PLS 引导突变重组以提高定向进化获得的牛肠激蛋白酶的稳定性

IF 3.8 3区化学 Q2 CHEMISTRY, PHYSICAL

ChemCatChem Pub Date : 2024-09-17 DOI:10.1002/cctc.202400943

Paul Dalby, Weina Li, Weinfeng Shen, Niccolo A. E. Venanzi, Cheng Zhang, Yiwen Li, Daidi Fan

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引用次数: 0

摘要

活性和热稳定性是酶的关键所在，但同时提高活性和热稳定性却具有挑战性。此前，我们利用定向进化鉴定出了具有更强可溶性表达和热稳定性的牛肠激酶（EKL）变体。对321个EKL变体的偏最小二乘法（PLS）分析揭示了单个突变的影响，并确定了表现最好的变体中的中性或有害突变。利用 PLS 排名，我们创建了突变更少、稳定性更强的新变体。大多数原始变体和 PLS 引导的变体都表现出活性-稳定性的权衡。然而，两个新的三重和四重突变体同时提高了活性和稳定性，超过了权衡极限。重组 PLS 引导的突变可能消除了中性或有害突变，从而提高了稳定性。MD 模拟将残基特异性动力学与稳定性联系起来，精确定位了易聚集区域附近的关键结构区域。我们的研究结果验证了 PLS 是增强酶特性的有效策略，是对定向进化的补充。

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PLS‐guided Mutant Recombination to Improve the Stability of Bovine Enterokinases Obtained by Directed Evolution

Activity and thermostability are critical yet challenging to improve simultaneously in enzymes. Using directed evolution, we previously identified bovine enterokinase (EKL) variants with enhanced soluble expression and thermal stability. Partial least squares (PLS) analysis of 321 EKL variants revealed the impact of individual mutations and identified neutral or detrimental mutations in top‐performing variants. Leveraging PLS rankings, we created new variants with fewer mutations and enhanced stability. Most original and PLS‐guided variants exhibited an activity‐stability trade‐off. However, two new triple‐ and quadruple‐mutants improved both activity and stability, surpassing the trade‐off limit. Recombining PLS‐guided mutations likely eliminated neutral or harmful mutations, enhancing stability. MD simulations linked residue‐specific dynamics with stability, pinpointing critical structural regions near aggregation‐prone areas. Our findings validate PLS as a potent strategy to enhance enzyme properties, complementing directed evolution.

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来源期刊

ChemCatChem 化学-物理化学

CiteScore

8.10

自引率

4.40%

发文量

511

审稿时长

1.3 months

期刊介绍： With an impact factor of 4.495 (2018), ChemCatChem is one of the premier journals in the field of catalysis. The journal provides primary research papers and critical secondary information on heterogeneous, homogeneous and bio- and nanocatalysis. The journal is well placed to strengthen cross-communication within between these communities. Its authors and readers come from academia, the chemical industry, and government laboratories across the world. It is published on behalf of Chemistry Europe, an association of 16 European chemical societies, and is supported by the German Catalysis Society.