同进化残留物的计算设计,以提高酸酸酶的稳定性和活性
Jiaen Song1, Jun Qiao2, Zhongyi Cheng1
1Key Laboratory of Industrial Biotechnology (Ministry of Education), School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
Biochemical and biophysical research communications
|January 31, 2025
概括
研究人员增强了关键的工业酶 - - 化酶 (NHase),以提高活性和稳定性,使用一种新的共同进化的残留物重新设计策略. 这种优化的NH酶促进了高效的化到胺生物转化.
科学领域:
- 生物催化和酶工程 生物催化和酶工程
- 蛋白质结构与功能之间的关系.
- 工业生物技术 工业生物技术
背景情况:
- 化酶 (NHase) 对于将化转化为胺至关重要,其高活性和热稳定性对于工业应用至关重要.
- 传统的突变策略不如重新设计共进化的残留物以提高向酶的效率.
- 提高酶的稳定性需要精确地准关键的功能站点进行计算设计.
研究的目的:
- 开发一个优化的策略来重新设计共进化的残留物,以提高酸酸酶 (NHase) 的稳定性.
- 为了提高来自伪心热 JCM3095 (PtNHase) 的NHase的特异性活性和热稳定性.
- 为设计其他强大的工业酶建立框架.
主要方法:
- 在PtNHase中分析了80个共进化的残留对,以确定21个可指定热点对.
- 虚拟和组合突变和折叠自由能量计算的应用.
- 对突变进行查和代组合,以获得最佳的酶变异.
- 分子动力学模拟以阐明改善活动和稳定性的机制.
主要成果:
- 在PtNHase中确定了21个热点共同进化的残留物对.
- 从8379个理论突变中产生了27个阳性突变候选.
- 开发出最佳突变A3 (αG86Y/αK57L/αE183F) 具有显著增强的特定活性 (1656.8至2370.1U/mg) 和热稳定性 (在65°C的半衰期从20.1至62.3分钟).
- 通过使用A3.3的全细胞催化剂,证明了烯酸转化为烯胺的改善生物转化.
- 分子动力学揭示了A3.3中的稳定活性位和增强基质通道灵活性.
结论:
- 对共进化的残留物进行重新设计的优化策略有效地提高了NHase活性和热稳定性.
- 开发的强大的NHase突变体 (A3) 显示出在化物生物转化中的工业应用的巨大潜力.
- 该研究为其他工业酶的合理设计和工程提供了有价值的框架.
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