3.8. Degradation Products Analysis of Recombinant AlyS02

In order to clarify the mode of action of AlyS02 against sodium alginate, 0.5% (w/v) sodium alginate was used as the substrate, and completely degraded with excess enzyme (5 U per mg of substrate) at 30 °C. The reaction mixture was continuously stirred and the degradation products were detected at regular time intervals. Then, the mixture solution was boiled for 10 min after incubation, and centrifuged at 12,000× g for 15 min to remove the unsolved materials. For removing proteins and other undegraded macromolecules, the supernatant was loaded on a centrifugal filter 3 K device (Millipore, Burlington, MA, USA). Then the filtrate was collected for detection.

First, the products were analyzed with a solvent system (1-butanol/acetic acid/water 2:1:1, v/v/v) using a TLC plate (TLC silica gel 60 F254, Merck KGaA, Darmstadt, Germany). After spraying with sulfuric acid/ethanol reagent (1:4, v/v), the TLC plate was heated at 80 °C for 30 min to visualize the spots. The mixture of disaccharide and trisaccharide was applied as a marker, and the total uronic acid concentration was 0.2% (w/v).

Next, a Superdex ^TM peptide 10/300 gel filtration column (GE Healthcare, Boston, MA, USA) was used for gel filtration chromatography analysis, and the complete degradation products were monitored at a wavelength of 235 nm by the ultraviolet detection system. The mobile phase was 0.2 M NH₄HCO₃ at a flow rate of 0.6 mL/min on an HPLC platform (LC-20A, Shimadzu, Japan). To further determine the composition of the end product, the filtrate was collected and mixed with methanol (1:1, v/v) and then quantitatively injected into an ESI-MS instrument (Bruker Esquire HCT, Billerica, MA, USA). The DPs of the enzymolysis products were profiled in negative-ion mode under the following conditions: calibration dynamics, 2; cone voltage, 20.00 V; capillary voltage, 4.00 kV; desolvation temperature, 350 °C; source temperature, 150 °C; desolvation gas flow, 500 L/h; and cone gas flow rate, 50 L/h; scan range, 100–1500 m/z.