鳞杯伞α-半乳糖苷酶的提取纯化及酶学性质研究

Purification and characterization ofα-galactosidase from Clitocybe squamulosa

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。Mn^(2+)、Cd^(2+)、Cu^(2+)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。

Theα-galactosidase from Clitocybe squamulosa was purified by ion exchange chromatography and gel filtration chromatography and a 50 kDa monounitα-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 anα-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.Mn^(2+),Cd^(2+)and Cu^(2+)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.