High-Throughput\nScreening for Enhanced Thermal Stability\nof Inherently Salt-Tolerant l‑Glutaminase and Its\nEfficient Expression in Bacillus licheniformis

In addressing the challenges posed by extended fermentation\ncycles\nand high-salt conditions in high-salt liquid-state fermentation soy\nsauce (HLFSS) production, a high-throughput screening method was devised\nto identify thermally stable l-glutaminase mutants. This\nstudy yielded mutants A146D and A51D, exhibiting enhanced thermal\nstability. Computer-aided analysis revealed that these mutations introduced\nadditional forces, compacting the protein structure and lowering the\nGibbs free energy, thereby improving thermostability. Furthermore,\nthe incorporation of aspartic acid augmented the negative surface\ncharge, contributing to superior salt tolerance compared to the wild\ntype (WT). Notably, in a 25% NaCl buffer, A146D and A51D demonstrated\nhalf-lives of 72.57 and 71.31 day, respectively, surpassing the WT’s\n59.08 day. In a 5 L bioreactor, the optimal mutant A146D achieved\na remarkable enzymatic activity of 2800.78 ± 98.1 U/mL in recombinant Bacillus licheniformis fermentation broth, setting\na new benchmark. This research offers valuable insights and a foundation\nfor the modification and application of l-glutaminase in\nthe food industry, particularly in HLFSS brewing.