Study Of Culture Analysis By Capillary Electrophoresis

Metabolic engineering combines systematic analysis of metabolic pathways with molecular biology and uses recombinant technique to improve cellular properties by designing and implementing rational genetic modification. In recent years, metabolic flux analysis has become one of the major tools in metabolic engineering. The aim of metabolic flux analysis is to quantify all the metabolic fluxes in the metabolism of a microorganism. In order to study the metabolic fluxes, the ingredients of the culture must be stable and clearly clarified; the change of environment and metabolites could be measured instantly and accurately during the metabolism. Quantitative and qualitative analysis of carbon and nitrogen resources are vitally important because these two resources are the key ingredients in cell culture. Various methods have been developed for this purpose, but are still not satisfactory. Therefore, synthetic and semi-synthetic culture, whose ingredients are highly stable and wholly identified, must be used and a set of highly sensitive, accurate, stable, specialized measurements must be developed for the study of metabolic fluxes.In this research, we built a series of CE (capillary electrophoresis) methods for quantitative and qualitative measuring sugar and amino acids in culture. These methods were applied preliminarily to analyzing the content of sugar and free amino acids in fermentation broth. Results showed that CE could be used in qualitative or quantitative analysis of sugar and amino acids with high speed, accuracy, and specification. Thus these methods could be applied to the research of metabolic fluxes of microorganism because of its promising ability of purification, analysis and reliability.We applied methods of CE with Borax-SDS-Tris buffer, to analyze various amino acids derived from PITC, and the results showed high resolution. The parameters such as pH, voltage, temperature, and the concentration of borax, SDS and Tris has been optimized. This methodcould be applied to assay amino acids concentration in bacitracin fermentation broth.For CE analysis of sugar, we used both indirect and derived methods. Comparative results showed that using boras buffer system and indirect UV detection could achieve better results in analyzing non-reducing sugar. And according to different concentrations of reducing sugar, we could choose two derived methods, either by 1-Naphthylamin or DHZ. The analysis conditions of non-reducing sugar by indirect UV detection and that of reducing sugar by DHZ derived assay were optimized. We also applied these methods to monitoring changes of amino acids and sugar during the fermentation of Bacitracin and obtained good results.