摘要
在单因素试验基础上,通过正交试验研究pH值、提取时间和料液比3个因素对巴西松子中蛋白酶活力的影响,得出巴西松子中蛋白酶的最佳提取缓冲液为pH9.0的硼酸-硼砂缓冲液、提取时间为60min、料液比为1:8(m/V);采用(NH4)2SO4沉淀、DEAE-Sepharose FF阴离子交换层析分离纯化巴西松子中的一种蛋白酶,结果表明:纯化后蛋白酶比活力提高到了8.61倍,回收率为21.65%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明:该蛋白酶分子质量为33kD。酶学性质结果表明:该蛋白酶最适pH值为9.0,属碱性蛋白酶;反应的最适温度为50℃;金属离子Mn2+对该蛋白酶活性有强烈的激活作用,而Ca2+、Mg2+和Cu2+对酶活性有抑制作用。
An orthogonal array design was used to optimize conditions for the extraction of protease from Brazilian pine nuts. Boric acid-borate buffer was found to be the best solvent for the extraction of protease, and its optimum pH was 9.0. The optimum extraction time and solid-to-solvent ratio were 60 min and 1：8 （m/V）, respectively. Pure protease was obtained from crude extract using salting out with ammonium sulfate followed by DEAE-Sepharose FF column chromatography. After purification, the specific activity of protease increased 8.61-fold and the activity recovery was 21.65%. As shown by SDS-PAGE, the molecular mass of the enzyme was 33 kD. Enzymatic characterization demonstrated that it was an alkaline and its optimum reaction pH and temperature were 9.0 and 50 ℃, respectively. In addition, Mn2＋ had a strong activating effect on the enzyme, whereas Ca2＋, Mg2＋ and Cu2＋ could inhibit its activity.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2013年第1期239-243,共5页
Food Science
关键词
巴西松子
蛋白酶
分离纯化
酶学性质
Brazilian pine nut
protease
purification
protease activity
