重组大肠杆菌热稳定性过氧化氢酶的纯化及性质研究

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\|Sephadex A\|50离子交换层析、HiPrep16/10 Phenyl疏水作用层析、Superdex200 HR 10/30凝胶层析提纯后得到电泳纯的酶，比酶活达到15629U/mg。此酶的最适温度为70℃，最适pH70，在60℃保温60min酶活力基本不变，在pH3～8的范围内比较稳定。此酶的Km和Vmax分别为775mmol/L和278mmol\5min\+\{-1\}·mg-1。1mmol/L的Zn2+、Ba2+、Mn2+可使该酶完全失活，KCN、NaN\-3、Na\-2S\-2O\-4、巯基乙醇对酶活力有抑制作用，50mmol/L的EDTA不影响酶活性。

Purification and Properties of Thermostable Catalase in Engineered E. coli

A 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 16/10 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 degrees C and 7.0 respectively. The catalase is stable below 60 degrees C and at pH range 3-8. The residual activity of the catalase was about 60% after treated at 70 degrees C for 60 minutes and 80 degrees C for 10 minutes. The apprant Km and Vmax value of the catalase were 7.75 mmol/L and 27.8 mmol.min-1.mg-1 respectively. The affects of some metal ions and compounds on this enzyme were shown. Zn2+, Ba2+, Mn2+ of 1 mmol/L could completely inactivate the enzyme, EDTA of 50 mmol/L had no affect on activity.