Figure 7.

The production of trisaccharide from alginate substrates by AlyM2. (A), the end degradation products of AlyM2 towards PM, PG, PMG, and sodium alginate. A 200 μL mixture containing 80 μg/mL AlyM2 and 2 mg/mL substrate was incubated at 30 °C for 12 h in the buffer containing 50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl. The degradation products were analyzed by gel filtration chromatography and monitored at 210 nm using a UV detector. Saturated mannuronate oligosaccharides from DP1 to DP6 were taken as the standards. (B), the effect of degradation time on the products. The reactions were carried out under the same conditions as described in Figure 7A for 0, 3, 6, or 12 h. The degradation products were analyzed by TLC. Lane M, saturated oligosaccharides standards including monomer, dimer, trimer, tetramer, and pentamer. (C), determination of the production of trisaccharides from sodium alginate degradation by AlyM2. The degradation reaction was performed in a 500 mL reaction mixture containing 1 g sodium alginate and 40 mg AlyM2 in the buffer containing 50 mM Tris-HCl (pH 8.0) and 0.5 M NaCl at 30 °C for 6 h. The degradation products were separated by gel filtration and monitored using a refractive index detector. A standard curve of trisaccharide concentration versus peak area was drawn by using commercial saturated mannuronate trisaccharide at the concentrations of 0–6 mg/mL as standards. The trisaccharide amount released from sodium alginate by AlyM2 was determined based on the standard curve.