Dual Strategy for Enhancing the Production Efficiency of Protein-Glutaminase in <i>Escherichia coli</i> Nissle 1917

Protein-glutaminase (PG) is a novel deamidase that has significant potential applications in the improvement of the functional properties of food proteins. However, the low production level of PG restricts its industrial applications. A dual strategy was applied to enhance the production efficiency of PG in heterologous expression systems. Strategy one was to improve the PG expression level by constructing the T7RNA polymerase-P_T7 plasmid in probiotic <i>Escherichia. coli</i> Nissle 1917, coupled with optimizations in the translation initiation region and fermentation processes, thereby increasing the PG yield 18.11-fold. Strategy two was to improve the PG activity by reshaping the substrate binding pocket, resulting in the acquisition of mutant PG-M8 with the highest specific activity. Combining the above two strategies, the PG yield reached 73.62 U/mL. This yield demonstrates its potential for industrial applications.