巴西松子中蛋白酶的分离纯化及酶学性质

食品科学 ›› 2013, Vol. 34 ›› Issue (1): 239-243.

巴西松子中蛋白酶的分离纯化及酶学性质

肖丽，应铁进*，蔡路昀，韩晓旭

浙江大学生物系统工程与食品科学学院

收稿日期:2011-11-03 修回日期:2012-12-16 出版日期:2013-01-15 发布日期:2013-01-07
通讯作者: 肖丽 E-mail:372015873@qq.com

Purification and Properties of Protease from Brazilian Pine (Araucaria angustifolia (Bert) O. Ktze) Nuts

Received:2011-11-03 Revised:2012-12-16 Online:2013-01-15 Published:2013-01-07

摘要/Abstract

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。

关键词: 巴西松子, 蛋白酶, 分离纯化, 酶学性质

Abstract: 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.

Key words: Brazilian pine nut, protease, purification, protease activity

中图分类号:

肖?丽，应铁进*，蔡路昀，韩晓旭. 巴西松子中蛋白酶的分离纯化及酶学性质[J]. 食品科学, 2013, 34(1): 239-243.

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