FT IR The extracellular protein and exopolysaccharide precipitates were

Activated sludge was washed 3 times with methanol to remove any lipid material before EPS extraction. Then A-740003 EPS extracted as described above. The protein in EPS was removed by TCA precipitation. The TCA (80%, w/v) was added to the EPS solution. The final concentration of TCA was adjusted to 4% for weakening the effect of TCA on polysaccharide. The mixture was incubated overnight at 4 °C after mixed thoroughly. Then the mixture was centrifuged at 10,000g for 30 min at 4 °C, and the precipitate was rejected. The supernatant was freeze-dried and redissolved to 1 mL using milliQ water. Next the petiole polysaccharide was precipitated using ethanol. 3 mL of ethanol (95% v/v) was added to the solution, and after 24 h at 4 °C, the precipitate was recovered by centrifugation (10,000g, 4 °C, 30 min). The precipitate was redissolved in milliQ water and centrifuged at 10,000g for 30 min at 4 °C to remove insoluble substances. The supernatant was dialysed (molecular weight cutoff 3500) against milliQ water with multiple changes for 72 h at 4 °C. The retentate, namely the polysaccharide, was freeze-dried for further experiments.