化学酶法对硫酸软骨素类多糖的定量分析

硫酸软骨素(chondroitin sulfate,CS)类多糖是一类具有多种临床治疗作用的糖胺聚糖。在硫酸软骨素裂解酶作用下,软骨素中葡糖醛酸和N-乙酰半乳糖胺之间的糖苷键发生断裂,可生成不饱和二糖。微生物发酵生产软骨素过程中,多糖定量往往需经过多次纯化以及使用昂贵的仪器进行检测,过程繁琐。文章研究偶联了专一性的酶解法与蒽酮-硫酸法,快速测定复杂培养液中的软骨素类多糖质量浓度。该方法通过超滤去除单糖等小分子杂质后,将多糖用专一性硫酸软骨素裂解酶分解为寡糖,再通过超滤去除大分子杂质,得到的寡糖过滤液使用蒽酮-硫酸法快速测定多糖质量浓度。该多糖定量方法可以选择不同特异性的裂解酶,适用于复杂培养液中其他多糖的快速定量分析。

Chondroitin sulfate (CS) and its derivatives are a kind of glycosaminoglycan with various clinical therapeutic effects. CS lyase can act on the glycosidic bond between glucuronic acid and N-acetylgalactosamine in CS to generate unsaturated disaccharides. The concentration determination of chondroitin produced from microbial fermentation requires several steps of purification and the employment of expensive equipment, which is very cumbersome. In this study, the coupling of specific enzymatic hydrolysis and anthrone-sulfuric acid method was carried out to determine the chondroitin concentration in the complex culture medium conveniently. The small molecular impurities, including monosaccharides, were removed by ultrafiltration. Then, the polysaccharide was enzymatically lyzed into oligosaccharides, followed by removing macromolecular impurities via ultrafiltration again. The concentration of obtained oligosaccharide filtrate was then quickly determined by the anthrone-sulfuric acid method. According to the specificity of the lyase action, this method can also be applied to the quantification of other polysaccharides in a complex culture medium.