Simultaneously optimizing multiple properties of β-glucosidase Bgl6 using combined (semi-)rational design strategies and investigation of the underlying mechanisms
Simultaneously optimizing multiple properties of β-glucosidase Bgl6 using combined (semi-)rational design strategies and investigation of the underlying mechanisms
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DOI：
10.1016/j.biortech.2023.128792
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发表时间：
2023-02-28
影响因子：
11.4
通讯作者：
Liu, Yuhuan
中科院分区：
文献类型：
--
作者：
Li, Shuifeng;Cao, Lichuang;Liu, Yuhuan
-葡萄糖苷酶在纤维素糖化过程中的性能由热稳定性、活性和葡萄糖耐受性决定。然而，它们之间的冲突使得同时优化三个属性具有挑战性。本研究以Bgl6-M3为研究起点，报道了一例此类病例。首先，利用计算机辅助工程策略获得4个增强热稳定性的突变体（突变体M7）。其次，M7的底基质结合袋被重塑，产生两个增加活性但降低葡萄糖耐量的突变（突变体M9）。然后重新设计活性位点空腔的一个关键区域，导致三个突变，提高葡萄糖耐量和活性。最后，获得了耐热性（半衰期为20倍）、活性（kcat/Km为5.6倍）和葡萄糖耐量（δ IC50为200 mM）同时提高的突变体M12。通过结构分析和分子动力学模拟阐明了性能改善的机理。总的来说，本文采用的策略和对潜在机制的新见解可能为其他酶的多性质工程提供指导。
The performance of beta-glucosidase during cellulose saccharification is determined by thermostability, activity and glucose tolerance. However, conflicts between them make it challenging to simultaneously optimize three properties. In this work, such a case was reported using Bgl6-M3 as a starting point. Firstly, four thermostability-enhancing mutations were obtained using computer-aided engineering strategies (mutant M7). Secondly, sub-strate binding pocket of M7 was reshaped, generating two mutations that increased activity but decreased glucose tolerance (mutant M9). Then a key region lining active site cavity was redesigned, resulting in three mutations that boosted glucose tolerance and activity. Finally, mutant M12 with simultaneously improved thermostability (half-life of 20-fold), activity (kcat/Km of 5.6-fold) and glucose tolerance (Delta IC50 of 200 mM) was obtained. Mechanisms for property improvement were elucidated by structural analysis and molecular dynamics simulations. Overall, the strategies used here and new insights into the underlying mechanisms may provide guidance for multi-property engineering of other enzymes.