Silica gel 60 aluminum plate

The reactivity of the recombinant enzymes was analyzed using the following substrates: polysaccharides [gum arabic AGP, larch AGP, and α-d-Galp-(1→3)-l-Ara-free gum arabic AGP] and gum arabic AGP-related oligosaccharides (S5-GA, S5-A, S5, S4-A, S3-GA, S3-A, and S3). In the case of the polysaccharides, each substrate (final concentration, 1.0%) was incubated with 2.2 nM BlArafA, 2.2 nM BlArafB, or 1.4 nM BlArafE in 40 μL of 50 mM sodium acetate buffer (pH 6.0) for 16 h. For the gum arabic AGP-related oligosaccharides, each substrate (final concentration, 0.01 mM) was incubated with 0.88 nM BlArafA, 0.90 nM BlArafB, or 0.57 nM BlArafE in 100 μL of 50 mM sodium acetate buffer (pH 6.0) for 16 h. The reaction products were analyzed by TLC or HPAEC-PAD. In the TLC analysis, the reaction products were spotted on a silica gel 60 aluminum plate (Merck, Darmstadt, Germany) with a 7:1:2 (vol/vol/vol) 1-propanol/ethanol/water solvent mixture, and the separated sugars were visualized by spraying an orcinol-sulfate reagent on the plates. In HPAEC-PAD analysis, a CarboPac PA-1 column (φ, 4 by 250 mm; Dionex Corp., Sunnyvale, CA, USA) was used with the flow rate of 1.0 mL/min using the following gradient: 0 to 5 min, 100% eluent A (0.1 M NaOH), 5 to 30 min, 0% to 100% eluent B (0.5 M sodium acetate in 0.1 M NaOH), and 30 to 35 min, 100% eluent B.

A 10-μl reaction mixture for thin-layer chromatography (TLC) analysis of dansylated substrates contained 50 mM sodium acetate buffer (pH 4.5), 50 μM substrate, and 1 μL of the expressed recombinant enzyme. After 12 h at 30°C, the reaction mixtures were spotted on a Silica Gel 60 aluminum plate (Merck, Darmstadt, Germany) and developed with a 3:1:1 solvent (v/v/v) of 1-butanol/acetic acid/water and finally visualized with UV light. For orcinol-stained TLC analysis, the 100-μL reaction mixture contained 50 mM sodium acetate buffer (pH 4.5), 35 μM substrate, and 2 μL of an expressed enzyme, and the reaction was conducted at 30°C for 12 h. Spotted silica gels were developed with a 2:1:1 solvent (v/v/v) of ethyl acetate/acetic acid/water. Sugars were visualized by spraying an orcinol—sulfate reagent onto the silica gel plate [31 (link)]. For high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis, oligosaccharides in the 100-μL reaction mixture were analyzed with a CarboPac PA-1 column. The column was eluted at a flow rate of 1.0 mL/min with the following gradient: 0–5 min, 100% eluent A (0.1 M NaOH); 5–30 min, 0–100% eluent B (0.5 M sodium acetate and 0.1 M NaOH); and 30–35 min, 100% eluent B.