Determination of Intracellular Concentration of Acyl‐Coenzyme A Esters for Metabolic Profiling Clostridium acetobutylicum
Determination of Intracellular Concentration of Acyl‐Coenzyme A Esters for Metabolic Profiling Clostridium acetobutylicum
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DOI：
10.1002/cjoc.201090183
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发表时间：
2010-06
影响因子：
5.4
通讯作者：
W. Deng;Jiayi Cheng;Tonghua Li;Jingwu Kang
中科院分区：
文献类型：
--
作者：
W. Deng;Jiayi Cheng;Tonghua Li;Jingwu Kang
建立了高效液相色谱法测定丙酮丁醇梭菌胞内酰基辅酶A酯的方法。本实验采用Zobax-C_（18）色谱柱，以乙腈-磷酸盐缓冲液（pH 5.0）为移动的相，对乙酰辅酶A和丁酰辅酶A两种重要中间体进行基线分离。样品经冻融、三氯乙酸沉淀后，可直接进样测定乙酰辅酶A、丁酰辅酶A，回收率均大于73%。利用该方法，获得了CA的酰基辅酶A酯的代谢物谱。比较了模式菌株ATCC 824和突变菌株EA 2018之间的代谢产物谱，突变菌株EA 2018产生的丁醇比模式菌株ATCC 824高。为了解CA选择性生产溶剂的机理提供了有益的信息。
A method for determination of intracellular acyl-coenzyme A esters in Clostridium acetobutylicum (CA) by high performance liquid chromatography (HPLC) was developed and validated. In our experiment, two important intermediates acyl-coenzyme A esters including acetyl-CoA, butyryl-CoA could be baseline separated on a Zobax-C18 column with mobile phase composed of the acetonitrile and phosphate buffer (pH 5.0). Samples treated with freeze-thaw and protein precipitated by addition of trichloroacetic acid could be directly injected for determination of acetyl-CoA, butyryl-CoA with a recovery higher than 73%. With this method, the metabolite profiling of the acyl-coenzyme A esters of CA was obtained. The comparison of the metabolite profiling between the model strain ATCC 824 and a mutant strain EA 2018, which produces higher butanol than the former was performed. Some useful information was concluded for understanding the mechanism of CA for selectively producing the solvent.