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

作　　者：李成付[1] 李凯[1] 李加友[1] 焦庆才[1] 刘茜[1] 易立涛[1] 

机构地区：[1]南京大学生命科学学院医药生物技术国家重点实验室,南京210093

出　　处：《微生物学通报》2008年第6期846-850,共5页Microbiology China

基　　金：国家技术创新基金(No.02CJ-13-01-16)

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46kD,该酶非变性情况下的分子量约为190kD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pH5～8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。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 Darginine. ADI catabolism was dependent on metal ions. At their adequate concentration, Mn^2＋, Mg^2＋ and Co^2＋ were the effective promoter, while superfluous Zn^2＋and Co^2＋ inhibited ADI activity. L-citmlline 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.

关 键 词：粪肠球菌NJ402 精氨酸脱亚胺酶 分离纯化 酶学性质 

分 类 号：Q93[生物学—微生物学]