Abstract: In order to improve its L-asparaginase activity, the L-asparaginase gene of Bacillus licheniformis was molecularly modified by directed evolution. Mutants S10, S16 and S21 were screened out of more than 19100 mutants by two rounds of error-prone PCR and one round of DNA shuffling, whose specific activities were increased by 106%, 74% and 43%, respectively, as compared to that of the wild type, together increased Kcat/Km. The amino acid sequence of S10 showed three mutations, K43E, N67S and I269L. The results of three-dimensional simulation showed that amino acid mutations at position 43 for glutamic acid and at position 67 for serine may improve substrate affinity and catalytic efficiency, thereby increasing enzymatic activity. The circular dichroism analysis showed that the mutant enzymes contained less α-helix and more random coil than the wild enzyme, indicating a slight decrease in rigidity and an increase in flexibility. This study indicates that directional evolution can effectively improve the L-asparaginase activity from B. licheniformis.

Key words: L-asparaginase, directed evolution, enzyme activity