纳豆激酶NKⅡ分离纯化及其酶促动力学研究

Separation, purification and enzymatic kinetics of nattokinase NKⅡ

该实验主要采用疏水柱层析分离纯化纳豆激酶NKII,聚丙烯酰胺凝胶电泳(SDS-PAGE)结果为单一蛋白条带,分子量为28 ku,酶活回收率56.51%,纯化倍数17.90,比活力达48 407.77 IU/mg。采用纤维蛋白平板法及显色底物法测定酶活力,结果表明,两种方法相关性高。以S-2251为底物的动力学研究表明,米氏常数Km值为0.379 6 mmol/L,最大反应速度Vm值为0.059 8 mmol/(L·min),Ca2+、Mg2+对酶活稳定性效果明显。

The nattokinase NKII was separated and purified by hydrophobic chromatography. The purified enzyme showed a single protein band in the SDS-PAGE and the molecular weight was 28 ku. The enzyme overall yield was 56.51%, the purification factor was 17.90 and the specific activity of NKII was 48 407.77 IU/mg. The activity of NK was detected by the methods of fibrin plate and chromogenic substrate method. Results proved that two methods had high correlation. When S-2251 was used as substance, the kinetic parameter Km was 0.379 6 mmol/L and Vm was 0.059 8 mmol/（L-min）, Ca2＋ and Mg2＋ showed significant effect on enzyme activity stability.