Combinatorial engineering for efficient production of protein-glutaminase in Bacillus subtilis

Protein-glutaminase (EC 3.5.1.44, PG) converts protein glutamine residues in proteins and peptides into glutamic acid residue, and markedly improves the solubility, emulsification, and foaming properties of food proteins. However, the source bacteria, Chryseobacterium proteolyticum, have low enzyme production ability, inefficient genetic operation, and high production cost. Therefore, it is critical to establish an efficient expression system for active PG. Here, combinatorial engineering was developed for high-yield production of PG in Bacillus subtilis. First, we evaluated different B. subtilis strains for PG self-activation. Then, combinatorial optimization involving promoters, signal peptides, and culture medium was applied to produce active recombinant PG in a B. subtilis expression system. Through combinatorial engineering, PG enzyme activity reached 3.23 U/mL in shaken-flask cultures. Active PG with the yield of 7.07 U/mL was obtained at 40 h by the P_SecA-YdeJ combination in fed-batch fermentation, which is the highest yield of PG in existing reports.