Cherniak R, Morris L C, Anderson B C, Meyer S A
Laboratory for Microbial and Biochemical Sciences, Georgia State University, Atlanta 30303.
Infect Immun. 1991 Jan;59(1):59-64. doi: 10.1128/iai.59.1.59-64.1991.
Cryptococcus neoformans was cultured in a chemically defined medium. The culture was adjusted to 0.25% formaldehyde or autoclaved after 5 days of growth at 35 degrees C, and a cell-free supernatant was obtained by centrifugation. Solid calcium acetate was added to the supernatant to give a 5% solution, and the pH was adjusted to approximately 5 with glacial acetic acid. The polysaccharide (PS) was precipitated by the addition of 3 volumes of 95% ethanol. The PS was dissolved in 0.2 M NaCl, and insoluble calcium salts were solubilized by the addition of several drops of glacial acetic acid. The PS solution was treated by ultrasonic irradiation for 15 min. This concurrently decreased the molecular weight of the PS and reduced the viscosity of the solution. The ultrasonically irradiated PS was precipitated by differential complexation with hexadecyltrimethylammonium bromide at 23 degrees C, the complex was dissolved in 1 M NaCl, and the glucuronoxylomannan was precipitated by adding 3 volumes of ethanol. The glucuronoxylomannan was dissolved in 1 M NaCl and then ultrasonically irradiated for 2 h to reduce the molecular mass to a limiting value of approximately 100 kDa (GXMS). The purified GXMS was centrifuged, dialyzed, and finally recovered by lyophilization. GXMS was chromatographed on DEAE-cellulose at reasonable concentrations without the complication of high solution viscosity. The sugar composition and structure of GXMS were determined by gas-liquid chromatography, permethylation gas-liquid chromatography-mass spectrometry, and 13C nuclear magnetic resonance spectroscopy. The improved solution characteristics of GXMS were ideal for the determination of its chemical and serological properties.
新型隐球菌在化学成分明确的培养基中培养。培养物在35℃生长5天后，用0.25%甲醛进行处理或高压灭菌，然后通过离心获得无细胞上清液。向上清液中加入固体乙酸钙，制成5%的溶液，并用冰醋酸将pH值调节至约5。通过加入3倍体积的95%乙醇沉淀多糖（PS）。将PS溶解于0.2 M氯化钠中，并通过加入几滴冰醋酸使不溶性钙盐溶解。PS溶液经超声辐照15分钟。这同时降低了PS的分子量并降低了溶液的粘度。超声辐照后的PS在23℃下通过与十六烷基三甲基溴化铵的差异络合沉淀，将络合物溶解于1 M氯化钠中，再通过加入3倍体积的乙醇沉淀葡糖醛酸木甘露聚糖。将葡糖醛酸木甘露聚糖溶解于1 M氯化钠中，然后超声辐照2小时，将分子量降低至约100 kDa的极限值（GXMS）。纯化后的GXMS经离心、透析，最后通过冻干回收。GXMS在合理浓度下于DEAE - 纤维素上进行色谱分析，不存在高溶液粘度的问题。通过气液色谱法、全甲基化气液色谱 - 质谱法和13C核磁共振光谱法测定GXMS的糖组成和结构。GXMS改善后的溶液特性对于确定其化学和血清学性质非常理想。
