Department of Technical Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
Department of Technical Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
Methods Enzymol. 2020;644:63-93. doi: 10.1016/bs.mie.2020.04.022. Epub 2020 May 5.
Rieske non-heme iron dioxygenases (ROs) are promising candidates to perform dihydroxylation reactions, since they are capable to incorporate both atoms of molecular oxygen into vicinal non-activated CH bonds, endowing valuable products for pharmaceutical and chemical applications. ROs harbor attractive features such as, striking activity in combination with remarkable regio- and stereo-selectivity, wide reaction spectrum, and broad substrate scope. In order to identify, characterize, and enhance targeted features of dioxygenases and related oxygen dependent enzymes via enzyme engineering and evolution approaches, proper screening and analytical methods are essential to detect and to analyze the expected dihydroxylation activity. This chapter presents different methodologies suitable for the study of dihydroxylation reactions. Detailed descriptions of our established analytical protocols for both gas and liquid chromatography, as well as a colorimetric assay to detect dioxygenase activity are provided. In addition, a novel and reliable system for real-time detection of oxygen consumption, in vivo, is reported.
Rieske 非血红素铁双加氧酶（ROs）是进行二羟基化反应的有前途的候选者，因为它们能够将分子氧的两个原子掺入相邻的非活化 CH 键中，赋予具有药物和化学应用价值的有价值的产物。ROs 具有吸引人的特点，例如与显著的区域和立体选择性、广泛的反应谱和广泛的底物范围相结合的惊人活性。为了通过酶工程和进化方法来识别、表征和增强双加氧酶和相关氧依赖酶的靶向特征，适当的筛选和分析方法对于检测和分析预期的二羟基化活性至关重要。本章介绍了适用于二羟基化反应研究的不同方法学。提供了我们针对气相和液相色谱以及用于检测双加氧酶活性的比色测定法建立的分析方案的详细描述。此外，还报道了一种用于实时检测体内氧消耗的新颖可靠系统。
