The thermostable α-amylase derived from (BLA) has multiple advantages, including enhancing the mass transfer rate and by reducing microbial contamination in starch hydrolysis. Nonetheless, the application of BLA is constrained by the accessibility and stability of enzymes capable of achieving high conversion rates at elevated temperatures. Moreover, the thermotolerance of BLA requires further enhancement. Here, we developed a computational strategy for constructing small and smart mutant libraries to identify variants with enhanced thermostability. Initially, molecular dynamics (MD) simulations were employed to identify the regions with high flexibility. Subsequently, FoldX, a computational design predictor, was used to design mutants by rigidifying highly flexible residues, whereas the simultaneous decrease in folding free energy assisted in improving thermostability. Through the utilization of MD and FoldX, residues K251, T277, N278, K319, and E336, situated at a distance of 5Å from the catalytic triad, were chosen for mutation. Seventeen mutants were identified and characterized by evaluating enzymatic characteristics and kinetic parameters. The catalytic efficiency of the E271L/N278K mutant reached 184.1gL s, which is 1.88-fold larger than the corresponding value determined for the WT. Furthermore, the most thermostable mutant, E336S, exhibited a 1.43-fold improvement in half-life at 95 ℃, compared with that of the WT. This study, by combining computational simulation with experimental verification, establishes that potential sites can be computationally predicted to increase the activity and stability of BLA and thus provide a possible strategy by which to guide protein design.

中文翻译：

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地衣芽孢杆菌 α-淀粉酶的计算设计，以提高其高温活性和稳定性

源自 (BLA) 的热稳定 α-淀粉酶具有多种优点，包括提高传质速率和减少淀粉水解过程中的微生物污染。尽管如此，BLA 的应用受到能够在高温下实现高转化率的酶的可及性和稳定性的限制。此外，BLA的耐热性还需要进一步增强。在这里，我们开发了一种计算策略，用于构建小型智能突变体库，以识别具有增强热稳定性的变体。最初，采用分子动力学（MD）模拟来识别具有高灵活性的区域。随后，计算设计预测器 FoldX 用于通过刚性化高度灵活的残基来设计突变体，而折叠自由能的同时降低有助于提高热稳定性。通过利用 MD 和 FoldX，选择距离催化三联体 5Å 的残基 K251、T277、N278、K319 和 E336 进行突变。通过评估酶学特征和动力学参数，鉴定并表征了 17 个突变体。E271L/N278K突变体的催化效率达到184.1gL·s，比WT测定的相应值大1.88倍。此外，与WT相比，最耐热的突变体E336S在95℃下的半衰期提高了1.43倍。这项研究通过将计算模拟与实验验证相结合，确定了可以通过计算预测潜在位点以提高 BLA 的活性和稳定性，从而提供指导蛋白质设计的可能策略。