Adam O, Vercellone A, Paul F, Monsan P F, Puzo G
Departement Glycoconjugués et Biomembranes, Laboratoire de Pharmacologie et Toxicologie Fondamentales du Centre National de la Recherche Scientifique, Toulouse, France.
Anal Biochem. 1995 Mar 1;225(2):321-7. doi: 10.1006/abio.1995.1161.
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.
已研究了Triton X-114相分离技术从克雷伯氏菌属I-714胞外多糖（EPS）中去除脂多糖（LPS，内毒素）的潜力。评估了经典纯化和化学解毒方法在去除残留LPS方面的有效性，同时保持EPS的结构和功能完整性。超速离心、Detoxi-Gel和离子交换色谱法均未去除内毒素，除了在脱氧胆酸钠缓冲液中于60℃进行的凝胶过滤色谱法。在这种情况下，纯化的EPS级分的生物活性显著降低，碱性水解处理后也是如此。此外，乙酸解毒程序会水解EPS。作为一种替代方法，低温下在Triton X-114中对EPS进行相分配提供了一种快速、温和且高效的去除LPS的方法，如鲎试剂（LAL）活性降低100倍且生物活性仅降低2倍所示。在4℃下用Triton X-114缓冲液进行凝胶过滤色谱法以及在75℃下用更具亲水性的Triton X-100非离子去污剂进行相分配导致LAL活性有类似程度的降低。Triton X-114分配的这种新应用是用于疫苗生产的革兰氏阴性细菌EPS化学解毒的非降解替代方法。在筛选生物活性之前对内毒素污染的多糖进行纯化也应受益于该技术。使用Triton X-114的提取方案可轻松用于大规模纯化过程。
