高频多位点突变对碱性蛋白酶定向进化影响作用的研究

Proteases are important biocatalysts in industrial applications. Although they can be produced by many microbe, proteases often require intensive modification to get desired performance for the applications. Directed evolution is powerful algorithm for protease reengineering. However the commonly used screening formats such as agar plate (halo formation) or microplate-based detection system have a low throughput and need to repeat construction of mutant libraries with low mutational loads and screening process many times to get an improved variant. In contrast, a flow cytometry-based ultra-high throughput screening system will enable a novel opportunity in directed enzyme evolution under high mutational loads in a single run. In the study, a subtilisin Carlsberg protease will be investigated by the flow cytometry-based evolution strategy using a library with a high mutational load and screened for increased activity at low temperature. The developed evolution strategy might highly improve the screening efficiency due to cooperative effects of high amino acids changes for the improved activity, and can likely be modified for other proteases and applied for additional properties.

蛋白酶是一类重要的生物催化剂，具有广泛的工业应用价值。通过定向进化手段，可以在体外进行蛋白质（酶）的工程改造。目前常用的进化策略是通过多轮筛选低突变频率的突变体库，重复构建低频率突变体库和筛选过程获得改造突变子，在筛选效率和效果上有待提高。本研究将利用前期工作建立的蛋白酶流式细胞仪超高通量筛选系统，以工业洗涤业对低温蛋白酶的需求为应用导向，探索新型进化策略，即单轮高通量筛选万级容量以上的高频率突变体库，获得具有多位点突变产生协同效应的进化蛋白酶，并对获得的突变子进行关键氨基酸位点分析，推测与蛋白酶低温酶活力相关的作用机制，为缩短改造周期，有效进行蛋白酶的定向进化改造工程提供理论和实验基础。

蛋白酶是一类重要的生物催化剂，具有广泛应用价值。定向进化是获得特异性能蛋白酶的有效途径。本研究针对蛋白酶的高效进化策略问题，开发了高效枯草芽孢杆菌转化方法，达到100万个转化子/μg质粒转化效率，建立了蛋白酶高频率突变体库。研制了基于液滴微流控高通量筛选平台和仪器装置，可以进行单细胞的液滴包埋、孵育和检测，具备10万个/分钟的检测速度，液滴分选速度达到5000个/每分钟。获得了低温时蛋白酶酶活力提高的突变子5个，探索了基于活性位点中心底物反应腔结构柔性和底物作用能力的作用机制。研究为后续蛋白酶的定向进化改造工程提供了良好的研究平台和基础。