Accurate yet efficient high-throughput screenings have emerged as essential technology for enzyme engineering via directed evolution. Modern high-throughput screening platforms for oxidoreductases are commonly assisted by technologies such as surface display and rely on emulsification techniques to facilitate single-cell analysis via fluorescence-activated cell sorting. Empowered by the dramatically increased throughput, the screening of significantly larger sequence spaces in acceptable time frames is achieved but usually comes at the cost of restricted applicability. In this work, we tackle this problem by utilizing roGFP2-Orp1 as a fluorescent one-component detection system for enzymatic H₂O₂ formation. We determined the kinetic parameters of the roGFP2-Orp1 reaction with H₂O₂ and established an efficient immobilization technique for the sensor on Saccharomyces cerevisiae cells employing the lectin Concanavalin A. This allowed to realize a peroxide-sensing shell on enzyme-displaying cells, a system that was successfully employed to screen for H₂O₂ formation of enzyme variants in a whole-cell setting.

中文翻译：

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开发使用荧光过氧化物传感器roGFP2-Orp1筛选氧化酶活性的细胞结合检测系统。

通过定向进化，准确而高效的高通量筛选已成为酶工程学的重要技术。用于氧化还原酶的现代高通量筛选平台通常由表面展示等技术辅助，并依靠乳化技术通过荧光激活的细胞分选促进单细胞分析。通过显着增加的通量，可以在可接受的时间范围内筛选出较大的序列空间，但通常以限制适用性为代价。在这项工作中，我们通过利用roGFP2-Orp1作为酶促H ₂ O ₂形成的荧光单组分检测系统来解决这个问题。我们确定了H与roGFP2-Orp1反应的动力学参数₂ O ₂并使用凝集素伴刀豆球蛋白A建立了一种将传感器固定在啤酒酵母细胞上的有效固定技术。这使得在酶展示细胞上实现了过氧化物敏感壳，该系统已成功用于筛选H ₂ O ₂在全细胞环境中形成酶变体。