Laminaripentaose

The cell wall contents, chitin, β-1,3-glucan and mannoproteins, of M. acridum were determined according to the previous study [36 (link)]. Briefly, hyphae of the WT, ΔMaAreB and CP strains were respectively inoculated in a 40 mL 1/4SDY liquid medium (1/4 SDAY without agar) and cultured at 28 °C with 220 rpm for 3 days. Then, the hyphae were harvested and washed with 2% SDS four times, followed by lyophilizing for 24 h. Five milligrams (dry weight) hyphae were consumed for each determination. For determination of the chitin content, hydrochloric acid was used to hydrolyze fungal cell wall, and glucosamine was used as the standard. For determination of β-1,3-glucan content, sodium hydroxide was used to treat the fungal cell wall to obtain the alkali-insoluble fraction, followed by degrading with zymolyase 100-T, and laminaripentaose (Sigma, St. Louis, MO, USA) was used as the standard. To detect the content of mannoprotein, sodium hydroxide was used to extract the mannoprotein in each strain. The standard curve of bovine serum albumin was made via Folin-phenol reagent method.

Yeast strain Clavispora NRRL Y-50464 obtained from the Agricultural Research Service Patent Culture Collections (Peoria, IL, USA) was used in this study. Cell cultures were maintained and precultured using yeastpeptone (YP) medium containing 10 g yeast extract, 20 g peptone, and 50 g glucose in one liter distilled water. Escherichia coli TOP10 and Pichia expression and transformation kits from Invitrogen (Carlsbad, CA, USA) were applied for gene cloning and selection procedures. An YP medium amended with 5% cellobiose was used for gene expression assays. All oligosaccharides were purchased from Sigma-Aldrich (St. Louis, MO, USA) including cellobiose, cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, laminaritetraose, laminaripentaose, laminarin, α-lactose, lichenan, salicin, and gentiobiose, and metal ions and chemicals KCl, CaCl₂, ZnCl₂, MgCl₂, CuCl₂, CoCl₂, HgCl₂, FeCl₂, FeCl₃, BaCl₂, PbCl₂, LiCl, NiCl₂, MnCl₂, SDS, triton X-100, 2-furaldehyde (furfural), and 5-(hydroxymethyl)-2-furaldehyde (HMF).