Sugar d column

To investigate cleavage patterns from the hydrolysis products of the purified enzymes, 5 mM maltooligosaccharide substrates (Mal3, Mal4, and Mal5) were dissolved in 50 mM sodium acetate buffer (rEf-AmyI: pH 5.5; rEf-AmyII: pH 5.0), and aliquots of the enzyme solution were added to 200 μL of each substrate solution. Enzyme reactions were performed at 37 °C for various times, and portions of the reaction mixture were then withdrawn and mixed with the same volume of chilled acetonitrile (−20 °C) to terminate the reaction. The resulting solutions were then applied to a Sugar-D column (4.6 × 250 mm; Nakalai Tesque, INC.) and were eluted with 70% acetonitrile at a flow rate of 1.0 mL/min. Substrates and products were monitored with an RI detector (Jasco Co., Tokyo, Japan).

Glucose and cellobiose concentrations in culture supernatants were measured by HPLC (Prominence; Shimadzu, Kyoto, Japan) equipped with an electrochemical detector (Coulochem III; thermo scientific, MA, USA). Supernatants were separated using a Sugar-D column (250 mm long, 4.6 mm inner diameter; Nacalai tesque) and the mobile phase was 80 % acetonitrile at a flow rate of 500 μL/min. The sample injection volume was 1 μL.

The method for analyzing carbohydrate deposition was performed as described by Ruan et al. [68 (link)]. Approximately 0.2 g of fiber was ground in liquid nitrogen. The samples were extracted with 4 mL of preheated 80% ethanol for 5 min at 80°C. After cooling, the samples were centrifuged at 12,000 × g for 10 min. The supernatants were collected, while the pellets were resuspended in 2 mL of 50% ethanol and centrifuged again as described above. The resulting pellet was re-extracted with 2 mL of water and re-centrifuged. The total 8 mL of supernatant collected was mixed with an equal volume of chloroform and shaken vigorously. The aqueous phase was collected, dried in a vacuum and redissolved in 1 mL of water. The sucrose, glucose and fructose contents were measured and analyzed using an anion-exchange HPLC system (Agilent 1100 series; Agilent technologies, Inc., Santa Clara, CA, USA), which consisted of a G1311A pump and a G1362A refraction index detector. Sugar compounds were separated on a Sugar-D column (4.6 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan), using acetonitrile/water (75: 25, v/v) as the mobile phase at the flow rate of 1.0 mL/min. The injection volume was 40 μL. Quantification of each sugar was accomplished by comparing the peak areas of the samples with those of standard solutions.