粪肠球菌SK32.001精氨酸脱亚胺酶的分离纯化及酶学性质研究

Purification and characterization of an arginine deiminase from Enterococcus faecalis SK32.001 producing citrulline

对Enterococcus faecalis SK32.001所产的精氨酸脱亚胺酶（ADI）进行分离纯化并对其酶学性质进行研究。实验结果表明,通过细胞破碎,硫酸铵沉淀,Hi Prep Q FF 16/10阴离子交换层析,Sephadex G-75等纯化方法获得电泳纯精氨酸脱亚胺酶,分子量约为42ku,催化最适温度和p H分别为50℃和6.5,在30~40℃和p H5.5~7.5时较稳定。不同浓度的Zn2＋对酶活性影响较大。1mmol/L的Zn^2＋和10mmol/L的Co^2＋、Ca^2＋、Mg^2＋对酶活有较大的促进作用,10mmol/L的Cu^2＋对酶的抑制作用最强。精氨酸脱亚胺酶在最适反应条件测定其米氏常数为1.33mmol/L,最大反应速度为2.41μmol/min。

The crude enzyme of the arginine deiminase（ADI） from Enterococcus faecalis SK32.001 had been purified and the basic properties of ADI were investigated. The result of SDS-PAGE showed that ADI was purified to homogeneity level by fraction precipitated with ammonium sulfate,HiPrep Q FF 16/10 and Sephadex G- 75 gel filtration chromatography. The molecular mass of the purified ADI was estimated to be about 42ku. The optimum temperature and pH of the purified ADI were 50% and 6.5,respectively. ADI activity kept stability under the temperature of 30-40℃ and at pH range of 5.5-7.5. Among the metal irons,different concentrations of Zn^2＋ could obviously affect the activity, lmmol/L Zn^2＋, 10mmol/L Co^2＋, 10mmol/L Ca^2＋ and 10mmol/L Mg^2＋ were the effective promoter while 10mmol/L Cu^2＋ had poor improvement in the activity. The Michaelis constant was 1.33mmol/L and the maximum velocity was 2.41μmol/min.