基于结构的设计,以提高阿尔多-基因还原酶KdAKRR的催化性能
Chen Dai1,2,3, Hai-Xing Cao1,2,3, Jia-Xin Tian1,2,3
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, People's Republic of China.
工程化生物催化剂称为阿尔多-基因还原酶 (AKRs) 显示增强的活性和稳定性,用于合成性酒精. 一种经过修改的KdAKR酶显著提高了催化效率和立体选择性,有助于制药生产.
科学领域:
- 生物催化和酶工程 生物催化和酶工程
- 有机化学和制药合成 有机化学和制药合成
- 蛋白质结构与功能之间的关系.
背景情况:
- 阿尔多基因减少酶 (AKR) 是制造药品中使用的性酒精的关键生物催化剂.
- 优化AKR活动,立体选择性和稳定性是高效合成的关键.
- 来自Kluyveromyces dobzhanskii (KdAKR) 的依赖NADPH的AKR是蛋白质工程的目标.
研究的目的:
- 通过向突变发生来增强KdAKR的催化活性和立体选择性.
- 使用共识序列方法来提高KdAKR的热稳定性.
- 为未来的酶设计提供AKR结构-功能关系的见解.
主要方法:
- 用于修改KdAKR基底结合口袋周围的循环区域,采用了位点定向的突变发生.
- 应用了一种共识序列方法来增强酶的热稳定性.
- 进行了酶动力学,立体选择性测试 (dep) 和稳定性测试 (t1/2).
主要成果:
- 最好的突变物M6对 (5S) -CHOH的催化效率增加了67倍,并增强了R选择性 (>99.5%dep)
- 与野生型KdAKR相比,M6在40°C的半衰期长6.3倍.
- 在8小时内,M6有效地将200g/L的 (5S) -CHOH转化为纯 (3R,5S) -CDHH,实现时空产量为300.7g/L/day.
结论:
- 工程设计的KdAKR (M6) 在催化效率,立体选择性和热稳定性方面明显优于野生类型.
- 这项研究为修改AKR以提高生物催化性能提供了有价值的框架.
- 这些发现有助于开发有效的酵素途径,以治疗性药物中间体.
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