Intein-Mediated Proenzyme Activation for Protein-Glutaminase Production in Bacillus subtilis

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 Escherichia coli by inserting different inteins between the pro-peptide and mature PG and the C-terminal activation efficiency was evaluated in vitro. Subsequently, Mth RIR1 (RIR1) and Mxe GyrA (Mxe) were selected for construction of the IMA system in various Bacillus subtilis strains. Finally, the PG activity increased to 37.2 U/mL after promoter optimization and fed-batch fermentation in B. subtilis WB600. This approach enables one-step and nonprotease-dependent PG production in B. subtilis and establishing an alternative strategy for other proenzyme activation.