To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetrameric enzymes were obtained. Compared to the wild-type tetrameric L-asparease, these mutants remained 19.7%, 42.3%, and 92% of the enzyme activity, respectively. Moreover, the examination of enzyme properties revealed that their k_ cat and K_M changed in varying degrees, and the optimum pH shifted towards acidic pH, while the dependence of the activity of enzyme on Mg 2+ concentration and thermostability increased. Therefore this strategy provides a novel approach to directed evolution of enzymes. To enhance the relative movement of domains, we inserted a random sequence of fifteen-peptide into the three domains of L-aspartase. By means of directed screening, the three isoforms of monomeric, dimmeric and tetrameric enzymes were obtained. Compared to the wild- type tetrameric L-asparease, these mutants were 19.7%, 42.3%, and 92% of the enzyme activity, respectively. Moreover, the examination of enzyme properties revealed that their k_cat and K_M changed in varying degrees, and the optimum pH shifted towards acidic pH, while the dependence of the activity of enzyme on Mg 2+ concentration and thermostability increased.