74(P51) Erwinia carotovoraリポ多糖の全体構造の解析(ポスター発表の部)

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An R-type lipopolysaccharide (LPS) from Erwinia carotovora FERM P-7576 was studied after strong alkaline degradation and mild acid hydrolysis. The resulting products were analyzed by fast atom bombardment MS, one- and two-dimensional ^1H and ^<13>C NMR spectroscopy, dephosphorylation and methylation analysis. With the structure of lipid A and the core moiety, the complete LPS structure was established and confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS of the native LPS. 1. Introduction A rough mutant E. carotovora FERM P-7576 excreted LPS spontaneously into the cultural medium. Structural studies of LPS are important for better understanding its role in the bacterium-plant interaction and specific phase behavior of LPS-containing membranes. 2. Materials and Methods Extracellularly produced LPS was isolated and purified from the cell free culture solution. LPS was acid hydrolyzed to lipid A and core oligosaccharide in 0.1M H_2SO_4 at 100℃ for 20 min. The lipidA and oligosaccharide was characterized by component analysis, methylation analysis, mass and NMR spectrometry. The ionization matrix employed for the positive ion FAB-MS measurement of the lipid A was an admixture of glycerol, thioglycerol, and meta-nitrobenzyl alcohol (1: 1: 1, v/v). The core oligosaccharide was measured in glycerol and thioglycerol mixture (1: 1). MALDI-MS of the native LPS was performed in the linear TOF mode. The LPS sample was dispersed in aqueous triethylamine, treated with a cation-exchange resin and admixed with 2,4,6-trihydroxy-acetophenone for ionization matrix. 3. Results Component and methylation analysis of the oligosaccharide indicated terminal galactose, phosphate linked heptose, 5-linked Kdo, etc. ^1H-NMR indicated eight anomeric sugar protons, other peaks were assigned by two dimensional methods. Positive ion FAB-MS of the lipid A yielded informative ions appeared by cleavage of glycosidic linkage of diglucosamine backbone and further loss of acyl moiety (Fig. 1). The negative ion MALDI-TOF mass spectrum of the native LPS yielded molecular ions and fragment ions that reflected the heterogeneity of LPS with respect to the core region and to the number of the secondary O-linked fatty acid residues within lipid A (Fig. 2). Fragment ions resulted from cleavage of lipid A and showed the heterogeneity with respect to the degree of acylation of lipid A and to the number of sugar residues in the core. By these data the complete structure of the E. carotovora rough-type LPS could be proposed as illustrated in Fig. 3.
天然有機化合物討論会の論文
1998-08-31

74(P51) Erwinia carotovoraリポ多糖の全体構造の解析(ポスター発表の部)

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