N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质

通过硫酸铵分级沉淀、疏水层析及阴离子交换层析等三步 ,有效地从一菌株NO .2 2 6 2中纯化了N 氨甲酰基 D 氨基酸酰胺水解酶。结果表明 ,酶活性回收约 2 0 %,纯化了 8 4倍。天然PAGE与SDS PAGE分析表明 ,该酶分子为同源四聚体 ,单体分子量约为 3 5kD。酶催化反应的最适pH为 7 7～ 8 0 ,最适温度为 45℃。以N 氨甲酰 DL 丙氨酸为底物时 ,Km =1 3×1 0 - 3 mol L ,Vmax=0 .3 3mol min。二价金属离子Ni2 + 有激活作用 ,Zn2 + 有明显的抑制作用 ,而Co2 + 对酶活无影响。该酶N 末端 8个氨基酸残基依次为TRQKILAF。

Purification and Some Properties of D-Carbamoylase

A D-Carbamoylase produced by a strain NO. 2262 was purified to electrophoretic homogeneity with the recovery of 20% activity and the purification factor of 8 fold by three steps including (NH4)2SO4 fractionation, hydrophobic column and pre-packed Hitrap Q HR. It is indicated from the results of nativ-PAGE and SDS-PAGE analysis that the enzyme could be a homogeneous tetramer consisting of four 35 kD subunits. In addition, its optimal pH and optimal temperature are 8.0 and 45 degrees C respectively. The basic kinetic parameters of the enzyme are Km = 1.3 x 10(-3) mol/L and Vmax = 0.33 mumol/min with N-carbamyl-DL-Alanine as the substrate. The effect of bivalent metal ions on the enzyme was showed that Ni2+ could be as an activator, Zn2+ as a powerful inhibitor, while Co2+ had no any influence at all. Its N-terminal sequence is TRQKILAF in turn.