wall, exposing more of glucan, with the glucanase hydrolysis glucan structure; The glucanase BGL1, EXG and ENG synergies degradation β-1,3-glucan component. Glucanase and chitinase enzymatic hydrolysis of cell walls together, so that the fruiting bodies of the fungus completely autolyzed. This study is a significance exploring of fungi autolysis mechanism, providing an important reference value. Research on fungi autolysis mechanism has great significance to antifungal agents, anti-fungal transgenic plants, etc.