Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering
Approaching boiling point stability of an alcohol dehydrogenase through computationally-guided enzyme engineering
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DOI：
10.7554/elife.54639
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发表时间：
2020-03-31
期刊：
影响因子：
7.7
通讯作者：
Fraaije, Marco W.
中科院分区：
文献类型：
--
作者：
Aalbers, Friso S.;Furst, Maximilian J. L. J.;Fraaije, Marco W.
酶的不稳定性是限制酶研究和应用的重要因素。因此，快速有效地提高酶稳定性的方法是非常有吸引力的。在这项研究中，我们采用了计算方法（FRESCO）来指导乙醇脱氢酶的工程。在177个选择的突变中，25个突变引起表观解链温度的显著增加（Δ T-m >= +3 ℃）。通过组合突变，产生了10倍突变体，其T-m为94摄氏度（相对于野生型+51摄氏度），几乎达到水的沸点，并且是迄今为止FRESCO的最高增幅。阐明了10倍突变体的结构，这使得能够鉴定活性损害突变。在回复该突变后，与野生型相比，该酶没有显示出活性损失，同时显示出88 ℃的T-m（相对于野生型+45 ℃）。这项工作证明了通过计算库设计的酶稳定的价值。
Enzyme instability is an important limitation for the investigation and application of enzymes. Therefore, methods to rapidly and effectively improve enzyme stability are highly appealing. In this study we applied a computational method (FRESCO) to guide the engineering of an alcohol dehydrogenase. Of the 177 selected mutations, 25 mutations brought about a significant increase in apparent melting temperature (Delta T-m >= +3 degrees C). By combining mutations, a 10-fold mutant was generated with a T-m of 94 degrees C (+51 degrees C relative to wild type), almost reaching water's boiling point, and the highest increase with FRESCO to date. The 10-fold mutant's structure was elucidated, which enabled the identification of an activity-impairing mutation. After reverting this mutation, the enzyme showed no loss in activity compared to wild type, while displaying a T-m of 88 degrees C (+45 degrees C relative to wild type). This work demonstrates the value of enzyme stabilization through computational library design.