顺序注射固定化酶化学发光法在线检测甲醇酵母发酵液中的甲醇

On-line Monitoring of Methanol in Fermentation Broth of Pichia Pastoris by Chemiluminescence Using Sequential Injection Analysis with an Immobilized Enzyme Column

运用顺序注射分析技术,完成了样品的在线预处理,并采用固定化酶化学发光检测法测定甲醇的浓度,建立了一种在线监测甲醇酵母发酵诱导阶段发酵液中甲醇浓度的新方法。在选定的最佳条件下,甲醇浓度在0%～1.0%(V/V)范围内与化学发光强度呈良好的线性关系(R=0.9995),相对标准偏差(RSD)为1.9%(n=11),72 h内RSD为5.0%(0.2%甲醇溶液)。该法可在线自动完成过滤、稀释、测定等操作,分析速度快,长期稳定性高,能满足在线监测甲醇酵母发酵液中甲醇浓度的要求。

A sequential injection analysis system is described that incorporates an enzyme column containing immobilized alcohol oxidase, allowing on-line monitoring of methanol in fermentation broth of pichia pastoris. Luminol chemiluminescence was used for the detection of the hydroperoxide generated in the enzyme reaction. Sample filtration and dilution were carried out on-line. The calibration graph was linear in the range of methanol concentration 0%-1.0%（V/V）（R=0.9995）. The RSD for the determination of methanol at a concentration of 0.2%（V/V） was 1.9% （n=11） and 5.0% during a 72- h monitoring process. The system is characterized by short analysis time, simple operation and good stability, which meet with the requirements of on-line monitoring of methanol in fermentation process.