技术与方法理性设计改造牛肠激酶的热稳定性

doi:10.13523/j.cb.20160807

中国生物工程杂志

2016, Vol. 36

Issue (8): 46-54 DOI: 10.13523/j.cb.20160807

技术与方法

技术与方法理性设计改造牛肠激酶的热稳定性

郭超^1,2,3, 王志彦^1,2,3, 甘一如^1,2,3, 李丹^1,2,3, 邓勇^1,2,3, 于浩然^1,2,3, 黄鹤^1,2,3

1. 天津大学化工学院天津 300072;
2. 系统生物工程教育部重点实验室天津 300072;
3. 天津化学化工协同创新中心天津 300072

Engineering Thermostability of Bovine Enterokinase by Rational Design Method

GUO Chao^1,2,3, WANG Zhi-yan^1,2,3, GAN Yi-ru^1,2,3, LI Dan^1,2,3, DENG Yong^1,2,3, YU Hao-ran^1,2,3, HUANG He^1,2,3

1. Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China;
2. Key Laboratory of System Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, China;
3. Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China

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摘要：

以牛肠激酶作为研究对象，利用理性设计的方法提高其热稳定性。首先通过分子动力学模拟软件Gromacs v 4.5.5，FlexService以及B-FITTER软件预测出了肠激酶的柔性区Fragment 64～69，Fragment 85～90；然后结合β-转角序列统计学信息以及引入位置原有的残基不参与形成氢键的原则，确立了3个突变位点S67P，R87P以及Y136P；通过Quik Change^TM 定点突变的方法引入突变位点，并进行了酶热稳定性分析。结果表明，R87P突变体酶的失活半衰（t_1/2）和T₅₀¹⁰ 较野生型分别提高了3.1 min和11.8℃，同时，动力学常数（K_m/k_cat）测定结果显示酶活未受到显著影响。该策略有潜力应用于其他工业酶分子的热稳定性改造，为推动生物酶的工业化应用奠定基础。

关键词： 热稳定性; 肠激酶; 理性设计; 脯氨酸; β-转角

Abstract:

The rational design method was applied to increase the thermostability of bovine enterokinase. Molecular dynamics software Gromacs v4.5.5, FlexService and B-FITTER were used to predict the flexibility profile of the bovine enterokinase structures. Fragment 64~69 and Fragment 85~90 were confirmed to represent the flexible region. Subsequently, β-turn sequence statistics and stereoscopic criteria of introducing proline were combined to pinpoint appropriate substitution sites by prolines. Finally, site-directed mutations (S67P, R87P and Y136P) were introduced within the fexible region using Quik Change^TM method and the thermostability of wild-type and the enterokinase mutants were investigated. The result demonstrated that the half-life (t_1/2) and half inactivation (T₅₀¹⁰) temperature of the R87P mutant increased by 3.1 min and 11.8℃ when compared to that of the wild-type enzyme. Meanwhile, the catalytic efficiency (K_m/k_cat) of the R87P mutant enzyme remained nearly unchanged. This strategy has potential to be applied to engineer thermostability of other enzyme, which is beneficial for the wider application of biocatalysts in industry.

Key words: Prolines Thermostability Rational design method Enterokinase β-turn

收稿日期: 2016-01-06 出版日期: 2016-03-02

ZTFLH:

Q819

基金资助:

国家自然科学基金（31470967）、国家科技重大专项（2011ZX09201-301-05；2014ZX09508006-002-002）资助项目

通讯作者: 黄鹤 E-mail: huang@tju.edu.cn

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引用本文:

郭超, 王志彦, 甘一如, 李丹, 邓勇, 于浩然, 黄鹤. 技术与方法理性设计改造牛肠激酶的热稳定性[J]. 中国生物工程杂志, 2016, 36(8): 46-54.

GUO Chao, WANG Zhi-yan, GAN Yi-ru, LI Dan, DENG Yong, YU Hao-ran, HUANG He. Engineering Thermostability of Bovine Enterokinase by Rational Design Method. China Biotechnology, 2016, 36(8): 46-54.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160807 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I8/46

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