刺参2 种天冬氨酸蛋白酶的酶学性质及其对自溶的影响

doi:10.7506/spkx1002-6630-201814015

食品科学 ›› 2018, Vol. 39 ›› Issue (14): 99-105.doi: 10.7506/spkx1002-6630-201814015

刺参2 种天冬氨酸蛋白酶的酶学性质及其对自溶的影响

王玲1,2，年益莹1,2，薛鹏1,2，季晓彤1,2，崔钰婷1,2，张公亮1，侯红漫1，孙黎明1,2,*

（1.大连工业大学食品学院，辽宁?大连 116034；2.国家海洋食品工程技术研究中心，辽宁?大连 116034）

出版日期:2018-07-25 发布日期:2018-07-16
基金资助:
国家自然科学基金青年科学基金项目（31201300）；辽宁省优秀人才项目（LJQ2012048）

Two Aspartic Proteases in Sea Cucumber (Stichopus japonicus): Enzymatic Properties and Effect on Autolysis

WANG Ling1,2, NIAN Yiying1,2, XUE Peng1,2, JI Xiaotong1,2, CUI Yuting1,2, ZHANG Gongliang1, HOU Hongman1, SUN Liming1,2,*

(1. School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China; 2. National Engineering Research Center of Seafood, Dalian 116034, China)

Online:2018-07-25 Published:2018-07-16

摘要/Abstract

摘要： 对刺参体内2?种天冬氨酸蛋白酶——组织蛋白酶D（cathepsin?D，Cat?D）和组织蛋白酶E（Cat?E）的酶学性质进行探讨，并考察两者可能在刺参自溶中的参与作用。先用Tris-HCl缓冲溶液提取刺参体壁中的粗酶，采用特异性荧光底物法测定Cat?D和Cat?E的酶学性质。结果表明，Cat?D和Cat?E的最适pH值分别为5.0和4.0，分别在60?℃和40?℃呈现最大酶活性，两者在20～40?℃活性均较为稳定。Zn2＋、Cu2＋、Fe2＋、Fe3＋、Mn2＋可抑制Cat?D的活性，抑制率分别为86%、76.3%、29.2%、56.5%和48.5%。Fe3＋、Fe2＋、Cu2＋可抑制Cat?E的活力，抑制率分别为99.1%、82.2%和28.6%。Pepstatin?A、Z-Leu-Leu-Leu-H、苯甲基磺酸氟、1,10-菲啰啉能够抑制两者活性，抑制率分别约为98%～99%、65%～78%、30%～35%、19%～23%。二硫苏糖醇、L-Cys和乙二胺四乙酸则可将两者活性分别提升30.4%～31.1%、7.58%～9.64%、6.6%～7.9%。结果表明，刺参Cat?D和Cat?E为2?种具有一定金属离子敏感性和依赖性的天冬氨酸蛋白酶，其活性中心有丝氨酸和半胱氨酸参与其活性调节，且两者很有可能参与刺参自溶过程中蛋白质的降解。

关键词: 刺参, 天冬氨酸蛋白酶, 组织蛋白酶D, 组织蛋白酶E, 酶学性质, 自溶

Abstract: In this paper, the enzymatic properties of two aspartic proteases, cathepsin D (Cat D) and cathepsin E (Cat E), in sea cucumber were studied, and their involvement in the autolysis of sea cucumber was also investigated. Crude enzyme was extracted from the body wall of sea cucumbers with Tris-HCl buffer (pH 7.0). The specific substrate fluorescence method was used to determine the enzymatic properties of Cat D and Cat E. Results showed that Cat D and Cat E exhibited maximum activities at pH 5.0 and 60 ℃, and pH 4.0 and 40 ℃, respectively. Their activities kept stable at 20–40 ℃. Zn2+, Cu2+, Fe3+, and Mn2+ inhibited Cat D activity by 86%, 76.3%, 29.2%, 56.5% and 48.5%, respectively. Fe3+, Fe2+ and Cu2+ inhibited Cat E by 99.1%, 82.2% and 28.6%, respectively. Pepstatin A, Z-LLL, PMSF, and 1,10-phenanthroline inhibited the two proteases by 98%–99%, 65%–78%, 30%–35%, and 19%–23%, respectively. L-Cys, DDT and EDTA increased their activities by 30.4%–31.1%, 7.58%–9.64%, and 6.6%–7.9%, respectively. These results suggested that Cat D and Cat E were sensitive to metal ions and they were metal ion-dependent aspartic proteases, with cysteine and serine around the active center involved in modulating the enzymatic activities. Moreover, they might be involved in protein degradation during the autolysis of sea cucumber.

Key words: Stichopus japonicus, aspartic proteases, cathepsin D, cathepsin E, enzymatic property, autolysis

中图分类号:

TS254.4

王玲，年益莹，薛鹏，季晓彤，崔钰婷，张公亮，侯红漫，孙黎明. 刺参2 种天冬氨酸蛋白酶的酶学性质及其对自溶的影响[J]. 食品科学, 2018, 39(14): 99-105.

WANG Ling, NIAN Yiying, XUE Peng, JI Xiaotong, CUI Yuting, ZHANG Gongliang HOU Hongman SUN Liming. Two Aspartic Proteases in Sea Cucumber (Stichopus japonicus): Enzymatic Properties and Effect on Autolysis[J]. FOOD SCIENCE, 2018, 39(14): 99-105.

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刺参2 种天冬氨酸蛋白酶的酶学性质及其对自溶的影响

Two Aspartic Proteases in Sea Cucumber (Stichopus japonicus): Enzymatic Properties and Effect on Autolysis

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