*4.3. GOX Activity Measurements*

Enzyme activities were measured using 5 μg of purified recombinant GOX in 50 mM Tris-HCl, 0.1 mM FMN, pH 8.0 and different glycolate (0.05 to 10 mM), L-lactate (0.3 to 10 mM) and 2-hydroxy-octanoate (0.15 to 5 mM) concentrations by an enzyme-coupled reaction at 30 ◦C. H2O2 produced by GOX activity was quantified in the presence of 0.4 mM *o*-dianisidine and 2 U horseradish peroxidase by measuring the ΔA440nm using a Varian Cary 50 spectrophotometer. KM and kcat values were calculated using SigmaPlot 13.0 software, based on the curve fitting Michaelis–Menten equation: v0 = Vmax[S]/(KM + [S]).
