Li Mengli, Zhang Yijing, Zhang Tao, Miao Ming
State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu 214122, China.
State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China.
Int J Biol Macromol. 2025 Jan;284(Pt 1):138004. doi: 10.1016/j.ijbiomac.2024.138004. Epub 2024 Nov 23.
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.
精氨酸脱亚氨酶（ADI）在临床和工业应用中具有潜力，但其在生理条件下较低的热稳定性和催化效率限制了其用途。在这项工作中，对粪肠球菌SK32.001的ADI进行了合理设计。通过依次叠加单点正向突变体（F44W、N163P、E220L、N318E、A336G、T340I）构建了总共120个组合突变体，范围从两点突变到六点突变。其中，突变体S604、S700、S601和S606表现出更高的T值，而突变体S605、S547、S602、S607、S517和S557表现出增强的酶活性。值得注意的是，五点突变体S547（F44W/N163P/E220I/A336G/T340I）表现出显著的pH耐受性（pH 4.5 - 9.5，残留酶活性超过80%）。其比酶活性达到131.60 U/mg，比野生型酶高2倍。该酶的T值增加到64.04℃，比野生型酶高11.62℃。AlphaFold 2预测的结构表明，增加的刚性、新氢键的形成以及疏水残基的增加可能是酶活性和热稳定性增强的原因。这项研究表明，合理的设计策略可以有效地优化酶的性质，为具有工业相关性的微生物酶的开发提供见解。
