The Extraction And Purification Of Key Lipids In Membranes Of Escherichia Coli

Lipopolysaccharide (LPS) and phosphatidyl ethanolamine (PE) are the major components in cell membranes of most Gram-negative bacteria. Lipopolysaccharides, located in the outer membrane, consist of hydrophilic polysaccharides and hydrophobic lipid A. Phosphatidylethanolamines are the most aboundant lipids in cell membranes of Gram-negative bacteria (70%). The methods for separation and purification of lipopolysaccharide and phosphatidylethanolamine from Gram-negative bacteria are important. In this study, methods to separate and purify lipopolysaccharides containing different numbers of fatty acid chain by hydrophobic interaction chromatography columns, and to adsorb phosphatidylethanolamine specificly by the molecularly imprinted polymer were developed.The results are listed bellow.(1) Three species of lipopolysaccharides were extracted from Escherichia coli W3110, MLK1067, and MKV15b, respectively, and their lipid As were extracted and confirmed by ESI/MS. Three different hydrophobic interaction chromatography columns Phenyl-sepharose, Butyl-sepharose, and Octyl-sepharose were used for adsorption of the three different LPS species 6FA-LPS, 5FA-LPS and 4FA-LPS. The adsorption capacity of Butyl-sepharose for LPS is the highest while that of Octyl-sepharose is the lowest. The bound LPSs in three different columns were eluted by water, methanol, ethanol, isopropyl alcohol or propyl alcohol, respectively. The elution ratio by propyl alcohol was the highest. Lipopolysaccharides bound to Butyl-sepharose were the easiest to be eluted.(2) The effects of the concentration of sodium acerate and pH on the adsorption of Butyl-sepharose for lipopolysaccharides were studied. The optimum concentrations of sodium acetate for the highest adsorption ability to 6FA-LPS, 5FA-LPS, 4FA-LPS were 0.6 M, 0.8 M, 0.7 M, respectively; the optimum pH for the highest adsorption ability to 6FA-LPS, 5FA-LPS, 4FA-LPS were 4.7, 4.5, and 4.4, respectively.(3) The elution ranges of propyl alcohol for eluting lipopolysaccharides bound to Butyl-sepharose were analysed. The elution ranges of propyl alcohol for 6FA-LPS, 5FA-LPS and 4FA-LPS were 17-19%, 19-21%, 24-26%, respectively. When mixtures of lipopolysaccharides containing 6FA-LPS, 5FA-LPS, 4FA-LPS were separated on Butyl-sepharose, 4FA-LPS was eluted by 12-14% propyl alcohol, 5FA-LPS was eluted by 17-19% propyl alcohol, 6FA-LPS was eluted by 22-24% propyl alcohol.(4) The polymerization recipe including polymerization solution, polymerization volume, additive amount of template and adsorption solution were optimized. The best polymerization solution was chloroform;the best polymerization volume was 10 mL; the best additive amount of template was 7.5 mg; the best adsorption solution was chloroform. The structure characteristics of the molecularly imprinted polymer were also analysed by FT-IR and SEM.(5) Adsorption characteristics of the molecularly imprinted polymer were studied. The adsorption amount of molecularly imprinted polymer reached the saturation value when the reaction time was 9 h. The adsorption rate was proportional to the product between polymer concentration and template concentration. The aturation adsorption amount of molecularly imprinted polymer reached to the maximum value when the initial concentration of the template was 0.6 mg/mL. The adsorption was consistent with Langmuir isothermal adsorption model. According to this model, the theoretical maximum adsorption amount of molecularly imprinted polymer for the template is 5.244 mg/g. The made molecularly imprinted polymer could adsorp phosphatidyl ethanolamine specifically..