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Light-driven Enzymatic Decarboxylation
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基于功能循环工程的基础上,提高碳基还原酶的催化性能
Tao-Shun Zhou1,2, Xiang-Yang Li1,2, Xiao-Jian Zhang1,2
1The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, Zhejiang, P. R. China.
Biotechnology and bioengineering
|October 22, 2024
概括
酶工程增强了碳基还原酶 (EaSDR6) 来合成一个关键的vibegron中间体. 循环修改显著提高了催化效率和立体选择性,使高效的大规模生产成为可能.
科学领域:
- 生物催化和酶工程 生物催化和酶工程
- 有机合成 有机合成
- 药用化学 医学化学
背景情况:
- 维贝格龙是一种选择性β3-上腺素受体激动剂,需要一种特定的性前体, (2S,3R) - 氨基基 (1b).
- 这种中间体的高效合成对于 vibegron 生产至关重要.
- 野生类型的Exiguobacterium藻类碳基还原酶 (EaSDR6) 在催化 (rac) - 氨基乙醇 (1a) 的不对称还原方面存在局限性.
研究的目的:
- 为改进Exiguobacterium藻类碳缩酶 (EaSDR6) 的工程,以改善 (rac) - 氨基 Ester (1a) 的不对称降解到性前体 (1b).
- 为了提高酶的催化效率和立体选择性.
- 建立一个大规模生物合成vibegron中间体的框架.
主要方法:
- 在EaSDR6的循环工程中,创建了一个变体M5 (A138L/A190V/S193A/Y201F/N204A).
- 使用野生型 (WT) 和M5变体进行 (rac) - 氨基乙醇 (1a) 的不对称降解.
- 酶表征,结构对齐,分子动力学 (MD) 模拟和结合自由能计算.
- 在有机-水性双相条件下测试M5突变的性能,基板负荷较高.
主要成果:
- M5变种的催化效率增加了868倍 (kcat/Km = 260.3 s-1 mM-1),并且具有优越的立体选择性 (>99% e.e.,>99% d.e.) 与WT EaSDR6.6相比,这是一个很好的方法.
- MD模拟显示M5-1a复合物促进了更有效的核友性攻击,并更容易地采用了反应前状态.
- M5增强了疏水性相互作用,减少了不利的疏水性相互作用,导致对基质1a的亲和力增加.
- 在双相条件下,M5突变物在12小时内使用300g/L基底1a实现了>99%的转化.
结论:
- 功能循环工程显著提高了碳基还原酶的催化性能.
- 突变M5为vibegron中间合成提供了高效和立体选择性的生物催化剂.
- 这项研究为vibegron中间体的工业生物合成提供了一个强大的和可扩展的框架.
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