Arginine deiminase derived from Halothermothrix orenii has the activity of catalyzing the hydrolysis of arginine to citrulline, but its thermal stability needs to be further improved. After a three-dimensional structural analysis combined with the HotSpot Wizard webserver to identify hot spots for rational design to improve its thermal stability, 10 single-point mutants were selected. Through molecular cloning, expression purification, and denaturation temperature (Tm) measurement, two mutants T180Y and A190P with slightly reduced enzyme activity but improved thermal stability were obtained, and the double point mutant T180Y/A190P was orderly stacked. The Tm values of T180Y, A190P, and T180Y/A190P were increased by 2.5, 1.9, and 5.2 ℃, respectively, and their specific enzyme activities were 91.36%, 80.53%, and 78.41% of the wild type. In addition, the optimal temperature of the single point mutants T180Y and A190P was the same as that of the wild type, while the optimal temperature of the two-point combination mutant T180Y/A190P increased by 5 ℃. Compared with the wild enzyme, the residual enzyme activities of the three positive mutants were increased by 30%, 23%, and 41.8% after incubation at 50 ℃ for 180 min, respectively. After molecular dynamics simulation, the results show that the RMSF value of the 180 and 190 sites region was higher than that of the wild type, indicating that the mutation of these two sites reduces the flexibility and enhances the rigidity of the region, making it more thermally stable. On the other hand, the reasons for the improved thermal stability were further analyzed from the three-dimensional structure. The three-dimensional structure of T180Y showed that the larger side chain groups of Tyr filled the adjacent surface cavities, and there was an aromatic interaction (cation-π) between Tyr and Arg269, which is closer in space. While A190P is presumed to be due to the better rigid structure of the pyrrole ring possessed by proline and the enhanced hydrophobic and polar interactions between proline and adjacent amino acid residues. Subsequently, the mutant was applied to the production of citrulline, the production rate of the double point mutant T180Y/A190P was 12.5% higher than that of the wild type, and the citrulline production was increased by 5 g/L. This study provides a reference for the rational design of other industrial enzymes.
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