Preparation Of Deuterated Amino Acids And Their Application In The Lipopeptide Biosynthesis

Deuterated amino acids have been playing an important role in the study of reaction metabolisms and metabolic pathway. This dissertation summarizes the preparation deuterated amino acids by the method of isotope exchange. The lipopeptide biosurfactant common amino acids were selected for the study, optimized conditions established the best method of preparation and derivatization of deuterated amino acids. In addition, established aspartic acidβ-H deuterium kinetic equation under acid conditions, wihich can be used for infer the time of entire deuterated under different conditions. Prepare deuterated amino acid by the optimizing method, and use in microbiological culture media, explore the deuterated amino acids in lipopeptide biosurfactants microbial synthesis and influence. The results include as followings.By Gas Chromatography-Mass Spectrometry (GC/MS)testing, calculated the sum of peaks area of amino acids derivatives under different derivatization conditions and identified the best method of amino acid derivatives. The best conditions for the esterification:the reaction temperature is 120℃, the reaction time was 80 min and hydrochloride-butanol concentration is 3.00 mol/L; The best conditions for acylation:the reaction temperature is 60℃, the reaction time was 40 min. The best silylation conditions:reaction in 40℃and 60 min.Select Asp and Glu as the research object, compare amino acids and deuterated amino acids spectra under different deuterium reaction conditions by GC/MS and'H-NMR, identified best deuterium reaction conditions of two amino acids:in the acid catalyst condition, the reaction temperature is 120℃, the reaction time was 16 h. Established aspartic acidβ-H deuterium kinetic equation under acid conditions, wihich can be used for infer the time of entire deuterated and deuterated rate under different conditions.Prepared deuterated amino acid by the optimizing method as one of the medium components for microbial lipopeptide production. Bacillus subtilis TD7 were cultivated in different culture media, deuterated amino acids and undeuterated amino acids were added to each medium, respectively. Electrospray Ionization Mass Spectrometry (ESI-MS) and GC/MS were applied to analyze the type and composition of lipopeptides producted under different culture media. The results showed that the ratio between homologues had been changed after the addition of Glu in the medium, the content of surfactin C14 was more than surfactin C15. However, after adding Asp in the medium, the ratio between homologues had not been changed. The ratio between homologues was C14> C15> C13> C16 after the addition of deuterated Asp in the medium, and the C14 increased significantly. After adding deuterated Glu in the medium, the ratio between homologues had not been changed.