摘要
对产自乳酸菌Enterococcuse faecalis TN-9的蛋白酶,进行了硫酸铵沉淀,DEAE-Sephadex A-25以及DEAE Cellulofine A-500离子交换层析的3步纯化和特性研究。纯化酶Native PAGE显示1条蛋白带。SDS-PAGE和凝胶层析分子量分别为30 ku及69 ku。纯化酶最适作用温度为30℃,最适作用pH为7.5～8.0,在pH 6.0～9.5和45℃以下条件下稳定,在0℃下显示了6.1%的相对活性,60℃以上热处理完全失去酶活。该酶被EDTA-2Na,Hg^(2+)、Cu^(2+)、Ni^(2+)、Ag^(2+)、Co^(2+)及Pepstatin A不完全抑制。Zn^(2+)对蛋白酶具有明显的激活作用。纯化酶作用于偶氮酪蛋白的K_(rs)和V_(max)分别为0.098%和72 mg/(h·mg)。该酶为N末端VGSEVTLKNS的明胶酶(Gelatinase)的一种,性质属于低温蛋白酶。
Protease from lactic acid bacterium Enterococcus faecalis TN-9 was purified with three steps, ammonium sulfate precipitation, DEAE-Sephadex A-25, and DEAE Cellulofine A-500 ion exchange chromatography and studied its properties. Native PAGE analysis of the purified enzyme showed a single protein band. The molecular weight was 30 ku by SDS-PAGE analysis and 69 ku by get chromatography analysis respectively. The optimal reaction temperature and pH of the enzyme were at 30℃ and 7.5 - 8.0 respectively. It was stable at pH 6.0 - 9.5 and 45 ℃. Under 0℃ it showed 6.1% of relative activity. Heat treatment above 60℃ it totally lost its activity. The enzyme was incompletely inhibited by EDTA-2Na, Hg^2＋ , Cu^2＋ ,Ni^2＋ , Ag^2＋ , Co^2＋ , and Pepstatin. Zn^2＋ had obvious activation to the protease. Km, and Vmax of the purified enzyme were 0. 098% and 72 mg/（ h . mag） respectively. The enzyme was one of gelatinase with N-terminal sequence of VGSEVTLKNS. Its property belonged to cold-adapted protease.
出处
《微生物学杂志》
CAS
CSCD
2009年第5期20-25,共6页
Journal of Microbiology
