重组大肠杆菌热稳定性过氧化氢酶的纯化及性质研究  

Purification and Properties of Thermostable Catalase in Engineered E.coli

作　　者：王凡强[1] 王正祥[1] 邵蔚蓝[1] 刘吉泉[1] 徐成勇[1] 诸葛健[1] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,无锡214036

出　　处：《微生物学报》2002年第3期348-353,共6页Acta Microbiologica Sinica

摘　　要：将产热稳定性过氧化氢酶的重组大肠杆菌培养后菌体破碎得到的粗酶液经热处理、硫酸铵分级沉淀、DEAE SephadexA 5 0离子交换层析、HiPrep 1 6 1 0Phenyl疏水作用层析、Su perdex2 0 0HR 1 0 3 0凝胶层析提纯后得到电泳纯的酶 ,比酶活达到 1 5 6 2 9U mg。此酶的最适温度为 70℃ ,最适pH7 0 ,在 6 0℃保温 6 0min酶活力基本不变 ,在pH3～ 8的范围内比较稳定。此酶的Km 和Vmax分别为 7 75mmol L和 2 7 8mmol·min- 1·mg- 1。 1mmol L的Zn2 + 、Ba2 + 、Mn2 +可使该酶完全失活 ,KCN、NaN3 、Na2 S2 O4 、巯基乙醇对酶活力有抑制作用 ,5 0mmolA thermostable catalase in engineered bacterium E.coli was purified to electrophoretic homogenenity by heat treatment, ammonium sulfate fractionation precipitation, DEAE-A50 ion exchange chromatography, HiPrep*R16/10 Phenyl hydrophobic interaction chromatography and Superdex200 HR 10/30 size exclusion chromatography with 187^2-fold purification and 9^8% recovery. The optimum reaction temperature and pH of this recombinant catalase were 70℃ and 7^0 respectively. The catalase is stable below 60℃ and at pH range 3～8. The residual activity of the catalase was about 60% after treated at 70℃ for 60 minutes and 80℃ for 10 minutes. The apprant K-m and V-{max} value of the catalase were 7^75 mmol/L and 27^8mmol5min+{-1}5mg+{-1} respectively. The affects of some metal ions and compounds on this enzyme were shown. Zn+{2+}, Ba+{2+},Mn+{2+} of 1mmol/L could completely inactivate the enzyme, EDTA of 50mmol/L had no affect on activity.

关 键 词：重组大肠杆菌 热稳定性过氧化氢酶 纯化 性质 

分 类 号：Q55[生物学—生物化学]