The kinetic parameters of WT-CsCE and mutants for
lactose conversion were determined at 80 ◦C with lactose concentration
over the range of 60–900mM (Table 2). The catalytic
efficiency (kcat/Km) of mutant E161D/S180P/S351G decreased to
approximately 56% of the wild type enzyme with a 1.6-fold increase
in apparent Km value. Mutant E161D/N365P had a 1.29-fold
increase in kcat/Km, which could be ascribed mainly to the improved
substrate affinity, because the apparent Km value decreased to
417.90mM from 581.37mM of wild type enzyme.