Substrate-Independent High-Throughput Assay for the Quantification of Aldehydes

Abstract

The selective and direct reduction of carboxylic acids into the corresponding aldehydes by chemical methods is still a challenging task in synthesis. Several reductive and oxidative chemical methods are known to produce aldehydes, but most of them require expensive reagents, special reaction conditions, are two-step procedures and often lack chemoselectivity. Nature provides an elegant tool, so called carboxylic acid reductases (CARs) for the direct reduction of carboxylic acids to aldehydes. Discovery as well as engineering of novel CAR enzymes necessitates a robust, product selective high-throughput assay (HTA). We report a simple and fast HTA that allows the substrate-independent and chemoselective quantification of aldehydes (irrespective of their chemical structure) and is sensitive to the nM range. The HTA was validated by NMR and GC analyses and in microbial cells by reexamination of the substrate scope of CAR from Nocardia iowensis (CAR(Ni)). The results were fully consistent with reported data.