In this study, the phenomenon of the stability-activity trade-off, which is increasingly recognized in enzyme engineering, was explored. Typically, enhanced stability in enzymes correlates with diminished activity. Utilizing Rosa roxburghii copper–zinc superoxide dismutase (RrCuZnSOD) as a model, single-site mutations were introduced based on a semirational design derived from consensus sequences. The initial set of mutants was selected based on activity, followed by combinatorial mutation. This approach yielded two double-site mutants, D25/A115T (18,688 ± 206 U/mg) and A115T/S135P (18,095 ± 1556 U/mg), exhibiting superior enzymatic properties due to additive and synergistic effects. These mutants demonstrated increased half-lives (T_1/2) at 80 °C by 1.2- and 1.6-fold, respectively, and their melting temperatures (T_m) rose by 3.4 and 2.5 °C, respectively, without any loss in activity relative to the wild type. Via an integration of structural analysis and molecular dynamics simulations, we elucidated the underlying mechanism facilitating the concurrent enhancement of both thermostability and enzymatic activity.

中文翻译：

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基于共识序列的半理性设计平衡酶活性与稳定性的权衡

在这项研究中，探讨了酶工程中日益受到认可的稳定性与活性权衡现象。通常，酶稳定性的增强与活性的降低相关。利用刺梨铜锌超氧化物歧化酶（RrCuZnSOD）作为模型，基于源自共有序列的半理性设计引入单位点突变。根据活性选择初始突变体集，然后进行组合突变。这种方法产生了两个双位点突变体，D25/A115T (18,688 ± 206 U/mg) 和 A115T/S135P (18,095 ± 1556 U/mg)，由于加和和协同效应而表现出优异的酶特性。这些突变体在 80 °C 下的半衰期 ( T _1/2 ) 分别延长了 1.2 倍和 1.6 倍，熔解温度 ( T _m ) 分别升高了 3.4 和 2.5 °C，且活性没有任何损失相对于野生型。通过结构分析和分子动力学模拟的结合，我们阐明了促进热稳定性和酶活性同时增强的潜在机制。