| Surfactin is a family of lipopeptide produced by various strain.It is important in scientific and industrial fields such as medicine, food, cosmetics and enhancement of oil recovery due to its excellent surface-active and specific bio-active properties. The studies focused on the improvement of the extraction procedure, optimization of hydrolysis condition, the quantification method and the kinetics of esterification of surfactin. The results could be described as follow.The surfactin sample was obtained from Bacillus subtilis HSO121in our laboratory. The surfactin, with the purity of98.6%, was obtained after separation from the cell-free broth, acid precipitation, solvent extraction, and purification by reverse-phase HPLC through shortening the operation process and time. A series of isoforms of surfactin, with fatty acid chains of C13, C14and C15, respectively, was isolated by HPLC. And the surface activities of these surfactins were studied.An optimum hydrolysis condition, in6.0mol/L HCl at90℃for24h, was established. The relative dehydration rate was decreased from34%to5%when the reaction temperature changed from110℃to90℃. It showed that at90℃, the temperature lower than the water boiling point, more β-hydroxy fatty acids and less unsaturated fatty acids were produced and the operation might be more safe and convenient. When the partially hydrolysis was performed at80℃in0.1mol/L NaOH for60min, more linear surfactin could be obtained. A series of isoforms of linear surfactin, with fatty acid chains of C13, C14and C15, was isolated by HPLC. And their surface activities were studied.A new method for quantitative determination of each isoform in surfactin family has been established. The surfactin was firstly hydrolyzed in acid solution at90℃for24h, dried and treated with bio (trimethylsilyl) trifluoroacetamide at60℃for20min, and the derived hydrolysates were then analyzed by GC-MS. The composition of the peptide part and the fatty acid part of surfactin was recognized. The amount of surfactin,4.80×10-7mol, was detected through the working curves made with the amino acids and the relationship between surfactin varants, amino acid residues and fatty acid residues. The mole fractions of nine surfactin isoforms with different fatty acid chains were, n C12(0.32%), iso C13(4.89%), anteiso C13(6.27%), iso C14(23.05%), n C14(8.95%), iso C15(17.69%), anteiso C15(38.69%), iso C16 (0.07%), n C16(0.07%), respectively. The mass of these nine isoforms would be obtained according to each molecular weight and the matter quantity. Finally, the accurate mass of the surfactin in this original sample was493μg (the purity was98.6%). This approach was the only method which could quantify the accurate mass of the isoforms of surfactin and might be applied to the quantitative analyses for other families of lipopeptides as long as the sequence of amino acid residues in peptide was determined.The reaction, by which surfactins in methanol solution with acid was esterified into the mono-or dimethyl-esters, was studied. At4℃,25℃and45℃, the kinetic model of surfactin esterification in TFA methanol was established via the HPLC. Results showed that the reaction rate of mono-esterification of surfactin was much larger than the double-esterification; the longer the carbon chain was, the smaller the activation energy was. We obtained the storage time at the three temperatures, when the surfactin less than10%was esterified. The results might help confirm that the condition for storage and treatment of surfactin.The results of this thesis that enriched the methods of preparation, extraction and separation of surfactin in order to enhance the purity of the sample; provided a new method to determine the content of each isoform of surfactin which had important reference value to screen the microorganism species, determine the fermentation conditions and the physical and chemical properties; made clear the characteristics of the derivative reaction and provided firm foundation for structure modification of surfactin. |
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