白色念珠菌细胞壁酸碱不溶性(1,3)-β-D-葡聚糖的分离、提取和分析

Isolation, extraction and analysis of insoluble(1,3)-β-D-glucan in cell walls from Candida albicans by acid-alkali method

目的研究用酸-碱法制备白色念珠菌细胞壁酸碱不溶性β-葡聚糖,并对其物理化学性质和结构进行分析,获得结构明确的酸碱不溶性(1,3)-β-D-葡聚糖。方法用沙氏肉汤培养白色念珠菌,经离心收集和干燥后,先后用NaOH溶液和乙酸进行处理,提取酸碱不溶性葡聚糖,采用薄层层析分析、高碘酸氧化分析和红外光谱法测定多糖的基本结构,并计算用该种方法提取的酸碱不溶性(1,3)-β-D-葡聚糖的提取效率。结果与标准的(1,3)-β-D-葡聚糖红外光谱相比,提取的多糖红外光谱在890 cm-1有特征吸收峰,表明多糖为β-构型;经苯酚-硫酸法鉴定,样品显桔红色,为多糖阳性反应。多糖经薄层层析分析,比较样品与单糖标准品的Rf值,确定多糖的构型为D-型;多糖经高碘酸氧化后,测定消耗高碘酸量和甲酸生成量,从而测定其多糖的主链键型为1,3位键合。经计算酸-碱法提取的酸碱不溶性(1,3)-β-D-葡聚糖效率为20.9%。结论用酸-碱法可以从白色念珠菌细胞壁中提取到高纯度的酸碱不溶性(1,3)-β-D-葡聚糖,为建立新型高特异(1,3)-β-D-葡聚糖的检测试剂提供关键物质基础。

Objective To extract insoluble（1,3）-β-D-glucan from the cell wall of Candida albicans with acid-alkali method and analyze their physical and chemical properties. Methods Candida albicans was grown in sabouraud broth and collected by centrifugation. Glucans were extracted from drieded Candida albicans using NaOH and acetic acid. The basic structure of extracted polysaccharide was characterized with thin layer chromatography, periodate oxidation and IR spectroscopy, and the extraction efficiency of insoluble（1,3）-β-D-glucan was calculated. Results In comparison with standard（1,3）-β-D-glucan IR,extracted polysaccharides had characteristic absorption peaks in the 890 cm-1IR spectrum indicating the polysaccharide is β-configuration;result of the phenol-sulfuric acid assay indicate the sample contains polysaccharides;compared with Rfvalue of the standard monosaccharide,the polysaccharides configuration were determined as the D-type by thin layer chromatographic analysis.The amount of periodate consumption and formic acid production were measured by periodate oxidation method, and the result showed that the extracted polysaccharide was1,3-position bond. The extraction efficiency of insoluble（1,3）-β-D-glucan was 20.9% by acid-alkali method.Conclusion High purity（1,3）-β-D-glucan can be extracted from the cell wall of Candida albicans with acid-alkali method, and provide critical substance for establishing a novel specific method to detect（1,3）-β-D-glucan of Candida albicans.