He Wei, Ge Xizhen, Li Ying
Beijing Key Laboratory of Biomass Waste Resource Utilization, College of Biochemical Engieering, Beijing Union University, Beijing 100023, China; Beijing Key Laboratory of Bioactive Substances and Functional Foods, College of Biochemical Engineering, Beijing Union University, Beijing 100023, China.
Beijing Key Laboratory of Biomass Waste Resource Utilization, College of Biochemical Engieering, Beijing Union University, Beijing 100023, China; Beijing Key Laboratory of Bioactive Substances and Functional Foods, College of Biochemical Engineering, Beijing Union University, Beijing 100023, China..
Int J Biol Macromol. 2025 Jul;318(Pt 4):145156. doi: 10.1016/j.ijbiomac.2025.145156. Epub 2025 Jun 10.
CotA, a bacterial multicopper oxidase, exhibits exceptional thermal stability, alkali resistance, and substrate versatility, making it valuable for industrial biocatalysis, environmental remediation, and dye degradation. Extensive research has enhanced its activity and stability through directed evolution, semi-rational design, and rational design, yet the underlying structural features remain largely unclear. In this study, we performed site-directed mutagenesis on CotA from Bacillus subtilis and analyzed its effects on stability. Among the CotA single and double mutants we constructed, the T377I/T418G variant exhibited a 6.12-fold increase in activity compared to the wild type and a 50 % improvement in thermal stability at 80 °C. To uncover the relationship between the enzyme activity, stability, and the spatial structure of the CotA mutants, we conducted molecular dynamics (MD) simulations to analyze the catalytic structural features. The results indicated that the increase in ABTS-specific enzyme activity was linked to an expanded binding pocket, while the increased thermal stability was attributed to a higher proportion of random coils in the secondary structure. Our study provides new insights into the catalytic activity and thermostability structural features of CotA, laying the groundwork for its applications in industrial and environmental fields.
CotA是一种细菌多铜氧化酶，具有出色的热稳定性、耐碱性和底物通用性，这使其在工业生物催化、环境修复和染料降解方面具有重要价值。广泛的研究通过定向进化、半理性设计和理性设计提高了它的活性和稳定性，但其潜在的结构特征仍 largely不清楚。在本研究中，我们对枯草芽孢杆菌的CotA进行了定点诱变，并分析了其对稳定性的影响。在我们构建的CotA单突变体和双突变体中，T377I/T418G变体的活性相比野生型提高了6.12倍，在80℃时热稳定性提高了50%。为了揭示CotA突变体的酶活性、稳定性与空间结构之间的关系，我们进行了分子动力学（MD）模拟以分析催化结构特征。结果表明，ABTS特异性酶活性的增加与结合口袋的扩大有关，而热稳定性的提高归因于二级结构中无规卷曲的比例更高。我们的研究为CotA的催化活性和热稳定性结构特征提供了新的见解，为其在工业和环境领域的应用奠定了基础。
