基于域工程的高辅酶亲和度的仿真氨基脱酶
Jialin Li1,2,3, Xiaoqing Mu4,5,6, Tao Wu1,2
1Laboratory of Brewing Microbiology and Applied Enzymology, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.
Bioresources and bioprocessing
|April 22, 2024
概括
工程化学氨基脱酶 (cFLF-AmDH) 增强了NADH辅因子亲和力和催化效率,用于芳香的奇拉氨基合成. 这种域混合酶为工业应用提供了更好的稳定性和更广泛的基质范围.
科学领域:
- 生物催化和酶工程 生物催化和酶工程
- 蛋白质工程和设计
- 合成化学 合成化学
背景情况:
- 依赖NADH的氨氨酸胺脱酶 (F-AmDH) 合成芳香性氨酸,但具有较低的辅酶亲和力和催化效率.
- 由于F-AmDH的工业应用有限,因此需要提高酶性能.
研究的目的:
- 开发一种具有增强NADH亲和力和催化效率的化学氨基脱酶 (cFLF-AmDH).
- 通过域混合来提高F-AmDH的热稳定性和基质频谱.
主要方法:
- 通过结合F-AmDH的基质结合域和leucine amine dehydrogenase (L-AmDH) 的辅因子结合域,构建了一个嵌合酶 (cFLF-AmDH).
- 进行了动态分析,以评估NADH亲和力和催化效率 (kcat/Km).
- 评估了热稳定性和基质频谱.
- 利用分子动力学模拟来了解结构稳定性和辅酶结合.
主要成果:
- 与母体F-AmDH相比,cFLF-AmDH的NADH亲和力提高了两倍,催化效率提高了4.4倍.
- 观察到增强的热稳定性,在55°C时半衰期延长60%,以及更广泛的基质谱.
- 分子动力学模拟表明cFLF-AmDH的结构稳定性增加.
- 减少性氨化反应率增加了150%,在0.05mM的NAD+度下,转化率增加了150%.
结论:
- 域混合是一种有效的策略,用于创建具有增强辅因子亲和力,催化效率,特异性和热稳定性的酶.
- 开发的cFLF-AmDH表现出显著的改进,使其成为工业应用的有希望的生物催化剂.
- 这项研究强调了域工程作为一种可行的方法,用于产生具有量身定制的催化性能的酶多样性.
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