Superdex peptide 10 300 gl gel filtration column

The end products of degradation by recombinant VxAly7B-FL and recombinant VxAly7B-CM were prepared by exhaustive degradation with excess activity (10 U of enzyme with 3 mg of alginate). Mixtures were boiled for 10 min to terminate the reactions, filtered through 0.22-μm filters, and centrifuged at 12,000 r/min for 10 min. Supernatants were analyzed by a Superdex peptide 10/300 GL gel filtration column (GE Healthcare, Madison, WI, United States) equilibrated with 0.2 M NH₄HCO₃ and detected at 235 nm by FPLC. The flow rate was 0.2 mL/min, and fractions containing uronic acids were pooled, lyophilized, and resuspended in 1 mL of acetonitrile-water (1:1, v/v) mixture. Oligosaccharide samples were analyzed by negative ion ESI-MS with a range from 0 to 1,500 m/z. The scope with no significant product peaks is not shown.

To study the substrate specificity, alginate, polyM, and polyG (0.3% (w/v) in 20 mM PB plus 300 mM NaCl, pH 7.3) were used as substrates in the standard enzymatic assay described above. One mL (10 U) of purified enzyme was added to 9 mL of high-viscosity alginate substrate solution and incubated at 30 °C for 0, 1, 2, 5, 10, 20, 30, or 60 min. After boiling for 10 min to halt the reaction, the viscosity and UV absorbance at 235 nm of each reaction mixture were measured to determine the mode of action of recombinant proteins.
The reaction mixture containing 0.1 mL (10 U) purified enzymes and 0.4 mL substrate solution was incubated at 30 °C overnight. The end products were analyzed using a Superdex peptide 10/300 GL gel filtration column (GE Healthcare, Madison, WI, USA) equilibrated with 0.2 M NH₄HCO₃ and detected at 235 nm by fast protein liquid chromatography (FPLC). The end products were analyzed by negative ion electrospray ionization mass spectrometry (ESI-MS) from 0 to 1500 m/z. The scope with no significant product peaks is not shown.

A mixture (200 μL) with 0.043 μg of VaAly2 and 0.6 mg sodium alginate was incubated in the buffer [50 mM Tris–HCl, 300 mM NaCl (pH 9.0)] at 30°C for 24 h. After termination by boiling for 10 min and centrifugation at 10,000 × g for 10 min, 100 μL unpurified oligosaccharide products degraded by VaAly2 were collected at different reaction time points.
To analyze the degradation pattern during the whole reaction, the oligosaccharide solutions were assayed by a gel filtration column. First, the collected product mixture was passed through 0.22-mm filters and centrifuged at 10,000 × g for 15 min. Then, the final products were tested with a Superdex peptide 10/300 GL gel filtration column (GE Healthcare, Madison, WI, USA) equilibrated with 0.2 M NH₄HCO₃ and were further subjected to fast protein liquid chromatography (FPLC) analysis at 235 nm. Meanwhile, the oligosaccharide mixture was also examined by electrospray ionization mass spectrometry (ESI-MS) set in the negative ion mode with a scanning mass from 0 to 2,000 m/z.