*2.5. Fast Protein Liquid Chromatography and Electrospray Ionization Mass Spectrometry Analysis of the Degradation Products of AlgA*

To investigate the action patterns of AlgA, degradation of alginate by this enzyme was performed. The degradation products at different time intervals were analyzed through gel chromatography. Fast protein liquid chromatography (FPLC) analysis indicated that AOS with different degrees of polymerization (DP) gradually accumulated (Figure 6). Therefore, AlgA is an endo-type alginate lyase.

**Figure 6.** Patterns of the polysaccharide degradation products of AlgA. Enzymatic degradation products collected at 0.5, 2, 4, 6, and 10 h were subjected to gel filtration with a Superdex peptide 10/300 GL column. The absorbances of the products were monitored at 235 nm.

In addition, the alginate was completely digested with an excess of AlgA at 40 ◦C for 24 h. The products were separated through gel chromatography. The elution profiles (Figure 7) of the degradation products presented three major fractions (peaks 1, 2, and 3).

**Figure 7.** Final products of alginate, polyM, and polyG after degradation by AlgA. Oligosaccharide products were gel-filtered through a Superdex peptide 10/300 GL column and monitored at a wavelength of 235 nm.

To identify the final oligosaccharide products of AlgA degradation and to determine their DP, three major fractions were subjected to electrospray ionization mass spectrometry (ESI-MS). The molecular masses of oligosaccharides in peaks 1, 2, and 3 were determined to be 351.06, 527.09, and 703.12, respectively (Figure 8). These results indicated that the main degradation products are di-, tri- and tetra-saccharides. The relative contents of di-, tri- and tetra-saccharides in alginate were 61.19%, 15.59%, and 23.22%, respectively, whereas those of di-, tri- and tetra-saccharides in the polyM substrate were 58.30%, 34.26%, and 7.43%, respectively. Meanwhile, AlgA has limited activity toward polyG. Therefore, AlgA may be used in the production of mannuronic oligosaccharides from polyM blocks and the preparation of polyG blocks from sodium alginate via the degradation of polyM blocks. The products of mannuronic oligosaccharides and polyG blocks possess special biological activity and have potential applications in many fields. For example, mannuronate oligosaccharides can promote the secretion of multiple cytokines [7], and polyG demonstrates higher macrophage-stimulation activity than polyM [41].

**Figure 8.** Electrospray ionization mass spectrometry (ESI-MS) analysis of the final oligosaccharide products. (**A**) Fraction peak 1 separated through fast protein liquid chromatography (FPLC), (**B**) fraction peak 2 separated through FPLC, and (**C**) fraction peak 3 separated through FPLC.
