使细菌漆酶的柔性区域刚性化能够高温降解黄曲霉毒素B1
Rigidifying Flexible Regions of a Bacterial Laccase Enables High-Temperature Aflatoxin B1 Degradation.
摘要(中文译文)
黄曲霉毒素B1（AFB1）对全球食品和饲料安全构成严重威胁。基于漆酶（laccase）的酶促降解是一种有前景的AFB1绿色去除策略；然而，野生型酶在高温加工条件（>70 °C）下快速热失活，严重限制了其工业应用。本研究通过将计算结构生物学与半理性设计相结合的综合策略，对来源于B10的漆酶进行了热稳定性改造。通过将分子动力学（MD）模拟与折叠自由能（ΔΔG）计算相耦合，我们识别了与热不稳定性相关的关键柔性区域，并随后实施了迭代饱和突变。最佳单突变体R196C在80 °C热处理10 min后仍保留超过96%的相对活性。进一步的迭代突变叠加逐步增强了热稳定性：R90E/R196C双突变体在80 °C下的活性比R196C高1.25倍，而R90E/R196C/H54F三突变体又比双突变体进一步提高1.16倍。最终的四突变体R90E/R196C/H54F/R253I在80 °C下24 h内实现了86.9%的AFB1降解率。高温MD模拟（353.15 K下100 ns）表明，热稳定性的增强与构象柔性降低、回转半径（Rg）和溶剂可及表面积（SASA）减小以及有助于稳定蛋白质核心的卷曲向β-折叠转变有关。此外，工程化酶在……中实现了高效分泌表达，达到3.0 U/mL，且粗酶液在80 °C下保持超过70%的活性。总体而言，这些结果为用于AFB1脱毒相关应用的热稳定生物催化剂的理性改造和可规模化生产提供了实践基础，并为工业酶热稳定性的靶向增强提供了更广泛的见解。
Abstract
Aflatoxin B1 (AFB1) poses a serious threat to global food and feed safety. Laccase-based enzymatic degradation represents a promising green strategy for AFB1 removal; however, its industrial application is severely limited by the rapid thermal inactivation of wild-type enzymes under high-temperature processing conditions (>70 °C). Here, we engineered the thermal stability of a laccase from B10 through an integrated strategy combining computational structural biology with semi-rational design. By coupling molecular dynamics (MD) simulations with folding free-energy (ΔΔG) calculations, we identified key flexible regions associated with thermal instability and subsequently implemented iterative saturation mutagenesis. The best single mutant, R196C, retained more than 96% relative activity after heat treatment at 80 °C for 10 min. Further iterative mutational stacking progressively enhanced thermostability: the R90E/R196C double mutant showed 1.25-fold higher activity at 80 °C than R196C, and the R90E/R196C/H54F triple mutant showed a further 1.16-fold increase over the double mutant. The final quadruple mutant, R90E/R196C/H54F/R253I, achieved 86.9% AFB1 degradation at 80 °C after 24 h. High-temperature MD simulations (100 ns at 353.15 K) indicated that the enhanced thermostability was associated with reduced conformational flexibility, lower radius of gyration (Rg) and solvent-accessible surface area (SASA), and a coil-to-β-sheet transition that contributed to stabilization of the protein core. In addition, efficient secretory expression of the engineered enzyme was achieved in , reaching 3.0 U/mL, while the crude enzyme maintained more than 70% activity at 80 °C. Collectively, these results provide a practical basis for the rational engineering and scalable production of thermostable biocatalysts for AFB1 detoxification-related applications of AFB1 control, and offer broader insights into the targeted enhancement of thermal stability in industrial enzymes.
如何引用
AMA
Dongwei Xiong, Huiying Sun, Yuhang Sun, Peng Li, Miao Long. Rigidifying Flexible Regions of a Bacterial Laccase Enables High-Temperature Aflatoxin B1 Degradation.. Microorganisms. 2026; doi:10.3390/microorganisms14040856.
APA
Dongwei Xiong, Huiying Sun, Yuhang Sun, Peng Li, Miao Long (2026). Rigidifying Flexible Regions of a Bacterial Laccase Enables High-Temperature Aflatoxin B1 Degradation.. Microorganisms. https://doi.org/10.3390/microorganisms14040856
GB/T 7714
Dongwei Xiong, Huiying Sun, Yuhang Sun, Peng Li, Miao Long. Rigidifying Flexible Regions of a Bacterial Laccase Enables High-Temperature Aflatoxin B1 Degradation.[J]. Microorganisms, 2026 doi:10.3390/microorganisms14040856.
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