Facilitated isolation, purification, and analysis of glucuronoxylomannan of Cryptococcus neoformans

Abstract

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.