A high-performance CE application for a quick, reproducible, highly precise and sensitive determination of the lipopolysaccharide produced by Escherichia coli K4 (O5:K4:H4) and of its de-lipid A form is described. The two species were separated within 30 min on an uncoated fused-silica capillary, in normal polarity mode at 20 W, using an SDS buffer. Detected at 190 nm, the de-lipid A and the LPS species showed two peaks at distinctive migration times (10.45 and 16.10 min, respectively) and were quantified with high reproducibility and linearity (the correlation factors were 0.99 and 0.98, respectively) over the ranges from 60 to 600 ng (1-10 ng/nL) for de-lipid A lipopolysaccharide and from 150 to 600 ng (2.5-10 ng/nL) for the LPS. The described method was also employed in the contemporary analysis and the determination of the two E. coli K4 cell surface polysaccharides, the LPS and the K4, and of their defructosylated and de-lipid A species, respectively. The four molecules were detected and precisely quantified in complex matrices as fermentation broth supernatant or in samples withdrawn throughout the purification process, thus demonstrating the possibility to apply high-performance CE as a reliable analytical tool in biotechnological processes.

High-performance CE of Escherichia coli K4 cell surface polysaccharides

DE CASTRO, CRISTINA;PARRILLI, MICHELANGELO;RESTAINO, ODILE FRANCESCA
2009

Abstract

A high-performance CE application for a quick, reproducible, highly precise and sensitive determination of the lipopolysaccharide produced by Escherichia coli K4 (O5:K4:H4) and of its de-lipid A form is described. The two species were separated within 30 min on an uncoated fused-silica capillary, in normal polarity mode at 20 W, using an SDS buffer. Detected at 190 nm, the de-lipid A and the LPS species showed two peaks at distinctive migration times (10.45 and 16.10 min, respectively) and were quantified with high reproducibility and linearity (the correlation factors were 0.99 and 0.98, respectively) over the ranges from 60 to 600 ng (1-10 ng/nL) for de-lipid A lipopolysaccharide and from 150 to 600 ng (2.5-10 ng/nL) for the LPS. The described method was also employed in the contemporary analysis and the determination of the two E. coli K4 cell surface polysaccharides, the LPS and the K4, and of their defructosylated and de-lipid A species, respectively. The four molecules were detected and precisely quantified in complex matrices as fermentation broth supernatant or in samples withdrawn throughout the purification process, thus demonstrating the possibility to apply high-performance CE as a reliable analytical tool in biotechnological processes.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/364774
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