烟曲霉5’-磷酸二酯酶的分离纯化和酶学性质的研究

庞宗文; 刘洋; 杨舜; 方国辉; 冯旄; 徐勇

doi:10.13386/j.issn1002-0306.2013.20.063

烟曲霉5’-磷酸二酯酶的分离纯化和酶学性质的研究

Study on purification and characterization of 5'-phosphodiesterase from Aspergillus fumigatus

摘要

摘要: 研究了烟曲霉5’-磷酸二酯酶的纯化及性质。烟曲霉固体发酵提取的胞外核酸酶的粗酶液经0.22μm微滤膜过滤、截留分子量为10ku和4ku的膜超滤、硫酸铵沉淀、DEAE-纤维素离子交换层析和DEAE-Sepharose CL-6B层析等方法分离。结果表明,5’-磷酸二酯酶酶液的比活力为1036.76U/mg、纯化倍数为82倍,纯化回收率为7.82%。纯化的酶经SDS-聚丙烯酰胺凝胶电泳检测显示为单一条带,该酶经SDS-聚丙烯酰胺凝胶电泳和Sephadex G-100凝胶层析测定,其相对分子质量约为9.5ku,由单一亚基组成。酶学性质研究结果表明,该酶反应最适pH为5.0,在pH5.0⁹.0之间酶活稳定;最适反应温度为60℃,在低于55℃下酶活较为稳定;该酶的Km为13.60mg/mL,Vmax为0.71mmol（/mg·min）;Fe3+、Fe2+和Zn2+对该酶有抑制作用,而K+对该酶有轻微的促进作用。

Abstract: The 5'-phosphodiesterase was purified from the crude enzyme solution of Aspergillus fumigatus XD9 by microfiltration through 0.22 μ m membrane, ultrafiltration through 10ku and 4ku membrane followed by ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and DEAE-Sepharose CL6B ion exchange chromatography. This protocol improved 82-fold purification with the specific activity of 1036.76U/mg protein and 7.82% recovery of enzyme activity. The purified enzyme showed a single protein band on SDS-PAGE and the molecular mass was estimated to be 9.5ku. The enzyme showed an optimum pH of 5.0 and an optimum temperature of 60℃. The enzyme was stable in the pH range from 5.0 to 9.0 and up to 55℃. Km and Vmax for the purified enzyme were 13.60mg/mL and 0.71mmol/ (mg·min) , respectively, with RNA as substrate. Its activity was activated by K+and strongly inhibited by Fe2+, Fe3+and Zn2+.