食品与发酵工业 >

2022 , Vol. 48 >Issue 2: 71 - 78

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.027737

研究报告

凡纳滨对虾肌肉中组织蛋白酶L提取工艺优化及分离纯化

  • 唐振冬 ,
  • 邵海艳 ,
  • 张迪 ,
  • 刘书成 ,
  • 吉宏武 ,
  • 徐杰 ,
  • 太敏瑞 ,
  • 苏伟明 ,
  • 梁善昊 ,
  • 何旋
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  • 1(广东海洋大学 食品科技学院,广东省水产品加工与安全重点实验室,广东省海洋生物制品工程实验室,广东省海洋食品工程技术研究中心,水产品深加工广东普通高等学校重点实验室,广东 湛江,524088)
    2(海洋食品精深加工关键技术省部共建协同创新中心(大连工业大学),辽宁 大连,116034)
硕士研究生(吉宏武教授为通信作者,E-mail:Jihw62318@163.com)

收稿日期: 2021-04-16

  修回日期: 2021-05-18

  网络出版日期: 2022-02-28

基金资助

国家现代农业产业技术体系项目(CARs-48);国家重点研发计划资助(2019YFD0902003);广东普通高等学校海洋食品绿色加工技术研究团队(2019KCXTD011)

收起

Optimization of extraction and purification of cathepsin L from Litopenaeus vannamei muscle

  • TANG Zhendong ,
  • SHAO Haiyan ,
  • ZHANG Di ,
  • LIU Shucheng ,
  • JI Hongwu ,
  • XU Jie ,
  • TAI Minrui ,
  • SU Weiming ,
  • LIANG Shanhao ,
  • HE Xuan
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  • 1(College of Food Science and Technology, Guangdong Ocean University, Guangdong Provincial Key Laboratory of Aquatic Products Processing and Safety, Guangdong Province Engineering Laboratory for Marine Biological Products, Guangdong Provincial Engineering Technology Research Center of seafood, Key Laboratory of Advanced Processing of Aquatic Product of Guangdong Higher Education Institution, Zhanjiang 524088, China)
    2(Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Dalian 116034, China)

Received date: 2021-04-16

  Revised date: 2021-05-18

  Online published: 2022-02-28

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摘要

优化凡纳滨对虾肌肉中组织蛋白酶L提取工艺,分离纯化组织蛋白酶L并验证其对肌原纤维蛋白的降解作用。以凡纳滨对虾肌肉为原料,采用单因素试验、Plackett-Burman试验和双因素等重复试验对肌肉中组织蛋白酶L的提取工艺进行了优化。采用Tris-HCl缓冲液浸提、硫酸铵沉淀、Q-Seharose F.F阴离子交换层析、Sephacryl S-100凝胶过滤层析和SDS-PAGA等方法对组织蛋白酶L粗酶液进行分离纯化,并验证提纯后的组织蛋白酶L对肌原纤维蛋白的降解作用。最终确定对虾肌肉中组织蛋白酶L最佳提取工艺为:缓冲液体系(Tris-HCl缓冲液浓度40 mmol/L,半胱氨酸浓度6 mmol/L,苯甲基磺酰氟浓度0.4 mmol/L),料液比1∶8(g∶mL),pH 6.0。优化后组织蛋白酶L总酶活力值升至378.36 U,酶活力得率达到150.1 U/g,纯化后的组织蛋白酶L的比活力从0.34 U/mg提高到了101.15 U/mg,纯化倍数达到298.28倍,得率为16.50%,分子质量为46.4 kDa,并进一步验证了提纯后的组织蛋白酶L对肌球蛋白重链和肌动蛋白有降解作用。发现对虾肌肉中组织蛋白酶L对肌原纤维蛋白降解有明显作用,为进一步研究对虾肌肉中组织蛋白酶L的相关性质及其对肌肉的降解机制提供理论依据。

关键词： 凡纳滨对虾; 组织蛋白酶L; 分离纯化; 工艺优化; 蛋白降解

本文引用格式

唐振冬 , 邵海艳 , 张迪 , 刘书成 , 吉宏武 , 徐杰 , 太敏瑞 , 苏伟明 , 梁善昊 , 何旋 . 凡纳滨对虾肌肉中组织蛋白酶L提取工艺优化及分离纯化[J]. 食品与发酵工业, 2022 , 48(2) : 71 -78 . DOI: 10.13995/j.cnki.11-1802/ts.027737

Abstract

The purpose of the study was to optimize the extraction condition of cathepsin L from shrimp muscle (Litopenaeus vannamei), to isolate and purify, cathepsin L in extraction solutions, and to verify the effect of cathepsin L on the degradation of myofibril protein. The shrimp muscle was used as raw materials. The extraction condition of cathepsin L from muscle was optimized by single-factor tests, Plackett-Burman test and double-factor test. The cathepsin L in extraction solutions was isolated and purified by Tris-HCl buffer extraction, ammonium sulfate precipitation, Q-Seharose F.F anion exchange chromatography, SephacrylS-100 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The effect of cathepsin L on the degradation of myofibril protein was verified by the confirmatory experiment. The optimal extraction conditions of cathepsin L from shrimp muscle were determined as follows: buffer system containing Tris-HCl buffer of 40 mmol/L, L-Cys 6 mmol/L and PMSF 0.4 mmol/L, the ratio of solid to liquid at 1∶8(g∶mL), pH 6.0. Under the optimized conditions, the total enzyme activity value of cathepsin L increased to 378.36 U, and the yield of cathepsin L was 150.1 U/g. The specific activity of purified cathepsin L increased from 0.34 to 101.15 U/mg, the purification multiple was 298.28 times, the yield was 16.50%, and the molecular weight was 46.4 kDa. The confirmatory experiment further confirmed that the purified cathepsin L had significant influences on the degradation of actin and myosin heavy chain. cathepsin L had significant effects on the degradation of myofibril protein. It provides a theoretical basis for further study on the related properties of cathepsin L and its degradation mechanism in shrimp muscle.

Key words： Litopenaeus vannamei; cathepsin L; separation and purification; process optimization; protein degradation

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