细菌代谢产物中小分子有机酸高效毛细管电泳分析.doc

细菌代谢产物中小分子有机酸高效毛细管电泳分析 　 作者：胡耀华,钟文彬,周林,黄树林 【摘要】 　　目的 建立高效毛细管电泳法测定甲酸、乙酸、丙酸、丁酸、乳酸的分析方法，测定细菌代谢产物中的小分子有机酸。方法 采用毛细管：Beckman coulter未涂层50 μm×60 cm (有效长度50 cm);缓冲溶液：pH 10.5的10 mmol/L(含0.5 mmol/L CTAB)的硼砂溶液;检测波长214 nm，分离电压-20 kV，进样压力为1.0 psi，进样时间为10 s。结果 4 min内甲酸、乙酸、丙酸、丁酸、乳酸得到基线分离。线性范围分别为：甲酸3.8～30.7 mg/L (r=0.997 1);乙酸3.3～52.5 mg/L (r=0.996 1);丙酸3.1～49.5 mg/L (r=0.999 3);丁酸3～48 mg/L (r=0.992 5);乳酸7.5～60.5 mg/L (r=0.999 5)。平均回收率分别为: 甲酸99.29%(RSD=0.67%);乙酸98.90%(RSD=1.36%);丙酸99.65%(RSD=0.65%);丁酸99.35%(RSD=0.42%);乳酸98.06%(RSD=1.44%)。结论 该方法简单、快速、准确，可用于细菌发酵液中代谢产物有机酸的检测。 【关键词】 细菌代谢产物 小分子有机酸 高效毛细管电泳 Abstract:Objective To establish a method for determining formic acid,acetic acid,propionic acid,butyric acid and lactic acid in bacterial metabolites by the highperformance capillary electrophoresis. Methods Samples were analyzed on a Beckman Coulter (50 μm ×50 cm) uncoated fusedsilica capillary using 10 mmol/L borate buffer ( pH 10.5,containing 0.5 mmol/L CTAB). The UV detection wavelength was 214 nm,the separation voltage -20 kV and injection pressure 1.0 psi×10 s. Results Standard samples of formic acid,acetic acid,propionic acid,butyric acid and lactic acid were separated well in 4 minutes,and their linear ranges were 3.8-30.7 mg/L (r=0.997 1),3.3-52.5 mg/L (r=0.996 1),3.1-49.5 mg/L (r=0.999 3),3.0-48.0 mg/L(r=0.992 5),7.5-60.5 mg/L (r=0.999 5),respectively,their average recoveries were 99.29%,98.90%,99.65%,99.35% and 98.06%,respectively. The RSD values (n=6) of formic acid,acetic acid,propionic acid,butyric acid and lactic acid were 0.67%,1.36%,0.65%,0.42%,1.44%,respectively. Conclusion The method is simple,rapid and accurate for detecting the organic acids in bacterial metabolites in fermentation liquor. 　　Key words:metabolic product;organic acid;highperformance capillary electrophoresis 　代谢流量是细胞生理学的一个基本决定因素，也是代谢途径中最重要的参数。对细菌进行代谢流量分析，特别是代谢产物中小分子有机酸的分析，有助于通过基因工程手段定向地、有目的地改变限制细胞代谢的关键途径，以更好地理解和利用细胞代谢进行化学转化、能量转导和活性产物表达[1]。在发酵过程中，产酸发酵细菌能将有机物转化为以脂肪酸和醇为主的末端产物，同时生成新的细胞物质。产酸发酵速率较快，末端产