食品科学 ›› 2019, Vol. 40 ›› Issue (24): 15-20.doi: 10.7506/spkx1002-6630-20181210-117

• 食品化学 • 上一篇    下一篇

蓝圆鲹骨骼肌内源性脯氨酸内肽酶抑制剂的纯化及其抑制机理

钟婵,谢雪琼,孙乐常,张凌晶,刘光明,曹敏杰   

  1. (1.集美大学食品与生物工程学院,福建 厦门 361021;2.水产品深加工技术国家地方联合工程研究中心,福建 厦门 361021)
  • 出版日期:2019-12-25 发布日期:2019-12-24
  • 基金资助:
    国家自然科学基金面上项目(31772049;31471640);福建省科技计划项目(2017N5011)

Purification and Mechanism of Action of a Prolyl Endopeptidase Inhibitor from the Skeletal Muscle of Blue Scad (Decapterus maruadsi)

ZHONG Chan, XIE Xueqiong, SUN Lechang, ZHANG Lingjing, LIU Guangming, CAO Minjie   

  1. (1. College of Food and Biological Engineering, Jimei University, Xiamen 361021, China;2. National and Local Joint Engineering Research Center of Processing Technology for Aquatic Products, Xiamen 361021, China)
  • Online:2019-12-25 Published:2019-12-24

摘要: 对蓝圆鲹(Decapterus maruadsi)骨骼肌采用热处理、硫酸铵分级沉淀、DEAE-Sepharose弱阴离子交换柱层析、Sephacryl S-200 HR凝胶过滤层析和HiTrap Q HP强阴离子交换柱层析相结合方法,分离、纯化出一种内源性脯氨酸内肽酶抑制剂(prolyl endopeptidase inhibitor,PEPI)。研究了PEPI对脯氨酸内肽酶(prolyl endopeptidase,PEP)活性的影响及抑制机理。Tricine-十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析表明纯化的PEPI分子质量约为10 kDa,具有较强的热稳定性和酸碱耐受性,其为丝氨酸蛋白酶类PEP的专一性、可逆竞争型抑制剂,抑制常数Ki为0.34 μmol/L。PEPI与PEP形成复合物后,α-螺旋、无规卷曲比例增加,β-折叠比例减少,PEP活性中心构象的改变是酶活力被抑制的主要原因。

关键词: 蓝圆鲹, 脯氨酸内肽酶, 内源性抑制剂, 抑制动力学, 二级结构

Abstract: In the present study, an endogenous prolyl endopeptidase inhibitor (PEPI) was purified from the skeletal muscle of the marine fish blue scad (Decapterus maruadsi) by thermal precipitation, ammonium sulfate fractionation and a series of column chromatographies on DEAE-Sepharose, Sephacryl S-200 HRand HiTrap Q HP. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) showed that the molecular mass of PEPI was about 10 kDa. It was thermally stable and displayed tolerance to pH change. PEPI was a specific inhibitor to serine proteinase PEP and it worked in a reversible competitive manner with an inhibitory constant (Ki) of 0.34 μmol/L. A significant increase in α-helix and random coil and decrease in β-sheet were detected after formation of the PEP-PEPI complex. Quite possibly, the conformational change of PEP was the major reason for enzymatic activity inhibition.

Key words: blue scad, prolyl endopeptidase, endogenous inhibitor, inhibitory kinetics, secondary structure

中图分类号: