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An optimized method for the chiral resolution of enantiomers of amino acids in bacterial supernatants is reported. This LC-MS/MS method is performed using a chiral Teichoplanin LC column and does not require sample clean up or chemical derivitization. This method allows for the determination of the relative amounts of the D and L enantiomers of 20 proteinogenic amino acids. The detection limits and response factors for the 20 amino acids were determined. Calibrations over three orders of magnitude showed least squares coefficient values (R^2) greater than 0.996 for eighty percent of the amino acids and greater than 0.992 for the remainder. The amino acids and their enantiomers were identified based on their retention times and their unique Multiple Reaction Monitoring (MRM) transitions for each amino acid. L-Aspartic acid-2,3,3-d3 was used as the internal standard. Cultures of Sinorhizobium meliloti (a nitrogen-fixing soil bacterium) were grown on minimal media; thus, all amino acids were biosynthesized by the bacterium. After centrifugation, supernatants were freeze dried, reconstituted in a small volume of methanol/water with internal standard and injected onto the LC column. The amino acids detected in the bacterial supernatant and the concentrations of the enantiomers were reported as the L and D isomers respectively: arginine [L, 12.6 ± 3.1 μg/L; D, 10.1 ± 3.2 μg/L], serine [L, 7.2 ± 1.16 μg/L; D, n.d.], threonine [L, n.d.; D, 11.2 ± 2.7 μg/L] and valine [L, 15.5 ± 4.3 μg/L; D, 11.3 ± 3.7 μg/L], where the term n.d. means below detection limit. The limits for detection for all amino acids ranged from 1.3 μg/L - 5.1 μg/L. In media with no added phosphate, the amino acid profiles changed somewhat under these stress conditions. Arginine was no longer detected while alanine and proline were now observed; the concentrations of the amino acids were: alanine [L, 7.7 ± 1.2 μg/L; D, 13.4 ± 2.5 μg/L], proline [L, n.d.; D, 8.63 ± 1.3 μg/L], serine [L, 7.6 ± 1.2 μg/L; D, n.d.], threonine [L, n.d.; D, 10.2 ± 3.2 μg/L] and valine [L, 11.6 ± 2.3 μg/L; D, 10.1 ± 3.1 μg/L]. These data represent the mean values of three independent bacterial growth experiments conducted over a 3 month period; the data came from the analysis of five separate aliquots from each growth experiment. The percent standard deviation for these data ranged from 15% to 33% and averaged 24%. Under both the normal and stressed growth conditions of S. meliloti produced the L enantiomer of serine, the D enantiomer of threonine and racemic valine. While racemic arginine was observed under normal growth conditions, levels were below detection under stressed conditions; under stress conditions only the D enantiomer of proline was observed while alanine was found in 1:2, L:D ratio. / Thesis / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(理学) / 甲第25106号 / 理博第5013号 / 新制||理||1715(附属図書館) / 京都大学大学院理学研究科物理学・宇宙物理学専攻 / (主査)教授 杉本 茂樹, 教授 青木 慎也, 教授 橋本 幸士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

This dissertation describes the simulation and optimization of adsorptive and chromatographic separation processes. The first part focus on the simulation and comparison of operational modes in simulated moving bed (SMB) chromatography for separation and purification in bioprocesses. The second part includes the simulation of gas-phase adsorptive processes by pressure swing adsorption and temperature swing adsorption technologies. The applications of SMB chromatography are popular in separating and purifying enantiomers, petrochemicals, pharmaceuticals and biochemicals with higher yield and lower solvent consumption. We simulate and compare several operational modes of simulated moving bed (SMB) for a binary and a ternary bioprocess using Aspen Chromatography. These operational modes are able to improve the separation efficiency of the basic SMB process by our simulation and optimization. We compare their separation performances and identify heuristics that will guide the selection of operational modes across a variety of systems. Pressure swing adsorption (PSA) and temperature swing adsorption (TSA) are two of the main technologies for gas-phase adsorption separation processes. We simulate and demonstrate a PSA model for air separation system and a TSA model for CO2 capture system in Aspen Adsorption. We present their separation performance plots to provide the physical insights of these two systems. / Ph. D.