A NONDEGRADATIVE ROUTE FOR THE REMOVAL OF ENDOTOXIN FROM EXOPOLYSACCHARIDES

The potentiality of the Triton X-114 phase separation technique for th e removal of lipopolysaccharide (LPS, endotoxin) from Klebsiella sp. I -714 exopolysaccharide (EPS) has been investigated, Classical purifica tion and chemical detoxification methods were evaluated for their effe ctiveness in removing residual LPS, while preserving structural and fu nctional integrity of EPS, Ultracentrifugation, Detoxi-Gel, and ion-ex change 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 signi ficantly lowered, as was seen after alkaline hydrolysis treatment, Mor eover, the acetic acid detoxification procedure hydrolyzed EPS. As an alternative, phase partitioning of EPS in Triton X-114 at low temperat ure provided a fast, mild, and efficient method for the removal of LPS as shown by a 100-fold reduction in Limulus amebocyte lysate (LAL) ac tivity and only a 2-fold reduction in bioactivity. Gel filtration chro matography performed at 4 degrees C with Triton X-114 buffer and phase partitioning with the more hydrophilic Triton X-100 nonionic detergen t at 75 degrees C led to a similar decrease in LAL activity, This nove l application of Triton X-114 partitioning is a nondegradative alterna tive to the chemical detoxification of gram-negative bacterial EPS for vaccine production. Purification of endotoxin-contaminated polysaccha rides prior to screening for biological activity should also benefit f rom this technique. The extraction scheme using Triton X-114 can be ea sily used in large-scale purification processes, (C) 1995 Academic Pre ss, Inc.