关键词: 谷氨酸脱羧酶, 定向进化, 酶活性

Abstract: In order to improve the activity of glutamate decarboxylase (GAD) of Bacillus megaterium, its encoding gene was molecularly modified by directed evolution. Mutants A5-3, E2-4 and E3-11 were screened out from more than 13 000 mutants by two rounds of error-prone PCR, whose specific activities were increased respectively by 157%, 115% and 97% and whose Kcat/Km ratio was also increased compared to the wild-type strain. The amino acid sequence of A5-3 showed two mutations, A55D and D451E. From the results of 3-dimensional simulations, the alanine to aspartate mutation at position 55 may provide H+ to the enzymatic reaction, thereby accelerating the reaction rate. In mutant E2-4, the leucine to glutamine mutation at position 34 may improve the thermal stability of the enzyme. As for mutant E3-11, the mutation of alanine 325 to serine was conducive to the formation of more hydrogen bonds inside the protein, increasing the flexibility of the site and facilitating the interaction between amino acid residues. The circular dichroism analysis showed that the mutants had similar three-dimensional structure to the wild-type strain. Compared to the wild-type enzyme, the mutant enzymes contained less α-helix but more random coil, indicating a slight decrease in rigidity and an increase in flexibility. This study indicates that directional evolution can effectively improve the glutamate decarboxylase activity of B. megaterium, which will lay the foundation for its industrial application.

Key words: glutamate decarboxylase, directed evolution, enzyme activity