Engineering Leifsonia Alcohol Dehydrogenase for Thermostability and Catalytic Efficiency by Enhancing Subunit Interactions
Engineering Leifsonia Alcohol Dehydrogenase for Thermostability and Catalytic Efficiency by Enhancing Subunit Interactions
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DOI：
10.1002/cbic.202100431
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发表时间：
2021-09
期刊：
影响因子：
3.2
通讯作者：
Lu Zhu;Yang Song;Chenchen Chang;Hongmin Ma;Lu Yang;Z. Deng;Wei Deng;X. Qu
中科院分区：
文献类型：
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作者：
Lu Zhu;Yang Song;Chenchen Chang;Hongmin Ma;Lu Yang;Z. Deng;Wei Deng;X. Qu
Leifsonia alcohol dehydrogenase（LnADH）是一种很有前途的手性醇合成生物催化剂。然而，野生型LnADH在实际应用中的局限性包括活性低和稳定性差。在这项工作中，蛋白质工程，以提高其热稳定性和催化效率，通过改变亚基界面。经鉴定，残基T100和S148对热稳定性和活性具有重要意义，突变体T100 R/S148 I的解链温度（ΔTm）和对酮底物的催化效率提高了18.7 °C和1.8-5.5倍。解析野生型酶和T100 R/S148 L的晶体结构揭示了突变对稳定性和催化活性的有益影响。最稳定的突变体T100 R/S148 I具有工业应用前景，在50 °C下，酶与底物的比例仅为1：500，就可以生产200 g l −1 day−1手性醇。
Leifsonia alcohol dehydrogenase (LnADH) is a promising biocatalyst for the synthesis of chiral alcohols. However, limitations of wild‐type LnADH observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve its thermostability and catalytic efficiency by altering the subunit interfaces. Residues T100 and S148 were identified to be significant for thermostability and activity, and the melting temperature (ΔTm) and catalytic efficiency of the mutant T100R/S148I toward ketone substrates was improved by 18.7 °C and 1.8–5.5‐fold. Solving the crystal structures of the wild‐type enzyme and T100R/S148L revealed beneficial effects of mutations on stability and catalytic activity. The most robust mutant T100R/S148I is promising for industrial applications and can produce 200 g liter−1 day−1 chiral alcohols at 50 °C by only a 1 : 500 ratio of enzyme to substrate.