Institute of Applied Synthetic Chemistry, OC-163, TU Wien, Getreidemarkt 9, 1060 Vienna, Austria.
Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, University of Greifswald, Felix-Hausdorff-Str. 4, 17489 Greifswald, Germany.
Chem Rev. 2023 Mar 22;123(6):2832-2901. doi: 10.1021/acs.chemrev.2c00304. Epub 2023 Feb 28.
Many successful stories in enzyme engineering are based on the creation of randomized diversity in large mutant libraries, containing millions to billions of enzyme variants. Methods that enabled their evaluation with high throughput are dominated by spectroscopic techniques due to their high speed and sensitivity. A large proportion of studies relies on fluorogenic substrates that mimic the chemical properties of the target or coupled enzymatic assays with an optical read-out that assesses the desired catalytic efficiency indirectly. The most reliable hits, however, are achieved by screening for conversions of the starting material to the desired product. For this purpose, functional group assays offer a general approach to achieve a fast, optical read-out. They use the chemoselectivity, differences in electronic and steric properties of various functional groups, to reduce the number of false-positive results and the analytical noise stemming from enzymatic background activities. This review summarizes the developments and use of functional group probes for chemoselective derivatizations, with a clear focus on screening for enzymatic activity in protein engineering.
许多成功的酶工程案例都是基于在大型突变文库中创造随机多样性，其中包含数百万到数十亿种酶变体。由于速度快、灵敏度高，能够实现高通量评估的方法主要是光谱技术。由于荧光底物可以模拟目标的化学性质，或者与光学读取的偶联酶测定法结合使用，从而间接评估所需的催化效率，因此很大一部分研究依赖于这些方法。然而，最可靠的结果是通过筛选起始物质转化为所需产物来实现。为此，官能团测定法提供了一种快速、光学读取的通用方法。它们利用各种官能团的化学选择性、电子和空间性质的差异，来减少假阳性结果的数量，并降低源自酶背景活性的分析噪声。本文综述了用于化学选择性衍生化的官能团探针的发展和应用，重点是在蛋白质工程中筛选酶活性。
