山黧豆种子萌发特异性蛋白酶的分离纯化及酶学性质研究

doi:10.11733/j.issn.1007-0435.2018.06.030

草地学报 ›› 2018, Vol. 26 ›› Issue (6): 1497-1502.DOI: 10.11733/j.issn.1007-0435.2018.06.030

山黧豆种子萌发特异性蛋白酶的分离纯化及酶学性质研究

曲瑞红, 刘凤娟, 毕春晓, 宋瑶瑶, 胡鑫, 徐全乐

西北农林科技大学生命科学学院, 陕西杨凌 712100

收稿日期:2018-09-10 修回日期:2018-12-12 出版日期:2018-12-15 发布日期:2019-01-28
通讯作者: 徐全乐
作者简介:曲瑞红(1993-),女,硕士研究生,主要从事山黧豆抗逆分子生物学研究,E-mail:807222242@qq.com
基金资助:
中国博士后科学基金面上资助（2016M590975）；陕西省博士后科研项目（2016BSHEDZZ119）；中央高校基本科研业务费专项资金重点研究基地建设项目（lzujbky-2018-kb05）资助

Purification of Proteases from Germinated Seeds of Lathyrus sativus and Its Enzymatic Properties Study

QU Rui-hong, LIU Feng-juan, BI Chun-xiao, SONG Yao-yao, HU Xin, XU Quan-le

College of Life Sciences, Northwest A & F University, Yangling, Shaanxi Province 712100, China

Received:2018-09-10 Revised:2018-12-12 Online:2018-12-15 Published:2019-01-28

摘要/Abstract

摘要： 蛋白酶在种子萌发过程中参与了储藏蛋白水解等多种生理过程，为植物的生长发育提供氮源并在种子萌发的生化机制中有重要作用。本研究采用明胶酶谱法检测了山黧豆种子在不同萌发时间的蛋白酶表达情况。利用盐析、离子交换层析等方法从萌发6d的山黧豆种子中分离纯化了蛋白酶并对其基本酶学性质进行了研究。结果表明，山黧豆种子萌发特异性蛋白酶造成了种子萌发过程中高分子质量蛋白质的水解和较低分子质量蛋白质的积累。对纯化蛋白酶进行总蛋白含量、酶活力、酶比活力等检测表明，山黧豆蛋白酶的纯化倍数为6.58，蛋白得率为7.97%。山黧豆蛋白酶对金属离子的耐受性较高，添加Fe³⁺可以引起蛋白酶活性的显著增加。此外，该蛋白酶对血红蛋白和明胶的反应活性最高，其最适反应pH和温度分别为pH7和60℃。本研究为进一步研究山黧豆种子萌发过程中蛋白质水解与其内源毒素β-N-草酰-L-α，β-二氨基丙酸积累的相互关系奠定了基础。

关键词: 山黧豆, 萌发种子, 蛋白酶, 分离纯化

Abstract: Proteases play important roles in hydrolysis of storage protein during seed germination to provide nitrogen for plant development. In this study,the expression pattern of proteases in germinated seeds with different time of Lathyrus sativus was analyzed via SDS-gelatin-PAGE. And then,the germinating specific proteases were purified via salting out,ion exchange chromatography of DE52 successively from 6-day germinated seed. The results suggested that the germinating specific proteases contribute to the hydrolysis of storage proteins with high molecular weight and the accumulation of proteins with relatively lower molecular weight. The purification fold was 6.58 and yield was 7.97%. The enzymatic activities determination suggested that the purified proteases displayed higher tolerance to different metal ion but was activated remarkably via Fe³⁺. Moreover,the purified proteases displayed more activities to hemoglobin and gelatin,and the optimal temperature 60℃ and pH 7.0,respectively. This study will lay a solid foundation for the further investigation of the relationship between storage protein hydrolysis and accumulation of toxic β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid) during seed germination of Lathyrus sativus.

Key words: Lathyrus sativus, Germinated seeds, Protease, Purification

中图分类号:

曲瑞红, 刘凤娟, 毕春晓, 宋瑶瑶, 胡鑫, 徐全乐. 山黧豆种子萌发特异性蛋白酶的分离纯化及酶学性质研究[J]. 草地学报, 2018, 26(6): 1497-1502.

QU Rui-hong, LIU Feng-juan, BI Chun-xiao, SONG Yao-yao, HU Xin, XU Quan-le. Purification of Proteases from Germinated Seeds of Lathyrus sativus and Its Enzymatic Properties Study[J]. Acta Agrestia Sinica, 2018, 26(6): 1497-1502.

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山黧豆种子萌发特异性蛋白酶的分离纯化及酶学性质研究

Purification of Proteases from Germinated Seeds of Lathyrus sativus and Its Enzymatic Properties Study

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