Ribulose 5-Phosphate 3-Epimerase Assays

The method of determining the ribulose-5-phosphate 3-epimerase activity was described in a previous paper (Dante et al., 2003). All enzyme assays were performed at 30°C. The assay principle is based on the consumption of NADH, coupled with a series of enzyme-catalyzed reactions. Before the reaction, the mixtures were equilibrated for 3 min at the reaction temperature, and the reactions were started by the addition of the cell-free extract. The change in the A₃₄₀ due to NADH consumption was measured using a BioTek neo2SM multi-mode reader (BioTek, Vermont, America). The transketolase (TktA from E. coli) was purified according to a published method (Amanatuzzakiah et al., 2013). A BCA protein assay kit (Solarbio, Beijing, China) was used to determine the protein concentration. The enzyme activity assays were performed in triplicates, and the reported results represented the average of three independent experiments.