精氨酸脱亚胺酶(ADI)因其客观根除癌细胞和产生L-瓜氨酸的能力而引起了极大的兴趣。为了满足生产需求,本研究旨在提高ADI的酶活性和热稳定性。在这项研究中,通过计算机辅助位点特异性突变在粪肠杆菌的ADI中获得24个ADI突变体。值得注意的是,F44W的特定酶活性,N163P,E220I,E220L,N318E,A336G,T340I,N382F增加了,达到原始酶的1.33-2.53倍。这项研究证实,位点特异性突变对于优化酶功能至关重要。此外,F44W,N163P,E220I,T340I,和A336G突变体表现出良好的热稳定性。突变体F44W的最佳pH值增加到8,而突变体E220I,I244V,A336G,T340I,和N328F维持7.5的最佳pH。相反,M109L,N163P,E220L,I244L,N318E突变体的最佳pH为7。这项研究表明,活性增加的突变酶更可能包含位于与催化残基相关的四个环附近的突变位点,而二聚体连接位点的突变具有更高的增强酶稳定性的趋势。这些发现有助于ADI工业应用的发展及其改进。
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.