Enhanced thermostability and catalytic activity for arginine deiminase from Enterobacter faecalis SK32.001 via combinatorial mutagenesis.

Arginine deiminase (ADI) exhibits potential for clinical and industrial applications, yet its low thermostability and catalytic efficiency under physiological conditions limit its utility. In this work, the ADI of Enterococcus faecalis SK32.001 was rationally designed. A total of 120 combinatorial mutants, ranging from two-point to six-point mutations, were constructed by sequentially stacking single-point positive mutants (F44W, N163P, E220L, N318E, A336G, T340I). Among them, the mutants S604, S700, S601, and S606 exhibited higher T values, while the mutants S605, S547, S602, S607, S517, and S557 demonstrated enhanced enzymatic activity. Notably, the five-point mutant S547 (F44W/N163P/E220I/A336G/T340I) exhibited remarkable pH tolerance (pH 4.5-9.5, with over 80 % residual enzyme activity). Its specific enzyme activity reached 131.60 U/mg, which was 2-fold higher than that of wild enzyme. The T value of this enzyme increases to 64.04 °C, 11.62 °C higher than that of the wild-type enzyme. The structure predicted by AlphaFold 2 revealed that the increased rigidity, formation of new hydrogen bonds, and an increase in hydrophobic residues may account for the enhanced enzyme activity and thermostability. This research demonstrates that rational design strategies can effectively optimize enzyme properties, providing insights for the development of microbial enzymes with industrial relevance.