Fig. 2.
Characterization of purified D-LDH from P. vulgatus, overproduced in E. coli. a SDS/PAGE analysis of recombinant D-LDH using silver staining. Lane 1: PageRulerTM Prestained Protein Ladder (ThermoFisher Scientific). Lane 2: purified D-LDH (1.5 μg protein). b Michaelis–Menten kinetics. For the detection of NADH oxidation, purified D-LDH and pyruvate in different concentrations were added and the change in absorbance at 340 nm was analyzed at 37 °C. The enzyme assay (1 ml) contained 100 mM K-phosphate buffer, pH 7, and 250 μM NADH. The pyruvate concentration was varied between 0.02 and 9 mM. Nonlinear regression of the Michaelis–Menten data was used to calculate the kinetic constants Vmax and Km with the program GraphPad Prism