猪肺血管紧张素转化酶的提取与纯化

Extraction and purification of angiotensin converting enzyme from fresh hog lung

【目的】研究猪肺血管紧张素转化酶(ACE)的提纯方法,为其生产利用提供技术支撑。【方法】以新鲜猪肺组织为试验材料,匀浆离心后经硫酸铵分级沉淀透析,然后采用离子交换—超滤离心联合法对猪肺ACE粗提物进行纯化,测定ACE的分子量、酶活力、纯化倍数及酶活力回收率。【结果】通过离子交换—超滤离心联合法分离纯化猪肺ACE,可得到相对分子量182 kDa、酶比活力1.2476 U/mg的电泳级纯品ACE,其纯化倍数达357.31倍,酶活力回收率达12.28%。ACE催化反应动力学米氏常数(Km)为0.849mmol/L,最大反应速率(Vmax)为9.775nmol/min。【结论】采用离子交换—超滤离心联合法纯化猪肺ACE能够极大缩短分离与纯化时间,且获得较高的纯化倍数和酶活力回收率,是猪肺ACE的高效纯化方法。

[Objective]Extraction and purification of angiotensin converting enzyme （ACE） was studied to provide techni- cal support for its production and utilization. [Method]ACE crude extracts were purified by DEAE-Sepharose FF anion ex- change and ultrafihration after the fresh lung homogenate was centrifuged, precipitated and dialyzed using ammonium sulfate. ACE molecular weight, specific activity, purification fold and activity recovery were determined. [Result]Lung ACE obtained by SDS-PAGE showed ACE-pure and molecular mass was 182kDa. The ACE was purified to 357.31-fold with specific activity of 1.2476 U/mg and activity recovery of 12.28%. The Michaelis-constant of the ACE was 0.849 mmol]L and Vmax was 9.775 nmol/min. [Conclusion]The ion exchange-uhrafiltration is an efficient purification method for lung ACE, as it can greatly shorten the separation and purification time, and get a higher purification fold and activity recovery.