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改变7β-hydroxysteroid脱酶的辅助因子依赖性:通过辅助因子自我循环,提高单合成效率,使陈诺德氧胆酸转化为乌尔索德氧胆酸
Xiubing Xie1, Runyi Huang1, Wenchi Zhang2
1School of Biotechnology, Jiangnan University, Wuxi 214122, China.
International journal of biological macromolecules
|October 8, 2024
概括
工程设计的7β-基类固醇脱酶 (7β-HSDH) 增强了NADH的结合,使得ursodeoxycholic acid (UDCA) 从chenodeoxycholic acid (CDCA) 的高效单合成成为可能,同时减少了辅因子的使用.
科学领域:
- 生物化学 生物化学
- 酶工程是什么? 酶工程是什么?
- 代谢工程是代谢工程.
背景情况:
- 陈氧胆酸 (CDCA) 转化为ursodeoxycholic acid (UDCA) 对于胆酸代谢至关重要.
- 依赖NAD的7α-固醇脱酶 (7α-HSDH) 和依赖NADPH的7β-固醇脱酶 (7β-HSDH) 是这种生物转化中的关键酶.
- 优化辅因子回收对于UDCA的高效酶合成至关重要.
研究的目的:
- 为了提高Hyphomicrobium sp.的NADH结合亲和力. 7β-HSDH用于增强UDCA的单合成.
- 为了改造7β-HSDH,以提高辅因子依赖性和催化效率.
- 为同源的HSDH酶开发一种通用的辅因子依赖工程技术.
主要方法:
- 蛋白质结构建模和7β-HSDH的位点定向突变发生.
- 酶活性测定和动力分析 (kcat/Km) 来评估NADH结合亲和力.
- 模拟分子动力学以调查酶-连接体相互作用.
- 使用工程7β-HSDH和7α-HSDH进行UDCA的结合酶合成.
主要成果:
- 一种7β-HSDH的主导突变 (A22T/Q23E/L45A/N46E) 对NADH的kcat/Km增加了44.9倍,酶活性增加了65.2倍.
- 该工程酶催化了UDCA合成,从5g/L 7K-LCA和12.5mM NADH获得77.6%的产量.
- 结合酶系统仅使用0.5毫米的NAD+实现了90.1%的UDCA产量和54.1g/L/d的时空产量,证明了有效的辅因子循环.
结论:
- 工程7β-HSDH显著提高了NADH结合和催化效率,使得高效的UDCA生产成为可能.
- 开发的一合成策略减少了辅助因子的要求,并改善了整体工艺经济性.
- 这项工作为各种生物技术应用工程同类HSDH酶提供了一个多功能平台.
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