Enhancing the thermal stability and activity of the engineered self-sufficient P450SPα-SOX by switching the domains linker
通过更换结构域连接肽来增强工程化自给自足型P450SPα-SOX的热稳定性和活性
- 关键词：
- 来源：
- International Journal of Biological Macromolecules
- 类型：
- 学术文献
- 语种：
- 中文
- 原文发布日期：
- 2025-06-23
- 摘要：
- This work reports on the engineering of the linker between P450 SPα (CYP152B1) and sarcosine oxidase (SOX), with the aim of enhancing the structural rigidity of the fusion protein (SPα-SOX) and study the effect on its stability and catalytic performance. Differential scanning calorimetry shows that the construct bearing the rigid linker (SPα-rigidSOX) results in a higher energy barrier to unfolding (765 kcal/mol) compared to the previous fusion system (SPα-flexible-SOX) (561 kcal/mol), as well as a Tonset above 50 °C. Furthermore, residual CO-binding after heat treatment was investigated for both the fusion systems, and a 5.7 °C increase of the T50 of SPα-rigid-SOX is shown. Interestingly, a stabilized P420 semifolded state of the SPα is also observed after SPα-rigid-SOX incubation at high temperature (40°). The two fusion systems were studied at high temperature for the turnover of lauric acid: SPα-rigid-SOX shows a 98 % conversion yield using 5 mM substrate compared to the 24 % conversion of SPαflexibleSOX when the catalysis is carried out at 40 °C. Finally, the activity of the two constructs was tested using styrene as a substrate, and three products of catalysis were observed: styrene oxide (85 %), phenylacetaldehyde (0–3 %) and 2-phenylpropenal (12–15 %). Interestingly, 2-phenylpropenal is observed for the first time and only for the fusion enzymes. Also in this case, SPα-rigid-SOX outperformed SPα-flexible-SOX with a 3-fold higher conversion yield. Overall, we demonstrate that the rigid linker improves the fusion enzyme thermal stability and catalytic performance, both at high temperature and in mild conditions, resulting also in the production of new molecules of biotechnological interest.
- 所属专题：
- 173
