类蛋白反应修饰海地瓜酶解物及ACE抑制肽的制备

doi:10.7503/cjcu20130951

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (5): 965.doi: 10.7503/cjcu20130951

类蛋白反应修饰海地瓜酶解物及ACE抑制肽的制备

沈晴晴, 曾名湧, 赵元晖()

中国海洋大学食品科学与工程学院, 青岛266003

收稿日期:2013-09-26 出版日期:2014-05-10 发布日期:2014-03-26
作者简介:联系人简介: 赵元晖, 男, 博士, 讲师, 主要从事海洋生物功能活性物质的研究. E-mail:zhaoyuanhui@ouc.edu.cn
基金资助:
山东省自然科学基金(批准号: ZR2010CQ001)、国家自然科学基金(批准号: 31000830)及长江学者和创新团队发展计划(批准号: IRT1188)资助

Modification of Acaudina Molpadioides Hydrolysates by Plastein Reaction and Preparation of ACE Inhibitory Peptides^†

SHEN Qingqing, ZENG Mingyong, ZHAO Yuanhui^*()

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

Received:2013-09-26 Online:2014-05-10 Published:2014-03-26
Contact: ZHAO Yuanhui E-mail:zhaoyuanhui@ouc.edu.cn
Supported by:
† Supported by the Natural Science Foundation of Shandong Province(No.ZR2010CQ001), the National Natural Science Foundation of China(No.31000830) and the Program for Changjiang Scholars and Innovative Research Team in University of China(No.IRT1188)

摘要/Abstract

摘要：

采用类蛋白反应修饰海地瓜体壁蛋白酶解物, 制得修饰肽的血管紧张素转换酶(ACE)抑制活性与原酶解液的活性相比显著提高; 模拟消化实验结果表明, 修饰肽与胃肠蛋白酶作用后可增强其降压活性; 细胞毒性实验结果表明, 样品浓度在低于10 mg/mL时对犬肾细胞(MDCK)无毒性, 且低浓度可以促进细胞生长. 采用超滤、 Sephadex G-15凝胶柱层析和RP-HPLC等分离技术纯化修饰产物, 得到高活性ACE抑制肽(IC₅₀值为3.67 μmol/L), 序列结构为NQNFVQYTTNT. 紫外光谱分析结果表明, 修饰肽与ACE 结合可引起吸光度变化. 用SYBYL软件对抑制肽与ACE活性位点进行模拟对接, 发现该抑制肽能与ACE很好地结合.

关键词: 海地瓜, 类蛋白反应修饰, 分离纯化, ACE抑制肽

Abstract:

The effect of modification by plastein reaction on the angiotensin converting enzyme(ACE) inhibitory activity of Acaudina molpadioides hydrolysates and its peptides was researched to show that the ACE inhibitory activity of modified peptide decreased definitely, compared with the activity of original hydrolysates. The stability of the modified peptides to simulated gastrointestinal digestion was tested in vitro. The result showed that the ACE inhibitory activities of the peptides could be enhanced by digestive enzyme. Toxicity test showed no toxic to the MDCK cells while concentration was less than 10 mg/mL, and low concentration could promote the cells’ growth. A novel ACE inhibitory peptide was isolated from modified product using different methods including ultrafiltration, Sephadex G-15 gel filtration and RP-HPLC. The purified ACE inhibitory peptide was sequenced as NQNFVQYTTNT with an IC₅₀ value of 3.67 μmol/L. The accurate sites of ACE that the peptide bind to were determined through the computer assisted software SYBYL, and UV spectra analysis proved that some changes could be observed in the binding of ACE and peptides.

Key words: Acaudina molpadioides, Plastein reaction, ACE inhibitory peptides, Isolation and purification

中图分类号:

O629.72

TrendMD:

沈晴晴, 曾名湧, 赵元晖. 类蛋白反应修饰海地瓜酶解物及ACE抑制肽的制备. 高等学校化学学报, 2014, 35(5): 965.

SHEN Qingqing, ZENG Mingyong, ZHAO Yuanhui. Modification of Acaudina Molpadioides Hydrolysates by Plastein Reaction and Preparation of ACE Inhibitory Peptides^†. Chem. J. Chinese Universities, 2014, 35(5): 965.

图/表 6

Fig.1 Isolation and purification of modified Acaudina molpadioides peptides by plastein reaction and the IC50 values of different components^(A) a—h are 8 groups of modified peptides isolated by Sephadex G-15 chromatograph; (A') IC50 values of a—h components(P<0.01); (B) component d is further purified by RP-HPLC on Zorbax SB-C18 column(5 μm, 9.4 mm×250 mm); (B') IC50 values of components d1—d7(P<0.05).

Fig.2 Purity and identification of modified peptides and the sequence profile of purified fraction d1

Fig.3 ACE-binding peptide by docking algorithm of computer aided molecular design

Fig.4 UV-Vis absorption analysis of the ACE-binding peptide(a), peptide(b) and ACE(c)

Fig.5 Cytotoxicity experiments of modified peptide on MDCK cells

Table 1 Impact of gastrointestinal proteases of modified hydrolysates by plastein reaction on ACE inhibitory activity and free amino groups

Sample	Free amino group/(mmol·L^-1)	IC₅₀/(μg·mL^-1)
Control	3.06±0.03	122±7.2^a
Pepsin	3.32±0.06	56±8.4^b
Pepsin+Chymotrypsin	3.49±0.05	66±6.7^b^,^c
Pepsin+Chymotrypsin+Trypsin	3.54±0.03	74±5.7^c

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类蛋白反应修饰海地瓜酶解物及ACE抑制肽的制备

Modification of Acaudina Molpadioides Hydrolysates by Plastein Reaction and Preparation of ACE Inhibitory Peptides^†

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类蛋白反应修饰海地瓜酶解物及ACE抑制肽的制备

Modification of Acaudina Molpadioides Hydrolysates by Plastein Reaction and Preparation of ACE Inhibitory Peptides†

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Modification of Acaudina Molpadioides Hydrolysates by Plastein Reaction and Preparation of ACE Inhibitory Peptides^†