Enzyme Assays for Chalcone Isomerase and Synthase

The purified protein was quantified using a Protein Quantitative Kit (BCA; Trans Gen Biotech, Beijing, China). For the chalcone isomerase assay, the reaction was in a volume of 200 μl, containing 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 10 μg purified protein, and 100 μM naringenin chalcone. The substrate is finally added to the reaction and the reaction incubated for 1 min at room temperature. For chalcone synthase assay, the reaction was in a volume of 250 μl, containing 100 mM HEPES-KOH buffer (pH 7.0), 2 mM DTT, 10 μg purified protein, 200 μM malonyl CoA and p-Coumaryl CoA at a series of concentrations (20–200 μM). The reaction was carried out at 35°C for 30 min. For CHS/CHIL complex analysis, the reaction was carried out according to the procedure described by Ban (Ban et al., 2018 (link)). After extraction, the product detected at 280 nm. Three biological replicates were analyzed of each enzyme reaction.