Fig. 5
a Kinetic contribution of xylonolactonase to the in vitro pathway to glycolate using xylonolactone as a substrate. The effect of Cc XylC concentration to the glycolate production was measured at pH 7.0 at 22 °C and the production of glycolate was followed through generation of NADH from the enzymatic oxidation step from glycolaldehyde to glycolate. The assays were performed in microtiterplates containing 100 μl: 50 mM Tris HCl pH 7.0, 10 mM MgCl2, 0.5 mM DTT, 2 mM NAD, 1 mM xylonolactone, 2 µg Cc XylD, 2 µg Ec YagE, and 2 µg Ec AldA. The amount of Cc XylC in the reaction was varied 0 µg (black line), 2 µg (magenta line), 4 µg (cyan line), 8 µg (blue line), 16 µg (green line) and 24 µg (red line). b Effect of d-xylonolactone concentration on production of glycolate. The efficiency of the 4-enzyme pathway was followed through generation of NADH from the enzymatic reduction step from glycolaldehyde to glycolate. The assays were performed in microtiterplates containing 100 μl: 50 mM Tris HCl pH 7.0, 10 mM MgCl2, 0.5 mM DTT, 2 mM NAD, 0.5 mM xylonolactone (black line), 1 mM xylonolactone (red line), 2 mM xylonolactone (green line), 3 mM xylonolactone (blue line), 4 mM xylonolactone (cyan line) and 5 mM xylonolactone (magenta line), 4 µg Cc XylC, 2 µg Cc XylD, 2 µg Ec YagE, and 2 µg Ec AldA