生物技术进展 ›› 2017, Vol. 7 ›› Issue (4): 310-314.DOI: 10.19586/j.2095-2341.2017.0011

• 研究论文 • 上一篇    下一篇

海马总蛋白提取及其酶解条件优化

胡佳瑶,张梅妍,王振灵,魏娜,高炳淼*   

  1. 海南医学院, 海南省热带药用植物研究开发重点实验室, 海口 571199
  • 收稿日期:2017-02-21 出版日期:2017-07-25 发布日期:2017-03-20
  • 通讯作者: 高炳淼,副教授,研究方向为南药与海洋药物资源开发。E-mail: gaobingmiao@qq.com
  • 作者简介:胡佳瑶,本科生,研究方向为海洋药物。E-mail: 61587122@qq.com。
  • 基金资助:
    国家自然科学基金项目(81560611);海南省大学生创新创业训练计划项目(20150084);海南医学院创新性实验计划项目(hycx2014060)资助。

Extraction  and Optimization of Hydrolysis Conditions of Total Protein from Hippocampus

HU Jiayao, ZHANG Meiyan, WANG Zhenling, WEI Na, GAO Bingmiao   

  1. Hainan Provincial Key Laboratory of Research and Development of Tropical Medicinal Plants, Hainan Medical University, Haikou 571199, China
  • Received:2017-02-21 Online:2017-07-25 Published:2017-03-20

摘要: 为了提取海马总蛋白并确定最佳的酶解条件,将海马粉碎后采用水提法提取海马总蛋白。分别选用木瓜蛋白酶、碱性蛋白酶、中性蛋白酶和胰蛋白酶,在不同酶解pH、时间、温度和E/S条件下进行单因素和正交试验法对海马总蛋白的酶解条件进行优化。利用聚丙烯酰胺凝胶电泳和BCA法来分别检测不同条件下的酶解情况及其蛋白含量,根据蛋白水解度来确定其最佳的酶解条件。实验结果表明:从10 g干海马中提取总蛋白为1.056 g,蛋白浓度为0.106 g/mL。海马总蛋白的最佳水解酶为碱性蛋白酶,其最佳酶解条件为:pH 9,E/S为5%,温度为50℃,时间为3 h,获得海马总蛋白的最大水解度为96.9%。研究获得了海马总蛋白及其最佳酶解条件,并为海马多肽生物活性研究奠定了基础。

关键词: 海马, 蛋白, 酶解条件, 优化

Abstract: In order to extract total protein of Hippocampus and optimize conditions for enzymatic hydrolysis, we extracted the total protein of Hippocampus by the method of water extraction of dried Hippocampus. Papain, alkaline protease, neutral protease and trypsin were used respectively under the different pH, time, temperature and E/S conditions to conduct single factor test, and finally the enzymatic hydrolysis conditions were optimized by orthogonal test. The enzymatic hydrolysis condition and protein content under different conditions were determined by SDS-PAGE and BCA, respectively. The optimal enzymolysis conditions were determined according to the degree of proteolysis shown in the charts. The results showed that 1.056 g total protein was extracted from 10 g dried Hippocampus, and its protein concentration was 0.106 g/mL. The best enzyme was alkaline protease. The best enzymatic conditions were pH 9, E/S 5%, hydrolysis temperature 50℃and hydrolysis time 3 h, and the degree of hydrolysis of the Hippocampus protein was 96.9%. The optimal enzymatic hydrolysis conditions have been determined, and the study laid a certain foundation for the study of biological activity of hippocampal polypeptide.

Key words: Hippocampus, protein, hydrolysis conditions, optimization