A nondegradative route for the removal of endotoxin from exopolysaccharides

The potentiality of the Triton X-114 phase separation technique for the removal of lipopolysaccharide (LPS, endotoxin) from Klebsiella sp. I-714 exopolysaccharide (EPS) has been investigated. Classical purification and chemical detoxification methods were evaluated for their effectiveness in removing residual LPS, while preserving structural and functional integrity of EPS. Ultracentrifugation, Detoxi-Gel, and ion-exchange chromatography did not remove endotoxin, except gel filtration chromatography performed at 60 degrees C in sodium deoxycholate buffer. In this case, the bioactivity of the purified EPS fraction was significantly lowered, as was seen after alkaline hydrolysis treatment. Moreover, the acetic acid detoxification procedure hydrolyzed EPS. As an alternative, phase partitioning of EPS in Triton X-114 at low temperature provided a fast, mild, and efficient method for the removal of LPS as shown by a 100-fold reduction in Limulus amebocyte lysate (LAL) activity and only a 2-fold reduction in bioactivity. Gel filtration chromatography performed at 4 degrees C with Triton X-114 buffer and phase partitioning with the more hydrophilic Triton X-100 nonionic detergent at 75 degrees C led to a similar decrease in LAL activity. This novel application of Triton X-114 partitioning is a nondegradative alternative to the chemical detoxification of gram-negative bacterial EPS for vaccine production. Purification of endotoxin-contaminated polysaccharides prior to screening for biological activity should also benefit from this technique. The extraction scheme using Triton X-114 can be easily used in large-scale purification processes.