文章摘要
通过定点突变提高纳豆激酶纤溶活性和热稳定性
Enhancing Fibrinolytic Activity and Thermal Stability of Nattokinase by Site-Directed Mutagenesis
  
DOI:doi:10.3969/j.issn.1005-7021.2023.02.003
中文关键词: 纳豆激酶  定点诱变  纤溶活性  热稳定性  分子动力学模拟
英文关键词: nattokinase  site-directed  mutagenesis  fibrinolytic activity  thermal stability  molecular dynamics simulation
基金项目:辽宁省“兴辽英才计划”项目(XLYC2002045);辽宁省教育厅面上项目(LJKZ0088);沈阳市科技特派团项目(21-116-3-48)
作者单位
于晓淼 辽宁大学 生命科学院辽宁 沈阳 110036 
赵允章 辽宁大学 生命科学院辽宁 沈阳 110036 
王锋超 辽宁大学 生命科学院辽宁 沈阳 110036 
孙悦 辽宁大学 药学院辽宁 沈阳 110036 
刘宏生 2.辽宁大学 药学院辽宁 沈阳 1100363.辽宁省生物大分子计算模拟与信息处理专业技术创新中心辽宁 沈阳 1100364.辽宁省药物分子模拟与设计工程实验室辽宁 沈阳 1100365.沈阳市生物大分子计算模拟与信息处理重点实验室辽宁 沈阳 110036 
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中文摘要:
      纳豆激酶(Nattokinase, NK)是一种纤溶酶,可溶解血栓,常用于治疗心血管疾病(CVDs)。然而,野生型NK往往表现出很少的纤溶能力和较低的热稳定性,针对如何提高NK的热稳定性和活性开展研究。使用重叠延伸PCR法将NK中位于194位的Ser替换为Pro,为验证突变后的热稳定性,将野生型NK和突变体S194P均在不同温度下孵育相同时间,使用纤维蛋白板法测定二者酶活,验证热稳定性。成功构建了pET-26b-NKS194P,测量酶活结果显示,野生型NK和突变体S194P酶活分别达到101.30 IU/mL和123.23 IU/mL,结果表明突变体S194P的酶活比野生型NK高(18.10±2)%,将野生型NK和突变体S194P在60~65 ℃温度下孵育30 min后测量酶活,野生型NK在63 ℃时丧失酶活,突变体S194P在65 ℃时丧失酶活,耐热温度提高2 ℃。结果表明,突变体S194p的蛋白结构在突变后发生了改变,使NK提高了耐热温度。
英文摘要:
      Nattokinase (NK), a kind of fibrinolytic enzyme, can dissolve thrombi and be commonly used to treat cardiovascular diseases (CVDs). However, wild-type (WT) NK exhibits little fibrinolytic ability and low thermal stability. Aiming at how to enhance the thermal stability and activity of NK, this paper carry out study on them, Ser located at 194 sites in NK was replaced with Pro using overlap extension PCR method. In order to test and verify the thermal stability of WT NK after mutation, wild-type NK and S194P were incubated at different temperatures for the same time, and determined the enzyme activity of both using the fibrin plate method to test and verify their thermal stability. The results showed that a plasmid pET-26b-NKS194P was successfully constructed in this study. The results of enzyme activities test showed that WT NK and S194P reached 101.30 IU/mL and 123.23 IU/mL respectively, which showed that S194P activity was (18.10±2)% higher than that of the WT NK. The enzyme activity was measured after incubation of WT NK and S194P at 60~65 ℃ for 30 min. The wild-type NK lost its enzyme activity at 63 ℃ and the mutant S194P lost enzyme its activity at 65 ℃, and the heat-resistant temperature was increased by 2 ℃. In conclusion, protein structure of mutant S194P has changed after mutation, resulting in an increase in the heat-resistant temperature of nattokinase.
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