Digalacturonic acid

The EDA conjugated silk solutions (SF(S)-EDA) of 157 kDa MW SF were carbodiimide coupled with the carboxylic acids of digalacturonic acid (Sigma-Aldrich, St. Louis, MO) in the presence of EDC and NHS (Sigma-Aldrich, St. Louis, MO). Briefly, 2 wt% of the SF(S)-EDA solution was dissolved in 0.1M MES buffer at pH 6. digalacturonic acid (10X) was weighed and pre-dissolved in Ultrapure™ distilled water and added to the SF(S)-EDA solution. The pH was adjusted to 6 by dropwise addition of freshly prepared 1M NaOH solution. EDC (10X) and NHS (10X) were added to the reaction mixture at pH 6. The final MES buffer concentration of the reaction mixture was adjusted to 0.05M by addition of Ultrapure™ distilled water. The reaction was stirred at RT for 18h. After the reaction got over, the reaction solution was filtered in a sterile cell strainer with 40 μm mesh size (Thermo Scientific, Rockford, IL) and dialyzed against DI water for 72h with six water changes (1h, 2h, 4h, 24h, 48h, and 72h). The dialysis was performed with a dialysis tube (3,500 MWCO, Thermo Scientific, Rockford, IL). After dialysis, the solutions were frozen at −80°C overnight followed by lyophilizing for 72h. The lyophilized powders were stored at 4°C until further use.