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克隆,表达和分子进化Komagataella phaffii 基因减少酶与增强的催化活性

Mohd Imran Shah¹, Kavitha Ramaswamy¹, Sangita Venkataraman¹

¹Department of Biotechnology, Anna University, Chennai, Tamil Nadu, India.

Preparative biochemistry & biotechnology

|July 4, 2025

概括

工程化降解酶 (KpKR变体) 显示了药物合成效率的提高. 使用核糖体显示的定向进化产生了具有增强基和基活性的生物催化剂.

科学领域:

生物催化和酶工程生物催化和酶工程
合成化学合成化学
分子生物学分子生物学

背景情况:

光学活性醇对于制药至关重要,但传统合成缺乏效率和立体选择性.
降解酶提供了一个更绿色的替代品,但在工业应用中需要增强的催化性能.
工程现有酶是满足高效和选择性生物催化剂需求的关键.

研究的目的:

通过定向进化来改造 *Komagataella phaffii* 缩酶 (KpKR) 以提高催化性能.
识别和描述具有增强活性和工业合成特异性的新型KpKR变体.
为了证明结合易出错的PCR和核糖体显示用于快速酶工程的有效性.

主要方法:

使用易发生错误的PCR和核糖体显示,对*Komagataella phaffii*基因减少酶 (KpKR) 的定向进化.
动力分析,分子对接,同质建模和基质特异性测试用于表征进化的变体.
结构和序列分析以阐明改善催化活性的机制.

主要成果:

确定了五种进化的KpKR变体 (M1-M5),与微生物缩酶相比,它们具有更高的催化性能.
变体M5显示了增强的催化效率 (kcat/Km = 199.58 s−1mM−1) 对于基因;变体M1显示了高活性对于基质.
结构分析揭示了C端修改,有助于提高活性部位的可访问性和改善酶功能.

结论:

进化的KpKR变体是高效的生物催化剂,适合挑战性制药合成.
核糖体显示是快速酶工程的有效工具,产生具有工业相关性质的变体.
这项研究提供了强大的,工程化降解酶,用于可持续和选择性合成吉拉性酒精.

关键词:

易发生错误的突变性突变发生.同源性建模的同源性建模凯托降解酶 (ketoreductase) 是一种分子对接的分子对接.具有光学活性的酒精.核糖体显示器可以显示核糖体.

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