*3.8. Analysis of Reaction Products and Hydrolytic Pattern*

Low viscosity sodium alginate was used for analysis the reaction product of Aly08. In order to examine the hydrolysis pattern of Aly08, the products from different incubation times (0, 5, 15, 30, 60, and 120 min) were monitored at A235 using gel filtration chromatography with a SuperdexTM peptide 10/300 GL column with 0.2 M NH4HCO3 (flow rate: 0.6 mL/min) as an eluent on HPLC platform (LC-20A, Shimadzu, Japan) [46]. The mode of action was also analyzed using an Ostwald viscometer (No.1; Shibata Scientific Technology) with high viscosity sodium alginate as substrate. The equal component products (0.5 mL), which were degraded by Aly08 at 1, 5, 10, 15, and 30 min, were removed to characterize the viscosity and degradation products.

The hydrolytic degradation products were monitored using TLC method, wherein a reaction system containing 0.3% of high viscosity sodium alginate and Aly08 was constructed and the samples selected at different times (1, 2, 5, 10, 15, 30, 60, 90, and 120 min). The reaction products were analyzed using a TLC plate (TLC silica gel 60 F254, Merck KGaA, 64271 Darmstadt, Germany) with butanol/acetic-acid/water (2:1:1, by vol.) and color-developed with sulfuric acid/ethanol reagent (1:4, by vol.) after heating the TLC plate at 80 ◦C for 30 min. ESI-MS system (Thermo Fisher ScientificTM Q ExactiveTM Hybrid Quadrupole-OrbitrapTM, Waltham, MA, USA) was employed to further investigate the composition and degree of polymerization (DP) of the end products.
