A ninhydrin-based colorimetric assay for monitoring (S)-Norcoclaurine synthase activity through dopamine quantification

Abstract

(S)-Norcoclaurine synthase ((S)-NCS) catalyzes the stereoselective Pictet–Spengler condensation between dopamine and 4-hydroxyphenylacetaldehyde, forming (S)-norcoclaurine, a central precursor in benzylisoquinoline alkaloid biosynthesis. Beyond its biological role, (S)-NCS is a promising biocatalyst for the sustainable synthesis of chiral tetrahydroisoquinolines, which are valuable scaffolds in medicinal chemistry. However, broader application of this enzyme in biocatalysis remains limited by the lack of simple, selective, non-expensive, and high-throughput methods to quantify its activity. Herein, we describe a straightforward colorimetric method to monitor (S)-NCS activity by selectively detecting dopamine using ninhydrin. Our protocol combines ethanol-induced enzyme precipitation with mild incubation conditions, ensuring reliable, interference-free quantification by standard UV–Vis spectrophotometry. This low-cost, straightforward assay provides a robust alternative to conventional analytical methods. It has the potential to be adapted for high-throughput screening and may serve as a starting point for developing methods to monitor other enzymatic reactions that consume primary amines, facilitating studies in enzyme engineering and sustainable biocatalysis.