High-Throughput Fluorescence Assay for Ketone Detection and Its Applications in Enzyme Mining and Protein Engineering

Ketones are of great importance as building blocks in synthetic organic chemistry and biocatalysis. Most ketones cannot easily be quantitatively assayed due to the lack of visible photometric properties. Effective high-throughput assay (HTA) development is therefore necessary for ketone determination. Inspired by previous works of an aldehyde assay based on 2-amino benzamidoxime derivatives, we developed a colorimetric method for rapid a HTA of structurally diverse ketones by using para-methoxy-2-amino benzamidoxime (PMA). This PMA-based method is characterized by high sensitivity manner (μM) with low background, as checked by gas chromatography (GC). It can be used for quantitatively monitoring ketones by fluorescence screening in microtiter plates. Furthermore, this HTA method was employed in mining alcohol dehydrogenases (ADHs), and in directed evolution aimed at enhancing ADH activity in the catalytic transformation of alcohols to ketones. This work provides a general tool for ketone detection in biocatalyst development.