The AlyM was mixed with 2% alginate and incubated at 45°C for approximately 6 h to obtain the hydrolysis products. The hydrolysis products were separated and purified by Bio-Gel P4 Polyacrylamide Gel (Bio-Rad Laboratories, Inc. US), and each of the purified components was analyzed by 1H-NMR. The hydrolysis products were freeze-dried with a vacuum freezer dryer and analyzed by infrared spectroscopy (IR), negative ion ESI-MS, and nuclear magnetic resonance (NMR) spectroscopy (Jouanneau et al., 2010 (link); Jagtap et al., 2014 (link); Swift et al., 2014 (link)).
To determine the monosaccharide composition of hydrolysis products, the hydrolysis products were successively precipitated by ethanol with different volumes. The precipitates were freeze-dried with a vacuum freezer dryer and applied for the decomposition by 2 M TFA at 110°C for 4 h. The samples were detected by HPLC after PMP (1-phenyl-3-methyl-5-pyrazolone) pre-column derivatization (Wang et al., 2018 (link)). The analysis of HPLC using a XDB-C18 column (Agilent Technologies Inc., Santa Clara, CA, USA) under the following conditions: the mobile phase was comprised of 50 mM KH2PO4 (pH 6.9), the column temperature was 25°C, the flow velocity was 1 mL/min, and detection was achieved by the UV detector at 245 nm. The molecular weight (Mw) of the precipitates was detected by Gel Permeation Chromatography (GPC) using the column of PL aquagel-OH 30 (Agilent Technologies Inc., USA). The 200 mM NaNO3 with10 mM NaH2PO4 was used as mobile phase at a flow rate of 0.6 ml/min. The precipitates were dissolved in mobile phase and filtered through 0.22 μm filter. The inject volume was 20 μL.
To determine the monosaccharide composition of hydrolysis products, the hydrolysis products were successively precipitated by ethanol with different volumes. The precipitates were freeze-dried with a vacuum freezer dryer and applied for the decomposition by 2 M TFA at 110°C for 4 h. The samples were detected by HPLC after PMP (1-phenyl-3-methyl-5-pyrazolone) pre-column derivatization (Wang et al., 2018 (link)). The analysis of HPLC using a XDB-C18 column (Agilent Technologies Inc., Santa Clara, CA, USA) under the following conditions: the mobile phase was comprised of 50 mM KH2PO4 (pH 6.9), the column temperature was 25°C, the flow velocity was 1 mL/min, and detection was achieved by the UV detector at 245 nm. The molecular weight (Mw) of the precipitates was detected by Gel Permeation Chromatography (GPC) using the column of PL aquagel-OH 30 (Agilent Technologies Inc., USA). The 200 mM NaNO3 with10 mM NaH2PO4 was used as mobile phase at a flow rate of 0.6 ml/min. The precipitates were dissolved in mobile phase and filtered through 0.22 μm filter. The inject volume was 20 μL.
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