*4.7. Assay for KDG Kinase Activity*

KDG kinase activity was assayed as follows. The reaction mixture containing 2.5 mM KDG, 2.5 mM ATP, 5 mM MgCl2, 20 mM Tris-HCl (pH 7.4), 100 mM KCl, 1 mM DTT, and 10 μg/mL recFlKin was incubated at 30 ◦C. At reaction times, 1, 15, and 30 min, an aliquot (160 μL) of the reaction mixture was taken out and heated at 100 ◦C for 3 min to terminate the reaction. To the mixture, 240 μL of a buffer containing 84 mM Tris-HCl (pH 7.4), 167 mM KCl, 0.67 mM NADH, 2.5 μg/mL recFlAld, and 1 unit of LDH was added and the pyruvate released was determined by the LDH–NADH system. One unit (U) of KDG kinase activity was defined as the amount of enzyme that produced 1 μmol of KDPG per min. Temperature dependence of recFlKin was determined at 10–60 ◦C. Thermal stability of recFlKin was assessed by measuring the activity remaining after the incubation at 10–50 ◦C for 30 min. pH dependence of recFlKin was determined with reaction mixtures adjusted to pH 4.5–5.3 with 20 mM CH3COONa buffer, pH 5.6–7.3 with 20 mM PIPES-NaOH buffer, pH 7.1–8.8 with 20 mM Tris-HCl buffer, and pH 9.1–9.7 with 20 mM glycine–NaOH buffer. The activity assay was conducted three times and the mean value was shown with standard deviation in each figure.
