2.4 Activity assays and kinetic parameters
The activity was assayed by measuring the reduction of NAD(P) or the oxidation of NAD(P)H at 340 nm (ɛ = 6.22 mM−1cm−1) at 30 °C using a spectrophotometer (AMR-100 Microplate Reader, Hangzhou, China).[10] The standard assay mixture for Ps XR contained 50 mM galactose in 100 mM Tris-HCl (pH 8.0) buffer. The standard assay mixture forRl GDH, Pd PDH, and its mutants contained 50 mM galactitol in 100 mM Tris-HCl (pH 8.0) buffer. Reactions were started by adding 0.25 mL of a 10 mM NAD(P) or NADPH solution to a final volume of 1.0 mL. One unit is a protein that produces or consumes 1 µM of NADH/NADPH per second. The Km and kcatvalues were calculated using the Michaelis–Menten equation by nonlinear fitting using GraphPad Prism 9. The results are showed in Table 1 and Table 2.