*2.4. Extraction and Purification of Polysaccharides*

After 72 h of fed-batch, the culture was centrifuged (10,000× *g*, 30 min) and the supernatant replaced by distilled water. The cells were then autoclaved at 105 ◦C for 10 min to be disrupted. The supernatant—designated as the "total extract"—was collected after centrifugation (10,000× *g*, 30 min). IR120 H+ Amberlite resin was used to lower its pH to 3.5 and precipitate proteins. This precipitate was then removed from the solution by centrifugation (10,000× *g*, 30 min). The pH was adjusted to 7 using NaOH, and 3 volumes of ice-cold 95% ethanol were added to the total extract to precipitate the polymer. Finally, the precipitated polysaccharide was isolated using centrifugation (10,000× *g*, 30 min, 4 ◦C). The pellet was then dissolved in distilled water. Pure chondroitin or chondbiuronan was obtained by anion-exchange chromatography on a MonoQ (HR16/10, GE Healthcare, Uppsala, Sweden) column run on an NGC medium-pressure chromatography system (Bio-Rad, Hercules, CA, USA). The column was washed with 20 mL of 10 mM Tris HCl pH 7.6 buffer, then a linear gradient of 50 mL of 0 to 1 M NaCl in the same buffer was applied. The flow

rate was 2 mL·min−<sup>1</sup> and fractions of 4 mL were collected. The presence of GAGs was monitored by UV detection at 210 nm and confirmed using a colorimetric assay [16]. The presence of contaminants was detected at 260 and 280 nm. Polysaccharide containing fractions were pooled and desalted on HighPrep 26/10 (GE Healthcare, Uppsala, Sweden) desalting column in water. The sample was finally lyophilized and stored at −20 ◦C for downstream analysis.
