高效液相色谱法测定谷氨酸棒杆菌发酵液中17种氨基酸

[目的]建立一种简单高效、可同时检测发酵液中17种氨基酸的方法,并利用此方法对发酵液中的氨基酸进行定量分析。[方法]以2,4-二硝基氟苯为衍生试剂,使用Phenomenex Gemini NX C18(250.0 mm×4.6 mm,5μm)色谱柱,柱温30℃,流动相A为0.05 mmol/L乙酸钠溶液,流动相B为乙腈-水(1∶1,V/V),梯度洗脱,检测波长为360 nm。[结果]17种氨基酸在0.01~2.00 g/L线性相关良好,各相关系数在0.990以上,各氨基酸的加样回收率为87.4%~105.1%。采用该方法对发酵液中的氨基酸进行分析,有10种氨基酸积累,其中谷氨酸、瓜氨酸、精氨酸积累量较大。[结论]该研究建立了一种简单高效、可同时检测发酵液中17种氨基酸的方法,为精氨酸进一步代谢改造提供理论依据。

Determination of 17 Amino Acids in Corynebacterium glutamicum Fermentation Broth by High Performance Liquid Chromatography

[Objective]A simple and efficient method for the simultaneous analysis of 17 free amino acids was established and applied to analyze free amino acids in Corynebacterium glutamicum.[Method] The method used pre-column derivatization with 2,4-dintrofluorobenzene(DNFB).The amino acids were separated in a Phenomenex Gemini NX C18 column (250.0 mm×4.6 mm,5 μm) at 30 ℃.The mobile phase was a mixture of phase A:0.05 mmol/L sodium acetate buffer (pH 6.8) and phase B:1:1(V/V) acetonitrile/water through the gradient elution.Detection was performed using a UV detector at 360 nm.[Result]The method showed good linearity with above correlation coefficients of 0.990 when 17 kinds of amino acids concentrations were in the context of 0.01-2.00 g/L.The recoveries were between 87.4% and 105.1%.Applying the method to analyze the fermentation broth,it found that the broth had 10 kinds of amino acids accumulation and glutamic acid,citrulline and arginine among them accumulated a large amount.[Conclusion]The study establishes a simple and efficient method for the simultaneous analysis of 17 kinds of amino acids,provides theoretical basis for the metabolic transformation of the arginine.