一种快速测定发酵液中丙酮酸含量的简易方法
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摘要
建立快速测定发酵液中丙酮酸含量的方法。利用酶固定化技术,将丙酮酸氧化酶(POD)及其辅酶(FAD)固定于核微孔膜,制备丙酮酸酶膜,并组装成丙酮酸酶电极,分析丙酮酸的含量。pH 7. 4,反应温度32℃,测定时间30 s。在酶电极最佳条件下,丙酮酸质量浓度在5～1200 mg/L范围内线性关系良好,检测限为3 mg/L;方法加标回收率为97. 1%～101. 8%,RSD为0. 68%。除高浓度NH+4外,不受丙酮酸发酵液中其他物质干扰物影响。丙酮酸发酵的实际应用中与HPLC法比较,测定结果无明显差异(P> 0. 05)。方法适用于实际发酵生产中丙酮酸含量的实时测定。
To establish a simple method for the determination of pyruvic acid in fermentation broth.Pyruvate oxidase( POD) and coenzyme( FAD) were immobilized on the nuclear microporous membrane to prepare pyruvate membrane,and then to develop enzyme electrode for the determination of pyruvic acid. The test conditions were simple and easy to operate in pH 7. 4 at 32 ℃. The reaction time was30 s. Under the optimum conditions,the enzyme electrode showed a good linear relationship in the range of 5-1200 mg/L,with limit of detection of 3 mg/L. The recovery was from 97. 1% to101. 8%. High stability,good reproducibility and strong selectivity were all realized. The standard deviation( RSD) for the test of pyruvate standard solution was up to 0. 68%. In this experiment,no other substances could interfere with the determination of pyruvic acid except high concentration of NH+4 in the fermentation. There was no significant difference( P > 0. 05) in measurement resultsbetween this method and HPLC in fermentation. The method showed a series of characteristics such as simple conditions,easy operation,accurate and rapid determination,high sensitivity and low cost.The enzyme biosensor was a promising method with practical prospect for measurement of pyruvate in fermentation.
To establish a simple method for the determination of pyruvic acid in fermentation broth.Pyruvate oxidase( POD) and coenzyme( FAD) were immobilized on the nuclear microporous membrane to prepare pyruvate membrane,and then to develop enzyme electrode for the determination of pyruvic acid. The test conditions were simple and easy to operate in pH 7. 4 at 32 ℃. The reaction time was30 s. Under the optimum conditions,the enzyme electrode showed a good linear relationship in the range of 5-1200 mg/L,with limit of detection of 3 mg/L. The recovery was from 97. 1% to101. 8%. High stability,good reproducibility and strong selectivity were all realized. The standard deviation( RSD) for the test of pyruvate standard solution was up to 0. 68%. In this experiment,no other substances could interfere with the determination of pyruvic acid except high concentration of NH+4 in the fermentation. There was no significant difference( P > 0. 05) in measurement resultsbetween this method and HPLC in fermentation. The method showed a series of characteristics such as simple conditions,easy operation,accurate and rapid determination,high sensitivity and low cost.The enzyme biosensor was a promising method with practical prospect for measurement of pyruvate in fermentation.
引文
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[16] Kumaresh S. Soppimath. J. Control. Rel.,2001,70:1