Ri detector

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 Enzyme reactions were performed at 37 °C for various times, and portions of the reaction mixture were then withdrawn and mixed with the same volume of chilled acetonitrile (−20 °C) to terminate the reaction.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).

The composition of metabolites of the hydrolysis and fermentation experiments were measured by HPLC equipped with a RI detector (Jasco International Co., Tokyo, Japan). Sugars were analyzed using an ion-exchange Aminex HPX-87P column (Bio-Rad, Richmond, CA, USA), while ethanol, glycerol, and other metabolites were analyzed using Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) according to the method described by Shafiei et al. [11 (link)]. The methane and carbon dioxide contents of the biogas were measured using a gas chromatograph (Sp-3420A, Propack Q column, TCD detector, Beijing, Beifen Ruili Analytical instrument Co). Helium at flow rate of 25 mL/min was used as a carrier of gas. The column, injector, and detector temperature were controlled at 50, 90, and 140°C, respectively.
Except for the triplicate biogas production experiments, all other experiments were performed in duplicates, and the results are presented as averages of the obtained data.

The method reported by Mohammadi et al. [11 (link)] was employed to measure the concentrations of glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) in AIM. AIM (10 mg) was mixed with 0.3 mL of 72% (v/v) sulfuric acid for 90 min. Then, each sample was diluted by adding 8.4 mL of distilled water and heated at 121 °C for 1 h. The obtained AIM hydrolysates were reacted with NaNO₂ to produce 2,5-anhydromannose. Ammonium sulfamate was used to neutralize the excess NaNO₂. The obtained 2,5-anhydromannose and acetic acid were analyzed by High Performance Liquid Chromatography (HPLC, Jasco International Co., Tokyo, Japan) with an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA) equipped with a RI detector (Jasco International Co., Tokyo, Japan) using 0.6 mL/min eluent (5 mM sulfuric acid) at 60 °C. Finally, GlcN and GlcNAc yields were calculated using 2,5-anhydromannose and acetic acid concentrations [11 (link)].