Intein-Mediated Proenzyme Activation for Protein-Glutaminase Production in <i>Bacillus subtilis</i>

Protein-glutaminase (PG), as a novel food additive with a remarkable deamidation capacity, is naturally expressed as a proenzyme. Activation of the PG proenzyme is mostly dependent on proteases. To address this problem, an intein-mediated activation (IMA) system was developed for PG production using self-splicing inteins that can catalyze a single C-terminal cleavage. In this study, fusion proenzymes were expressed in <i>Escherichia coli</i> by inserting different inteins between the pro-peptide and mature PG and the C-terminal activation efficiency was evaluated <i>in vitro</i>. Subsequently, Mth RIR1 (RIR1) and Mxe GyrA (Mxe) were selected for construction of the IMA system in various <i>Bacillus subtilis</i> strains. Finally, the PG activity increased to 37.2 U/mL after promoter optimization and fed-batch fermentation in <i>B. subtilis</i> WB600. This approach enables one-step and nonprotease-dependent PG production in <i>B. subtilis</i> and establishing an alternative strategy for other proenzyme activation.