鳞杯伞α-半乳糖苷酶的提取纯化及酶学性质研究

采用离子交换层析和凝胶过滤层析对鳞杯伞子实体中的α-半乳糖苷酶进行纯化,得到了一种分子量为50 kDa的α-半乳糖苷酶,命名为CSG。纯化后的CSG纯化倍数为891.46倍,比活力为54.78 U/mg,得率为0.71%。通过BLAST比对液相色谱-串联质谱(LC-MS/MS)获得其肽段,发现其为GH27家族的α-半乳糖苷酶。CSG的最适pH为3.0,最适温度为50 ℃。在酸性范围pH 2.2-7.0和温度范围4-30 ℃有较好的稳定性。Mn²⁺、Cd²⁺、Cu²⁺对CSG有较强的抑制作用。半乳糖和蜜二糖对CSG的抑制类型为混合型抑制。化学修饰剂N-溴代琥珀酰亚胺显著降低CSG的活力,碳二亚胺对CSG具有显著的激活作用。该酶具有良好的蛋白酶抗性,且对棉子糖家族寡糖(RFOs)、瓜尔豆胶和赤槐豆胶均表现出良好的水解作用。

Purification and characterization of α-galactosidase from Clitocybe squamulosa

The α-galactosidase from Clitocybe squamulosa was purified by ion exchange chromatography and gel filtration chromatography and a 50 kDa monounit α-galactosidase named CSG was obtaind. The purification multiple of purified CSG was 891.46 times, the specific activity was 54.78 U/mg, and the yield was 0.71%. BLAST comparison of the peptide obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that the enzyme was an α-galactosidase of the GH27 family. The optimal pH of CSG was 3.0 and the optimal temperature was 50 °C. It had good stability in pH limits of 2.2-7.0 and temperature of 4-30 °C. Mn²⁺, Cd²⁺ and Cu²⁺ had strong inhibitory effect on the enzyme. Galactose and melibiose showed mixed inhibition to this enzyme. The chemical modifier N-bromosuccinimide significantly reduced the activity of CSG, and carbodiimide significantly activated CSG. The enzyme showed effective protease resistance and evidently hydrolyzed raffinose family oligosaccharides (RFOs), sophora locust gum and guar gum.