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中国生物工程杂志

China Biotechnology
China Biotechnology  2016, Vol. 36 Issue (8): 46-54    DOI: 10.13523/j.cb.20160807
    
Engineering Thermostability of Bovine Enterokinase by Rational Design Method
GUO Chao1,2,3, WANG Zhi-yan1,2,3, GAN Yi-ru1,2,3, LI Dan1,2,3, DENG Yong1,2,3, YU Hao-ran1,2,3, HUANG He1,2,3
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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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 ChangeTM method and the thermostability of wild-type and the enterokinase mutants were investigated. The result demonstrated that the half-life (t1/2) 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 (Km/kcat) 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 wordsProlines      Thermostability      Rational design method      Enterokinase      β-turn     
Received: 06 January 2016      Published: 02 March 2016
ZTFLH:  Q819  
Cite this article:

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.

URL:

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

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