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Construction of Analytical Platform for Tryptophan Biosynthesis Metabonomics Based on the LC-MS/MS

Author: MeiHui
Tutor: DaiJun
School: Jiangnan University
Course: Agricultural Products Processing and Storage
Keywords: Metabolomics E. coli High performance liquid chromatography - tandem mass spectrometry Quenching Washing Extract Metabolic Flux Analysis
CLC: R96
Type: Master's thesis
Year: 2010
Downloads: 176
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Abstract


With the continuous progress of the research of the microbial production of tryptophan, the microbial production of tryptophan to practical and in a dominant position. In order to further improve the production of tryptophan, microbial metabolomics have come into being. Microbial metabolomics studies related to a large number of metabolites in both qualitative and quantitative analysis, which requires the establishment of a technology platform to test the molecular effect of reconstruction be able to analyze the flow changes in metabolic networks. Based on a series of liquid chromatography and mass spectrometry parameters optimized, the establishment of of tryptophan synthesis metabolomics LC-MS/MS analysis method; this analysis method is applied to examine the E. coli metabolomics sample pre-treatment steps in order to optimize the best pre-treatment method, the establishment of tryptophan metabolomics analysis platform; take advantage of this platform to E. coli fermentation late metabolic flux analysis and metabolic node. The main conclusions are as follows: 1. Established a rapid, accurate simultaneous determination of Escherichia coli (E. coli) the 24 intracellular metabolic intermediate liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis method. Samples Waters Atlantis HILIC silica column separation, acetonitrile -10 mM / L ammonium acetate as the mobile phase gradient elution flow rate of 250μL/min. Electrospray ionization (ESI) mode of ionization, multiple reaction monitoring mode (Multiple reaction monitoring, MRM) monitoring, quantitative matrix standard solution. The results showed that: 24 metabolites in the range of 0.10 to 100μM / L showed a good linear relationship, the correlation coefficients were greater than 0.99; 24 metabolites detection limit (S / N gt; 3) 0.05 to 0.34μM / L three add a level of recovery was 83.7% ~ 108.1%, the relative standard deviation (RSD) were less than 10%. Comparative Study of the E. coli bacterial cells based on liquid chromatography and mass spectrometry (LC-MS/MS) quenching the washing and extraction of intracellular metabolites and cell disruption methods and crushing frequency and visits. Showing: results in the quenching process, the glycerol-NaCl solution can better maintain the integrity of the cells, to prevent the leakage of metabolites, 0.9% NaCl can better maintain the osmolality of the cell is a bacterial cell washing solution, preferably selection; After the samples were quenched and washed with cold methanol extract obtained by the method relatively highest extraction rate, and good reproducibility (RSD lt; 5%), liquid nitrogen freezing method for the extraction of intracellular metabolites sonicator The method is more thorough, and will not lead to changes in the heat-sensitive substances; intracellular metabolites by liquid nitrogen freezing and thawing four most intracellular metabolites. 3 through the establishment of production tryptophan E. coli metabolic flux balance model and metabolic node analysis, the PEP node and the the G6P node at the metabolic flow direction, in order to improve the yield of the tryptophan.

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