galactomannan and lichenin

Heterologous endo-xanthanase (EX) from the thermophilic planktomycete Thermogutta terrifontis strain was obtained using Penicillium verruculosum 537 (ΔniaD) expression system with the cellobiohydrolase 1 gene promoter. Homogeneous EX with a molecular weight of 23.7 kDa (pI 6.5) was isolated using liquid chromatography methods. This xanthan degrading enzyme also possesses the enzymatic activity towards CM-cellulose, β-glucan, curdlan, lichenan, laminarin, galactomannan, xyloglucan but not towards p-nitrophenyl derivatives of β-D-glucose, mannose and cellobiose. The temperature and pH optima of EX were 55°C and 4.0, respectively; the enzyme exhibited 90% of its maximum activity in the temperature range 50-60°C and pH 3-5.

Topics: Bacterial Proteins; beta-Glucans; Cellulose; Cloning, Molecular; Galactose; Glucans; Glycoside Hydrolases; Hot Temperature; Hydrogen-Ion Concentration; Mannans; Planctomycetales; Planctomycetes; Substrate Specificity; Talaromyces; Xylans

The formation of a glucan/chitin/glycoprotein cell wall matrix is vital for fungal survival, growth, and morphogenesis. The cell wall proteins are important cell wall components and function in adhesion, signal transduction, and as cell wall structural elements. In this report we demonstrate that Neurospora crassa GH72 glucan transferases function to crosslink cell wall glycoproteins into the cell wall. With an in vitro assay, we show that the glucan transferases are able to attach lichenin, a cell wall glucan with a repeating β-1,4-glucose-β-1,4-glucose-β-1,3-glucose structure, to cell wall glycoproteins. We propose that the pathway for attachment of lichenin to the glycoprotein has four steps. First, N-linked oligosaccharides present on the glycoproteins are modified by the addition of a galactomannan. As part of our report we have characterized the structure of the galactomannan, which consists of an α-1,6-mannose backbone with galactofuranose side chains. In the second step, the galactomannan is processed by members of the GH76 α-1,6-mannanases. In the third step, the glucan transferases cleave the lichenin and create substrate-enzyme intermediates. In the final step, the transferases transfer the lichenin to the processed galactomannan. We demonstrate that the N. crassa glucan transferases have demonstrate specificity for the processed galactomannan and for lichenin. The energy from the cleaved glycosidic bond in lichenin is retained in the substrate-enzyme intermediate and used to create a new glycosidic bond between the lichenin and the processed galactomannan. The pathway effectively crosslinks glycoproteins into the fungal cell wall.

Topics: Cell Wall; Chitin; Galactose; Glucans; Glycoproteins; Mannans; Neurospora crassa