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| 海参组织蛋白酶K的部分酶学性质及其对海参自溶的影响 |
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季晓彤1,2, 王玲1,2, 薛鹏1,2, 年益莹1,2, 周婉茹1, 张元峰3, 张公亮1, 候红漫1, 孙黎明1,2
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| (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;3.中美食品与农业创新中心, 山东 潍坊 261000) |
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| 摘要: |
| 采用特异性荧光底物法检测海参体壁组织蛋白酶K（CTSK）的酶学性质并初步探讨其对海参自溶的影响。结果表明：CTSK的最适pH为5.0，最适温度为50℃，在20~40℃之间酶活力稳定性较好；Zn2+、Fe2+、Fe3+、Cu2+均对CTSK产生一定抑制作用，抑制率均超过60%；而Ca2+和Mn2+对其活性影响不大，Mg2+可增强该酶活性、CA-074、Z-Leu-Leu-Leu-H（ZLLL）、碘乙酸、E-64、抗痛素、PMSF对CTSK的抑制率均在85%以上，EDTA和1，10-菲啰啉对该酶的抑制率分别为39.7%和16%，而DTT和L-Cys可将该酶活力分别提高至127.46%和238.3%。在海参匀浆物中分别加入特异性抑制剂CA-074和ZLLL，25℃水浴加热使其发生自溶，并通过SDS-PAGE检测CTSK对海参蛋白降解的影响。结果显示ZLLL能够在一定程度上抑制海参蛋白的降解。CTSK是一种巯基氨基酸含量较高的半胱氨酸蛋白酶，具有一定的金属离子依赖性，但又易被多种金属离子抑制；CTSK有可能直接参与海参自溶过程中蛋白质的降解。 |
| 关键词: 海参 组织蛋白酶K 酶学特性 自溶 |
| DOI：10.11841/j.issn.1007-4333.2017.09.09 |
| 投稿时间：2016-11-17 |
| 基金项目:国家自然科学基金项目（31201300）；辽宁省自然科学基金项目（2015020794）；辽宁省优秀人才项目（LJQ2012048） |
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| Characteristics of cathepsin K of sea cucumber Stichopus japonicus and its effects on autolysis |
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JI Xiaotong1,2, WANG Ling1,2, XUE Peng1,2, NIAN Yiying1,2, ZHOU Wanru1, ZHANG Yuanfeng3, ZHANG Gongliang1, HOU Hongman1, SUN Liming1,2
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| (1.School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China;2.National Engineering Research Center of Seafood, Dalian 116034, China;3.Zhong Mei Food and Agriculture Innovation Center, Weifang 261000, China) |
| Abstract: |
| Characteristics of cathepsin K (CTSK) in the body wall of sea cucumber and its influence on autolysis was studied.Specific fluorescence substrate (Z-Gly-Pro-Arg-MCA) was used to investigate some properties of CTSK.The results showed that:Maximum activity of CTSK was observed at pH5.0 and 50℃.Its activity kept stable at 20-40℃ and was inhibited by Zn2+,Fe2+,Fe3+ and Cu2+ with an inhibition rate more than 60%.Ca2+ and Mn2+ had no effect on its activity,however,Mg2+ enhanced the activity.CA-074,Z-Leu-Leu-Leu-H(ZLLL),iodoacetate,E-64,antipain,PMSF inhibited CTSK activity with an inhibitory rate over 85%.EDTA and 1,10-phenanthroline showed inhibition effect on CTSK at an inhibition rate of 16% and 39.7%,respectively.Compared with the control group,DTT and L-Cys promoted the activity up to a high percentage of 127.46% and 238.3%,respectively.CA-074 and ZLLL were then added into sea cucumber meat homogenate respectively,the mixture was incubated at 25℃ for the meat to develop autolysis.The effect of CA-074 and ZLLL on protein degradation of sea cucumber was examined by SDS-PAGE.The results indicated that ZLLL inhibited the degradation of protein to a certain degree.Sea cucumber CTSK was a typical cysteine protease with high content of sulphur amino acid,ion dependent and sensitive to many metal ions.It might directly participate in the protein degradation during autolysis of sea cucumber. |
| Key words: sea cucumber cathepsin K enzymatic characteristics autolysis |
