High-Throughput Screening for Enhanced Thermal Stability of Inherently Salt-Tolerant <scp>l</scp>-Glutaminase and Its Efficient Expression in <i>Bacillus licheniformis</i>

In addressing the challenges posed by extended fermentation cycles and high-salt conditions in high-salt liquid-state fermentation soy sauce (HLFSS) production, a high-throughput screening method was devised to identify thermally stable l-glutaminase mutants. This study yielded mutants A146D and A51D, exhibiting enhanced thermal stability. Computer-aided analysis revealed that these mutations introduced additional forces, compacting the protein structure and lowering the Gibbs free energy, thereby improving thermostability. Furthermore, the incorporation of aspartic acid augmented the negative surface charge, contributing to superior salt tolerance compared to the wild type (WT). Notably, in a 25% NaCl buffer, A146D and A51D demonstrated half-lives of 72.57 and 71.31 day, respectively, surpassing the WT's 59.08 day. In a 5 L bioreactor, the optimal mutant A146D achieved a remarkable enzymatic activity of 2800.78 ± 98.1 U/mL in recombinant <i>Bacillus licheniformis</i> fermentation broth, setting a new benchmark. This research offers valuable insights and a foundation for the modification and application of l-glutaminase in the food industry, particularly in HLFSS brewing.