端基法测定聚唾液酸平均聚合度

摘要

An end group assay method for determining the mean degree of polymerization of polysialic acid was developed. The total sialic acid content of polysialic acid was measured by resorcinol method and the end reducing group content of polysialic acid was determined by fluorescent method. Then the mean degree of polymerization of polysialic acid was calculated by dividing total sialic acid content by the end reducing group content. The fluorescence reaction was conducted in boric acid buffer (pH 9. 5) at 80℃ for 25 minutes. The fluorescence intensity of the reaction was linear with the content of the end reducing group of polysialic acid. The range of the confidence interval was in content of 1 - 20 mg/L sialic acids, giving a detection limit of 0. 36 mg/L and a RSD of 3.1% . The degree of polymerization of polysialic acid in the broth produced by Escherichia coli was 45. 76, with an inaccuracy of 3. 2% comparing with that of the HPGFC method. The end group analysis method was testified that it could be applied in the determination of the mean degree of polymerization of polysialic acid for fermentation process analysis and product quality characterization.%建立了端基法测定聚唾液酸平均聚合度的方法.采用间苯二酚比色法和丙二腈荧光法分别测定聚唾液酸中唾液酸总量和还原端唾液酸残基含量,两者之比即为平均聚合度.研究表明,在pH 9.5的硼酸缓冲液中,80℃水浴下聚唾液酸还原端唾液酸残基与丙二腈反应25 min后生成荧光物质,其荧光强度与还原端唾液酸残基含量呈线性正相关关系,线性范围在1～ 20 mg/L之间,变异系数和检出限分别为3.7％和0.36 mg/L.端基法测定大肠杆菌发酵液中聚唾液酸的平均聚合度为45.76,与高效液相凝胶色谱法比较,误差为3.2％.该法可以简便快速地测定发酵液中聚唾液酸的平均聚合度,有利于聚唾液酸生产过程分析及产品性能评估.