Rational design to improve the catalytic efficiency and stability of arginine deiminase

Abstract

Arginine deiminase (ADI) has garnered significant interest because of its ability to objectively eradicate cancer cells and produce L-citrulline. To meet the production demands, this study focused on enhancing the enzyme activity and thermal stability of ADI. In this study, 24 ADI mutants were obtained through computer aid site-specific mutation in the ADI of Enterobacter faecalis. Notably, the specific enzyme activities of F44W, N163P, E220I, E220L, N318E, A336G, T340I, and N382F increased, reaching 1.33–2.53 times that of the original enzyme. This study confirmed that site-specific mutations are critical for optimizing enzyme function. Additionally, the F44W, N163P, E220I, T340I, and A336G mutants demonstrated good thermal stability. The optimal pH for mutant F44W increased to 8, whereas mutants E220I, I244V, A336G, T340I, and N328F maintained an optimal pH of 7.5. Conversely, the M109L, N163P, E220L, I244L, and N318E mutants shad an optimal pH of 7. This study revealed that mutant enzymes with increased activity were more likely to contain mutation sites situated near the four loops associated with catalytic residues, whereas mutations at the dimer junction sites had a higher tendency to enhance enzyme stability. These findings contribute to the development of ADI industrial applications and its modifications.