N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质  

Purification and Some Properties of D-Carbamoylase

作　　者：袁静明[1] 石亚伟[1] 杨秀清[1] 连惠勇[2] 齐延红[2] 

机构地区：[1]山西大学生物工程中心,太原030006 [2]山西省生物研究所,太原030006

出　　处：《微生物学报》2002年第1期88-92,共5页Acta Microbiologica Sinica

基　　金：山西省重点行业科技发展项目 ( 9832 2 5 )

摘　　要：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。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 (NH 4) 2SO 4 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 35kD subunits. In addition, its optimal pH and optimal temperature are 8.0 and 45℃ respectively. The basic kinetic parameters of the enzyme are K m=1.3×10 -3mol/L and Vmax 0.33 μmol/min with N-carbamyl-DL-Alanine as the substrate. The effect of bivalent metal ions on the enzyme was showed that Ni 2+ could be as an activator, Zn 2+ as a powerful inhibitor, while Co 2+ had no any influence at all. Its N-terminal sequence is TRQKILAF in turn.

关 键 词：菌株NO.2262 N-氨甲酰基-D-氨基酸酰胺水解酶 纯化 性质 D-氨基酸 

分 类 号：Q556[生物学—生物化学]