大黄鱼肌原纤维结合型丝氨酸蛋白酶抑制剂的分离纯化及性质分析

doi:10.7506/spkx1002-6630-20250427-225

食品科学 ›› 2025, Vol. 46 ›› Issue (24): 90-97.doi: 10.7506/spkx1002-6630-20250427-225

• 基础研究 • 上一篇

大黄鱼肌原纤维结合型丝氨酸蛋白酶抑制剂的分离纯化及性质分析

霍卫森，张凌晶，陈玉磊，孙乐常，翁凌，黄建联，曹敏杰

（1.集美大学海洋食品与生物工程学院，福建厦门 361021；2.福建省冷冻调理水产品加工重点实验室，福建厦门 361028；3.安井食品集团股份有限公司，福建厦门 361028）

发布日期:2025-12-26
基金资助:
“十三五”国家重点研发计划“蓝色粮仓科技创新”重点专项（2018YFD0901004）；福建省冷冻调理水产品加工重点实验室开放课题（FJKLRCAPP2024-01）

Purification and Characterization of Myofibril-Bound Serine Proteinase Inhibitor from Larimichthys crocea

HUO Weisen, ZHANG Lingjing, CHEN Yulei, SUN Lechang, WENG Ling, HUANG Jianlian, CAO Minjie

(1. College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China; 2. Key Laboratory of Refrigeration and Conditioning Aquatic Products Processing, Fujian Province, Xiamen 361028, China; 3. Anjoy Foods Group Co., Ltd., Xiamen 361028, China)

Published:2025-12-26

摘要/Abstract

摘要： 目的：探究大黄鱼（Larimichthys crocea）肌原纤维结合型丝氨酸蛋白酶抑制剂（myofibril-bound serine proteinase inhibitor，MBSPI）的结构特性及其对肌原纤维结合型丝氨酸蛋白酶（myofibril-bound serine proteinase，MBSP）的抑制机理。方法：采用硫酸铵分级沉淀、DEAE-Sepharose和SP-Sepharose柱层析等从大黄鱼背部肌肉中纯化得到MBSPI；通过圆二色光谱、内源性荧光光谱分析MBSPI的结构特性；利用抑制动力学分析MBSPI对MBSP的抑制机理。结果：MBSPI是分子质量为55 kDa的单体蛋白，可与大鼠抗白姑鱼MBSPI多克隆抗体发生特异性免疫反应，一级结构与葡萄糖-6-磷酸异构酶高度一致。MBSPI主要由α-螺旋构成，热变性温度为53 ℃。当温度高于53 ℃时，MBSPI二级结构发生显著变化，表现为α-螺旋含量减少，无规卷曲和反平行β-折叠含量增加。MBSPI对MBSP呈竞争性抑制，半抑制浓度为0.03 µmol/L。MBSPI可有效抑制MBSP对肌原纤维蛋白中肌球蛋白重链、肌动蛋白和原肌球蛋白的降解。综上，大黄鱼肌肉中内源性抑制剂MBSPI可显著抑制由MBSP引起的肌原纤维蛋白自身降解，本研究可为鱼糜制品生产过程中水溶性蛋白的利用提供新思路。

关键词: 大黄鱼；肌原纤维结合型丝氨酸蛋白酶；抑制剂；葡萄糖-6-磷酸异构酶；抑制机理

Abstract: Objective: To investigate the structural characteristics of a myofibril-bound serine proteinase inhibitor (MBSPI) from Larimichthys crocea and to elucidate its inhibitory mechanism against MBSP. Methods: MBSPI was purified from the skeletal muscle of L. crocea using ammonium sulfate fractionation, DEAE-Sepharose, and SP-Sepharose ion-exchange chromatography. Its structural characteristics were analyzed by circular dichroism (CD) spectroscopy and intrinsic fluorescence spectroscopy. Inhibition kinetics was employed to determine the inhibitory mechanism. Results: MBSPI was identified as a monomeric protein with a molecular mass of 55 kDa and exhibited specific immunoreactivity with rat polyclonal antibody against white croaker-derived MBSPI. Its primary structure exhibited a high identity to that of glucose-6-phosphate isomerase (GPI). MBSPI was predominantly composed of α-helices and had a thermal denaturation temperature of 53 ℃. At temperatures above 53 ℃, its secondary structure underwent significant changes, Specifically, the α-helix content decreased and the random coil and antiparallel β-sheet contents increased. Kinetic analysis revealed that MBSPI exhibited a competitive inhibitory effect on MBSP with an IC50 of 0.03 µmol/L. MBSPI effectively inhibited MBSP-induced degradation of myosin heavy chain (MHC), actin, and tropomyosin in myofibrillar proteins. Conclusion: The endogenous inhibitor MBSPI from L. crocea skeletal muscle can significantly suppress MBSP-induced autolysis of myofibrillar proteins, providing a new strategy for the utilization of water-soluble proteins in surimi-based product processing.

