The effects of hydrogen peroxide on enterokinase catalysis were studied using several fusion proteins recombinantly produced from E. coli. It was demonstrated that hydrogen peroxide enhanced the rate of enterokinase cleavage reaction, leading to a faster release of the target peptide as discussed in patent WO07149053. Among the conditions tested, we observed that hydrogen peroxide could exert its effect on the cleavage of fusion proteins over a wide range of pH and temperature. This finding might provide a simple solution for the accelerated enterokinase cleavage of thermolabile fusion proteins at low temperature.