Fig. 6

Optimisation of the in vitro pathway for glycolate production starting from d-xylonolactone. The assays were performed at 22 °C in microtiterplates containing 100 ul: 50 mM Tris HCl pH 7.0, 10 mM MgCl₂, 0.5 mM DTT, 2 mM NAD, and 1 mM xylonolactone as the starting reaction conditions. The concentration of each enzyme in the pathway was varied in four separate experiments, where the concentrations of the other three enzyme were always fixed. The amount of glycolate produced was measured through NADH production (at 340 nm). The enzyme concentrations tested were: a Cc XylC (0–7 µg), Cc XylD (2 µg), Ec YagE (1.4 µg), Ec AldA (2 µg), b Cc XylC (6 µg), Cc XylD (0–9 µg), Ec YagE (1.4 µg), Ec AldA (2 µg), c Cc XylC (6 µg), Cc XylD (8 µg), Ec YagE (0–2.5 µg), Ec AldA (2 µg), d Cc XylC (6 µg), Cc XylD (7.8 µg), Ec YagE (0.7 µg), Ec AldA (0–7 µg)