A Novel Salt-Tolerant L-Glutaminase: Efficient Functional Expression, Computer-Aided Design, and Application

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(H105K) 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(H105K) functional expression box was contributed to Bacillus subtilis 168 by auxotrophic complementation, and the production in a 5-L bioreactor was improved to 2516.78 +/- 20.83 U mL(-1), 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.