Key words: Larimichthys crocea; myofibril-bound serine proteinase; inhibitor; glucose-6-phosphate isomerase; inhibitory mechanism

中图分类号:

TS254.1

霍卫森，张凌晶，陈玉磊，孙乐常，翁凌，黄建联，曹敏杰. 大黄鱼肌原纤维结合型丝氨酸蛋白酶抑制剂的分离纯化及性质分析[J]. 食品科学, 2025, 46(24): 90-97.

HUO Weisen, ZHANG Lingjing, CHEN Yulei, SUN Lechang, WENG Ling, HUANG Jianlian, CAO Minjie. Purification and Characterization of Myofibril-Bound Serine Proteinase Inhibitor from Larimichthys crocea[J]. FOOD SCIENCE, 2025, 46(24): 90-97.

[1]	陈一铭刘红英冯婷玉许家铭卢虹宇骆骥欣巫忠东盛洁王禹萱董平. 超声处理对河鲀鱼精核蛋白凝胶特性及抗氧化活性的影响[J]. 食品科学, 0, (): 0-0.
[2]	李红光, 马佳华, 毕聪慧, 陶裕, 徐杰, 王林彬, 邵廷, 杨鲁. 多糖基质虾青素酯微胶囊制备及其稳定性和消化吸收特性[J]. 食品科学, 2025, 46(16): 100-106.
[3]	杨玉莹, 李言初, 刘家彤, 王泽富, 韩宗元, 刘书成, 魏帅. 超声联合高压CO2处理对虾原肌球蛋白结构和致敏性的影响[J]. 食品科学, 2025, 46(16): 295-303.
[4]	董诺，翟瑞意，胡冰，李雨函，马堃，赵爽，韩玲钰，李婷婷. 磷酸化海参肽螯合锌对鲅鱼鱼糜凝胶特性的影响[J]. 食品科学, 2025, 46(10): 70-78.
[5]	高莉杨俊燕赵英虎季海霞陈佳慧郝媛石英郝瑞. 植物蛋白基黄色素微胶囊的制备及性能测定[J]. 食品科学, 0, (): 0-0.
[6]	杨玉莹李言初刘家彤王泽富韩宗元刘书成魏帅. 超声联合高压CO2处理对虾原肌球蛋白结构和致敏性的影响[J]. 食品科学, 0, (): 0-0.
[7]	蔡沛峰廖威龙冯骏胡利李彩虹徐德峰. 南美白对虾肌肉骨架蛋白特异性染色及其冷藏期间参与质构劣化的作用[J]. 食品科学, 0, (): 0-0.
[8]	于凡，郭莹莹，李娜，代心悦，朱文嘉，姚琳，江艳华，王联珠. 低分子质量岩藻聚糖硫酸酯的分离纯化、结构表征及体外抗氧化活性[J]. 食品科学, 2025, 46(2): 50-56.
[9]	刘华宇，廖彭莹，张天丰，张新锐，邓纭宁，李耀华，韦金锐，陈俊. 鳖蛋黄血管紧张素转化酶抑制肽分离纯化及活性分析[J]. 食品科学, 2025, 46(1): 40-48.
[10]	刘华宇廖彭莹张天丰张新锐邓纭宁李耀华韦金锐陈俊. 鳖蛋黄血管紧张素转化酶抑制肽分离纯化及活性分析[J]. 食品科学, 0, (): 0-0.
[11]	舒志强, 刘芳, 井月欣, 矫春娜, 季一枝, 王茂剑, 王共明, 张健. 海参多糖的结构、活性及构效关系研究进展[J]. 食品科学, 2024, 45(20): 354-365.
[12]	舒志强张健王共明刘芳井月欣矫春娜季一枝王茂剑. 海参多糖的结构、活性及构效关系研究进展[J]. 食品科学, 0, (): 0-0.
[13]	刘煜珺, 郑杰, 刘东竹, 于笛, 李龙, 傅志宇, 刘桂英, 周遵春. 海参皂苷结构特征及其生物活性和作用机制研究进展[J]. 食品科学, 2024, 45(18): 326-341.
[14]	王馥仪, 于双, 董新玉, 陈碧漪, 李莹, 赵前程, 桑雪. 海洋多糖的结构、组成及其抑制肥胖作用机制的研究进展[J]. 食品科学, 2024, 45(17): 335-347.
[15]	王馥仪于双董新玉陈碧漪李莹赵前程桑雪. 海洋多糖的结构、组成及其抑制肥胖作用机制的研究进展[J]. 食品科学, 0, (): 0-0.

大黄鱼肌原纤维结合型丝氨酸蛋白酶抑制剂的分离纯化及性质分析

Purification and Characterization of Myofibril-Bound Serine Proteinase Inhibitor from Larimichthys crocea

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价