粪肠球菌精氨酸脱亚胺酶酶学性质研究

The Research of Enzymology Characterization about Arginine Deiminase from Enterococcus faecalis

摘要:

经硫酸铵分级沉淀、Q-Sepharose Fast Flow阴离子交换层析、SephadexG-75凝胶柱层析从NJ402自溶细胞超声破碎液中提纯得到精氨酸脱亚胺酶(ADI), 纯化倍数为34.5, 活力回收率为31.4％, 经SDS-PAGE以及Native-PAGE测定结果表明, ADI亚基分子量约为46 kD, 该酶非变性情况下的分子量约为190 kD左右, 该酶为同四聚体结构。酶学性质研究结果表明：ADI催化最适温度和最适pH分别为50℃和6.5, 在45℃以下和pH 5~8之间有很好的稳定性。ADI是L-型脱亚胺酶, 具有严格的光学选择性, 适当浓度的Mn2+、Mg2+、Co2+对ADI催化活力的促进作用较大, 高浓度的Zn2+和Co2+对酶有一定程度的抑制作用, L-瓜氨酸对酶无抑制作用而L-鸟氨酸却表现出较强的抑制作用。ADI在最佳催化条件下作用于L-精氨酸的米氏常数为3.2686 mmol/L, 最大反应速度为2.44 μmol/min。

Abstract:

Arginine Deiminase(ADI)was purified to homogeneity using ammonium sulfate precipitation, Q-Sepharose Fast Flow anion exchange chromatography and SephadexG-75 gel filtration chromatography. This purification protocol resulted in a 34.5-fold purification of ADI with 31.4% final yield. A molecular weight of about 190 kD determined by native gradient polyacrylamide gel electrophoresis. The enzyme has only one kind of 46 kD subunit determined by SDS-PAGE. Combining the results from the two kinds of electrophoresis, the authors deduce that the enzyme may be a tetramer. The optimum pH and temperature for lipolytic activity of ADI was pH 6.5 and 50℃, respectively. It was extremely stable at 45℃ and retained 97.9% of its original activity for 30 min. The stability declined rapidly as soon as the temperature rose over 50℃. ADI was highly stable in the pH range from pH 5-8. ADI acted on L-arginine but not on D-arginine. ADI catabolism was dependent on metal ions. At their adequate concentration, Mn2+, Mg2+ and Co2+ were the effective promoter, while superfluous Zn2+and Co2+ inhibited ADI activity. L-citrulline did not act on ADI, but L-ornithine inhibited ADI activity. The degradation of L-arginine with ADI catalysis was according to simple Michaelis-Menten equation. The Michaelis constant was 3.2686 mmol/L and the maximum velocity was 2.44 μmol/min.