使用酵母显示器的聚合物降解酶的超高通量定向进化
Mario A Cribari1, Maxwell J Unger1, Ilona C Unarta1,2
1Department of Chemistry, University of Wisconsin─Madison, Madison, Wisconsin 53706, United States.
Journal of the American Chemical Society
|December 5, 2023
概括
研究人员开发了一种高通量酵母表面显示平台, 这种新方法选了超过1000万种酶突变,显著提高了聚乙烯二甲 (PET) 的降解能力.
科学领域:
- 生物技术
- 生物化学
- 聚合物科学
背景情况:
- 合成聚合物的酶降解为塑料回收提供了一种可持续的方法.
- 定向进化对于增强塑料等非自然基质上的酶活性至关重要.
- 现有的酶进化选方法的吞吐量有限,阻碍了快速发现.
研究的目的:
- 开发一个高通量平台,用于塑料降解酶的定向演化.
- 显著提高查能力以识别增强的酶变体.
- 提高聚乙烯二甲 (PET) 脱聚合的效率.
主要方法:
- 使用酵母表面显示以在酵母细胞上呈现单个酶突变物.
- 采用基于光的测定方法来检测合成聚合物探针裂时的酶活性.
- 使用光激活细胞分类 (FACS) 进行高通量分离活跃突变.
- 进行了DNA测序,以确定叶子和枝叶堆肥酶 (LCC) 的定向进化中的有益突变.
主要成果:
- 开发了一个能够选10^7个酶突变的平台,比以前的方法增加了3个数量级.
- 鉴定出突变,大大提高了LCC酶对固体PET薄膜的降解动力.
- 生物化学试验和分子动力学模拟表明,H218Y突变改善了酶基质与PET的结合.
结论:
- 酵母表面展示平台极大地加速了聚合物降解酶的发现和优化.
- 这种方法可以识别在固体塑料基板上显著提高活性的酶变体.
- 开发的平台为推进环保塑料回收技术带来了巨大的前景.
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