L-Aspartase was modified by directed evolution. After four rounds of error-prone PCR and three rounds of DNA shuffling, an evolved enzyme purified from the final round showed a 28-fold increased k(cat)/K-m and 4.6-fold decreased K-m. The thermostability and stable pH range were also enhanced. The DNA sequence of the evolved aspartase gene showed seven base changes, resulting in three amino acid changes from the native enzyme: N217K, T233R, V367G. The mechanism of the enhancement of activity was analyzed.