Cellopentaose

Identification and quantification of the released soluble sugars by HPAEC-PAD were performed in a Dionex ICS 3000 (Sunnyvale, CA, USA) equipped with a pulsed amperometric detector. 200 µL of sample (or appropriate dilution of samples in distilled water) was mixed with 50 µL of 0.5 M NaOH and 25 µL were applied to a Dionex CarboPac PA1 column (4 × 250 mm) preceded by the corresponding guard column (4 × 50 mm) at 30°C. Sugars were eluted with the buffers 0.1 M NaOH and 0.5 M sodium acetate +0.1 M NaOH as the eluents A and B, respectively. For glucose quantification the following multi-step procedure was used: isocratic separation (5 min, 95% A + 5% B), column wash (2 min, 99% B) and subsequent column equilibration (2.5 min, 95% A + 5% B). For analysis of cellodextrins, the same A and B buffers were used but the multi-step procedure was as follows: isocratic separation (5 min, 95% A + 5% B), separation gradient (8 min, 10–37% B), column wash (2 min, 99% B) and subsequent column equilibration (2.5 min, 95% A + 5% B). The flow rate was kept at 1 mL/min in all cases. Injection of samples containing glucose, cellobiose, cellotriose, cellotetraose, cellopentaose (Sigma-Aldrich) and cellohexaose (Seikagaku, Tokyo, Japan) at known concentrations (ranging from 5 to 100 µM) was used to identify and quantify the released sugars.

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).

A high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Dionex ICS-5000 system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Dionex CarboPac PA100 column (250 mm × 2 mm; Thermo Fisher Scientific) was used for the quantitative analysis of NeoDP4, NeoDP6, and AgaDP5 [33 (link)]. At a flow rate of 0.25 mL/min, a gradient comprising the following mobile phases at 25 °C were used: (A) double-distilled water, (B) 0.1 M sodium hydroxide, (C) 0.1 M sodium hydroxide with 0.2 M sodium acetate, and (D) 0.25 M sodium hydroxide with 1 M sodium acetate. Before running samples, the column was washed with 100% D for 15 min, a linear gradient to 100% C for 10 min, and subsequently to 90% A and 10% B. Then, the column was equilibrated with 90% A and 10% B for 20 min. After sample injection, the following gradient was applied: 0 to 10 min, isocratic 90% A and 10% B; 10 to 20 min, linear to 100% B; 20 to 65 min, linear to 50% B and 50% C; 65 to 80 min, linear to 100% C; and 80 to 90 min, linear to 100% D. Cellotetraose (Sigma-Aldrich), cellopentaose (Sigma-Aldrich), and cellohexaose (Sigma-Aldrich) were used as standards for the quantitative analysis of NeoDP4, AgaDP5, and NeoDP6, respectively (Supplementary Materials Figure S3).