文献类型：期刊文献

中文题名：^(13)CMFA过程中GC-MS分析菌体蛋白氨基酸的^(13)C标记丰度

英文题名：Analysis of Mass Isotopomer Distributions of Proteingenic Amino Acids Used GC-MS during ^(13)CMFA

作者：王建[1];王泽建[1];黄明志[1];钱江潮[1];储炬[1];张嗣良[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室/国家生化工程技术研究中心,上海200237

年份：2009

卷号：29

期号：7

起止页码：87

中文期刊名：中国生物工程杂志

外文期刊名：China Biotechnology

收录：CSTPCD;;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；

语种：中文

中文关键词：^13C代谢通量分析;菌体蛋白氨基酸;氨基酸衍生化;气相色谱-质谱;^13C标记丰度信息

外文关键词：^13C metabolic flux analysis Proteinogenic amino acids Amino acids derivatized GC-MS ^13C labelling pattern

摘要：基于13C标记的代谢通量分析(13CMFA)由于具有准确性和应用性已成为国际上代谢工程研究的热点,其中一个至关重要的问题就是分析菌体蛋白氨基酸的13C标记丰度信息。用含20%全标记葡萄糖([U-13C])和80%天然葡萄糖的合成培养基喂养维生素B12生产菌Pseudomonas denitrifican,然后在稳态条件下取20mg带13C标记菌体用1ml6mol/L盐酸95℃水解24h得到带13C标记的菌体蛋白氨基酸;氨基酸经分离、浓缩、真空干燥、MBDSTFA衍生化后得到的TBDMS衍生物可以用气相色谱-质谱(GC-MS)进行分析,最后分析质谱图得到15种菌体蛋白氨基酸的13C标记丰度信息。成功获得菌体蛋白氨基酸13C标记丰度信息的实验方法和样品处理技术,为13CMFA在国内进一步发展提供了参考意义。
^13C metabolic flux analysis （^13CMFA） have been the research hotspots of metabolic engineering internationally due to its accuracy and applicability. It is vital that the measurement of ^13C labelling pattern of proteinogenie amino acids for ^13C metabolic flux analysis. To acquire ^13C-labelling proteinogenic amino acids, Pseudomonas denitrifican which products Vitamin B12 was firstly fed with mimimal culture medium contained 20% U-^13C and 80% natural glucose, after the culture reached a steady state, then about 20 mg biomass was hydrolyzed by 1 ml of 6 mol/L hydrochloric acid for 24h at 110℃. Then amino acids was separated, concentrated, evaporated in a vacuum, and derivatized with MBDSTFA, TBDMS-derivatized amino acids can be observed by GC-MS last we get ^13C labelling pattern of fifteen aminio acids through mass spectrum. The experimental methods and sample preparation offers referential value for the development of ^13C metabolic flux analysis in our courtry.