Catechin detection in tea samples based on catechin-induced conformational changes in papain

Abstract

Catechin, the major ingredient in tea that affects its taste, varies based on the tea variety, climate, and extraction techniques. Although several techniques have been developed to determine catechin levels, an easy-to-use method is still lacking. Herein, the catechin levels were determined using papain for the first time. After incubating papain with catechin, the precipitate was discarded, and the remaining catechin-enzyme complex in the supernatant was determined by UV-visible absorption spectroscopy. Upon catechin binding papain’s main absorption band (at 280nm) was altered, reflecting conformation changes in their aromatic amino acids. It was supported by the kinetic measurements determined with N-α-benzoyl-arginine-p-nitroanilide (BAPNA) as a substrate. Complex formation between catechin and papain markedly affects the enzyme activity, resulting in the diminishes of the yellow-colored product observed at 410nm in a concentration-dependent manner. Under the optimum conditions, the analytical curve was linear in the concentration range 0.10 – 0.80mg/mL (r2 = 0.9893) with a detection limit of 0.44mg/mL. This developed biosensor demonstrated long-term stability (90 days) and reproducibility with a relative standard deviation below 10%. This usefulness of papain’s conformation changes was further demonstrated in the analysis of tea infusion samples, and the recoveries are in the acceptable range (79.1-81.2%).