A high-throughput pH-based colorimetric assay: application focus on alpha/beta hydrolases

作者：Paye Marietou F; Rose Harrison B; Robbins John M; Yunda Diana A; Cho Seonggeon; Bommarius Andreas S^*

来源：Analytical Biochemistry, 2018, 549: 80-90.

DOI：10.1016/j.ab.2018.03.009

• pH
• Colorimetric
• Alpha/beta hydrolase
• AEH
• CocE

Research involving alpha/beta hydrolases, including alpha-amino acid ester hydrolase and cocaine esterase, has been limited by the lack of an online high throughput screening assay. The development of a high throughput screening assay capable of detecting alpha/beta hydrolase activity toward specific substrates and/or chemical reactions (e.g., hydrolysis in lieu of amidase activity and/or synthesis instead of thioesterase activity) is of interest in a broad set of scientific questions and applications. Here we present a general framework for pH-based colorimetric assays, as well as the mathematical considerations necessary to estimate de novo the experimental response required to assign a 'hit' or a 'miss,' in the absence of experimental standard curves. This combination is valuable for screening the hydrolysis and synthesis activity of alpha/beta hydrolases on a variety of substrates, and produces data comparable to the current standard technique involving High Performance Liquid Chromatography (HPLC). In contrast to HPLC, this assay enables screening experiments to be performed with greater efficiency.

• 出版日期2018-5-15