基因工程菌发酵液中多种成分快速检测方法的建立

Development of a method for rapid determination of several components in fermentation liquids of recombinant bacterial strains

目的 建立一种快速检测基因工程菌发酵液中葡萄糖、甘油、甲醇和乙酸含量的方法。方法 采用HPLC法检测基因工程菌发酵液中葡萄糖、甘油、甲醇、乙酸的含量：使用AminexHPX-87H column（300 mm×7.8 mm,9μm）,以5 mmol/L H2SO4溶液作为流动相,在柱温35℃、流速0.60 ml/min的条件下,用示差折光检测器进行检测,采用外标法定量。对方法进行系统适用性、专属性、检测限、定量限、线性范围、准确性、精密性验证及初步应用。结果各物质色谱峰理论塔板数均大于8 500,各峰之间的分离度均大于4.5,阴性对照在相应位置处未见色谱峰;在标准曲线线性范围内,乙酸、葡萄糖、甘油、甲醇浓度与峰面积均呈良好的线性关系,相关系数在0.999 97～0.999 98之间;该方法检测葡萄糖、甘油、甲醇、乙酸的平均加样回收率分别为102.18%、103.84%、105.16%、102.82%;该方法重复6次检测发酵液,其中葡萄糖、甘油、甲醇、乙酸峰面积的RSD分别为0.15%、0.21%、0.23%、0.23%。该方法对重组大肠埃希菌和毕赤酵母发酵过程中葡萄糖、甘油、甲醇、乙酸的含量进行检测,结果能够满足发酵操作的要求。结论 建立的HPLC法系统适用性和专属性良好,加样回收率和精密性较高,不受培养基中蛋白胨、酵母粉和其他代谢产物的影响,可应用于基因工程菌发酵过程中3种碳源和乙酸含量的检测。

Objective To develop a method for rapid determination of glucose, glycerol, methanol and acetic acid contents in fermentation liquids of recombinant bacterial strains. Methods The glucose, glycerol, methanol and acetic acid contents in fermentation liquid of recombinant E. coli were determined by HPLC. Aminex HPX-87H column （300 mm × 7. 8 mm, 9 μm） was served as chromatographic column, while 5 mmol/L sulfuric acid solution as flow phase at a flow rate of 0. 60 ml/min. Refractometer was used at a column temperature of 35℃. Quantitative analysis was performed by using external standard. The developed method was verified for suitability, specificity, detection limit, quantitative limit, linear range, accuracy and precision, and preliminarily applied. Results All the theoretical plate numbers of chromato- graphic peaks of various components were more than 8 500, while the separation degree was more than 4. 5. However, no chromatographic peaks of negative controls were observed at the corresponding locations. The concentrations of acetate acid, glucose, glycerol and methanol showed good linear relationship to the peak areas within the linear range of standard curve, with correlation coefficients of 0. 999 97 - 0. 999 98. The spike recovery rates of glucose, glycerol, methanol and acetic acid samples determined by the method for 6 times were 102. 18%, 103.84%, 105. 16% and 102. 82%, while the RSDs of peak areas were 0. 15%, 0. 21%, 0. 23% and 0. 23%, respectively. The determination results of the above-mentioned components in recombinant E. coli and P. pastoris during fermentation proved that the method met the requirements for fermentation procedure. Conclusion The developed HPLC method showed high suitability, specificity, precision and spike recovery rate. The peptone, yeast powder and other metabolic products in media showed no influence on the determination result. The method may be used for the determination of three carbon sources and acetate acid contents during fermentation of recombinant bacterial strains.