Laminaritriose

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).

For detection of enzymatic reaction products, high-performance anion exchange column chromatography (HPAEC) with a pulsed amperometoric detector (PAD) equipped with a CarboPac PA10 guard column (4 × 50 mm) and a CarboPac PA10 analytical column (4 × 250 mm; Dionex Co.) was used. Enzymatic reaction was performed by incubation with equivalent volume of rAaBGL1 (20.0 nM) and each substrate in 20 mM sodium acetate buffer (pH 5.0) at 37°C. Reaction mixture was sampled at appropriate time, and added into equal volume of 0.2 M NaOH. Resultant mixtures were subjected to HPAEC-PAD using mobile phase of 100 mM NaOH with 10 mM sodium acetate. Glucose, cellobiose (Wako Pure Chemical Industries, Ltd.), cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, laminaritetraose, laminaripentase (Megazyme), gentiobiose, sophorose (SIGMA-ALDRICH, Co.) were used as standards.

3′-SialylLactose (3′SL) was obtained from Kyowa Hakko Kogyo Co, Ltd, Tokyo, Japan. 6′-SialylLactose (6′SL) and 2′-fucosylLactose (2′FL) were obtained from Glycom A/S, Lyngby, Denmark. Lactose, sodium cyanoborohydride, 2-aminobenzamide, laminaritriose and dimethylsulphoxide were obtained from Sigma-Aldrich, Buchs, Switzerland. Water was deionised of 18.2 MΩ quality produced by a Milli-Q Plus system (Millipore, Billerica, MA). All other chemicals were obtained from Merck, Darmstadt, Germany.