| Summary: | The low productivity in long fermentation duration and high-salt working conditions limit the application of L-glutaminase in soy sauce brewing. In this study, a novel L-glutaminase (LreuglsA) with eminent salt tolerance was mined and achieved more than 70% activity with 30% NaCl. To improve the robustness of the enzyme at different fermentation strategies, mutation LreuglsA<sup>H105K</sup> was built by a computer-aided design, and the recombinant protein expression level, an essential parameter in industrial applications, was increased 5.61-fold with the synthetic biology strategy by improving the mRNA stability. Finally, the LreuglsA<sup>H105K</sup> functional expression box was contributed to <i>Bacillus subtilis</i> 168 by auxotrophic complementation, and the production in a 5-L bioreactor was improved to 2516.78 ± 20.83 U mL<sup>−1</sup>, the highest production ever reported. When the immobilized cells were applied to high-salt dilute-state soy sauce brewing, the L-glutamate level was increased by 45.9%. This work provides insight into the salt-tolerant enzyme for improving the efficiency of industrial applications.
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