Determination of Alginate Oligosaccharides

The reactions were conducted at 37 °C for 24 h with a 100 μl solution of 50 mM Tris buffer pH 7.5, 0.4% (w/v) alginate (500 cps), and 0.01 mg/ml enzyme. After incubation, two volumes of ethanol were added to the solution. Then, the mixture was centrifuged at 15,000 g for 10 min and the supernatant was transferred to a tube. The concentration of the unsaturated alginate oligosaccharides was determined using the extinction coefficient of 6150 1/M cm^{44 (link)}. The conditions of the samples and mass analyser (Orbitrap Elite hybrid mass spectrometer; Thermo Scientific, Waltham, MA) are described in the Supplementary Information.