The nitrogen-fixing bacterium Burkholderia tropica, strain Ppe8, is an endophyte which was isolated from pineapple plants in Brazil. When grown on a liquid synthetic medium containing mannitol and glutamate, it produced copious amounts of exopolysaccharides (EPS), which in turn made the medium somewhat viscous. In order to isolate the EPS, cells were removed after centrifugation from a 1 L liquid culture and the supernatant was concentrated and then added to excess cold etanol (3 v/v). The resulting precipitated material was recovered, resolubilized and dialyzed against water in a 16 kDa MWCO membrane before freeze-drying. Retained acidic polysaccharide was then purified by anion-exchange chromatography in a fast run FPLC system. A portion of the EPS was carboxyreduced to give EPS-CR and both, native EPS and EPS-CR, were analyzed for their neutral monosaccharide composition after derivatization to alditol acetates and analysis by GC-MS. The native EPS contained Glc and Rha in a molar ratio of 1:1, while EPS-CR showed Glc and Rha in a 3:2 proportion. Methylation –GC-MS analysis revealed that EPS-CR had non-reducing terminal units of Glc, which proved to be from GlcA carboxyreduced unit from EPS. ¹³C-NMR analysis showed anomeric signals at d 94.7, 98.0, 99.0 and 103.1 for EPS and at d 95.7, 98.3, 100.7 and 103.2 for EPS-CR. Signals corresponding to C-6 of Rhap units arose from both polyssacharides at d 16.6. Acetyl substituents were also present, giving signals at d 20.2 (CH₃) and 173.6 (CO₂H). Complete ¹H and ¹³C assignments of all monosaccharide units were feasible after using COSY, TOCSY and HMQC analyses. Enantiomeric configurations were determined by GC-MS after total hydrolysis and derivatization to form acetylated 2-(-)-octylglycosides. Oligosaccharides obtained after partial hydrolysis of the native EPS were analyzed ESI-MS in the negative mode. The molecular ion found at m/z 809.2 was attributed to the pentasaccharide repeating unit of the EPS. Daughter ions at m/z 647.2, 501.2, and 339.2 corresponded to a tetra-, tri- and disaccharides respectively. Also, the fragment at m/z 851.3 showed to be the acetylated pentasaccharide. With these results we were able to determine the structure of the acidic exopolysaccharide as:

[®4)-b-d-Glcp-(1®2)-[a-D-GlcpA-(1®4)]-a-L-Rhap-(1®4)-a-D-Glcp-(1®3)-b-L-(Ac-2)Rhap-(1®] _n