黑曲霉N5-5单宁酶的纯化及酶学性质测定

摘要

采用中空纤维膜超滤和葡聚糖凝胶层析相结合的方法对黑曲霉N5-5单宁酶进行纯化,然后对纯酶性质进行测定.结果显示,黑曲霉N5-5单宁酶用该方法纯化后,可纯化近20倍,酶活力可回收23.30％.对纯酶作十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,可知黑曲霉N5-5单宁酶为分子质量64.2 kD的单肽链蛋白.纯酶的酶促反应最适温度为45℃,且在25～45℃范围内热稳定性良好;酶促反应最适pH值为5.0,且在pH 5.0～5.5范围内酸碱稳定性良好.另外,反应动力学测定结果表明,该酶对底物没食子酸丙酯的米氏常数Km为0.916 mmol/L,最大反应速率Vmax为0.877 mmol/ (L·min).%The crude tannase from Aspergillus niger N5-5 was purified by hollow fiber membrane ultrafiltration and Sephadex G-150 gel chromatography.The properties of the purified tannase were then determined.The results showed that the tannase from Aspergillus niger N5-5 could be purified about 20 folds with a 23.30％ recovery.The enzyme was a 64.2 kD protein with a single peptide chain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.The optimal temperature for the enzyme was 45 ℃,and it had good thermostability in the range of 25-45 ℃;the optirnal pH value was 5.0,and the enzyme displayed good pH stability in the pH range of 5.0-5.5.In addition,the results of reaction kinetics showed that the Km and vmax values towards the substrate propyl gallate were 0.916 mmol/L and 0.877 mmol/(L·min),respectively.