以设计为基础,构建自给自足的细胞染色体P450融合酶,以高效地氧化阿弗梅克
Chao Xiang1, Jia-Jia Mou1, Yue Jiang1
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China; Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, China; National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, China.
International journal of biological macromolecules
|February 28, 2026
概括
研究人员通过将P450s与氧化还原合作伙伴联系起来,设计了自给自足的细胞染色体P450融合酶. 这提高了氧化反应的生物催化剂效率,显示了工业应用中的活性和转化率的显著改善.
科学领域:
- 生物催化和酶工程 生物催化和酶工程
- 蛋白质工程是指蛋白质工程.
- 计算生物学 计算生物学
背景情况:
- 细胞染色体P450酶是强大的氧化生物催化剂,但由于氧化还原伙伴的低效电子转移而受到限制.
- 在工业氧化反应中,P450s与氧化还原伙伴 (RPs) 之间的短暂相互作用阻碍了它们的实际应用.
研究的目的:
- 通过共价连接P450s与RP来开发自给自足的P450融合酶,以克服电子转移的限制.
- 优化链接序列和长度,以提高酶活性和电子转移效率.
- 通过结合实验和计算方法,建立一个设计高效P450生物催化剂的框架.
主要方法:
- 通过将P450-Ema1-M212A与使用各种链接序列和长度的选定RP连接,构建融合酶.
- 计算建模,包括AlphaFold3多蛋白组件和分子动力学模拟,以预测和分析链接效应.
- 融合变体的实验性表征和动力分析 (kcat/Km),包括特定基质合成的转换率.
主要成果:
- 一种9-氨基酸链接剂 (F4变体) 显示出最佳的酶活性,稳定功能性构造并促进有效的电子转移.
- 与非聚变P450.0相比,F4聚变酶显著提高了催化效率 (kcat/Km).
- 在8小时内实现了90%的阿弗梅克丁转化为4′′-oxo-avermectin,展示了生物催化合成的卓越性能.
结论:
- 开发了一个半理性的,以实验驱动的框架,用于创建自给自足的P450融合酶.
- 证明了计算预测和蛋白质工程在提高生物催化剂性能方面的协同作用.
- 工程融合酶在氧化生物转化中的工业应用具有很高的潜力.
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