耐热对硝基苯磷酸酶的酶学性质

Enzymatic Characteristics of Thermostable p-Nitrophenylphosphatase

通过分离纯化和酶学性质测定 ,确定耐热对硝基苯磷酸酶 (p nitrophenylphosphatase)是金属酶蛋白 ,活性部位含有镁离子 ;该酶是中度耐热蛋白质 ,金属离子有助于提高其热稳定性 .该酶的Km 为 3 0 31mmol L ,Vmax为 313 5 μmol·min-1·mg-1;二级结构中大约有 74 5 %α螺旋 ,2 5 5 % β转角 .在pH 7 0～ 12 0范围内相当稳定 .该酶在SDS溶液中稳定性较差 ,在TritonX 10 0溶液中较为稳定 ,在Tween 2 0溶液中很稳定 .

Thermostable p-nitrophenylphosphatase (TNPPase) had been expressed in E. coli M15, and purified rapidly. It is sensitive to metal chelator and Mg ion is necessary to its activity. TNPPase exhibited a moderate degree of thermostability, and the thermostability of TNPPase increased significantly when treated with 1 mmol/L Mg, Mn, Cu, Ba, Ca, Ni and Zn ions. TNPPase is stable within a wide range of pH (pH 7.0～12.0). It catalyzed the hydrolysis of p-nitrophenylphosphate with a Michaelis constant 3.031 mmol/L and a V max 313.5 μmol·min -1 ·mg -1 . The α-helix percentage and β-turn percentages were 74.5 and 25.5 in the secondary structure of TNPPase, respectively. TNPPase is not stable in SDS solution, but stable in Triton X-100 and Tween 20 solutions.