Recombinant e coli malate dehydrogenase

Malate dehydrogenase (EC 1.1.1.37) activity was assayed in the forward direction (NADH production) as previously described (Dasika et al., 2015 (link)). Mass inference was obtained by comparison to a standard curve made with recombinant E. coli malate dehydrogenase (Sigma-Aldrich, cat# SRP6105). Reactions were assembled in 96-well flat-bottom plates kept on ice. Sixteen microliters of different amounts of Leuginase®, Aginasa® and recombinant E. coli malate dehydrogenase (Sigma-Aldrich, cat# SRP6105) were added to 100 μL of a reaction mixture containing 1 mM NAD, 1 mM Malic acid, 90 mM KCl, 100 mM Tris, pH 8.9. NADH concentration was immediately measured by fluorescence at 470 nm with excitation at 340 nm, at 37 °C, using a Synergy H1 Hybrid Reader (Biotek). Data were measured over time. Data presented refer to 2:08 min, when a plateau was reached for the highest concentrations.