Oligosaccharide generation by enzymatic hydrolysis.

Pn3P powder (20 mg) was incubated with 0.5 mg of Pn3Pase (31) at 37°C for 48 h. The reaction was stopped by heating at 100°C for 5 min and loaded onto a 120 ml Superdex 30 column (GE). Products were separated in phosphate-buffered saline (PBS) with a flow rate of 1 ml/min and monitored by refractive index and absorbance at 205 nm. Fractions (0.5 ml) were collected, and oligosaccharide peaks were combined and desalted into water on a 30-ml packed fine P2 column (Bio-Rad). Desalted oligosaccharides were lyophilized, and hexasaccharide and tetrasaccharide identities were confirmed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (31).