Alkaline Serine Protease AprE Plays an Essential Role in Poly-γ-glutamate Production during Natto Fermentation

Natto is a traditional Japanese food made from soybeans fermented by natto starter strains of Bacillus subtilis natto. It has been suggested that extracellular protease activity released by the bacteria are involved in the production of poly-gamma-glutamate (gamma-PGA) during natto fermentation. One of the natto starters, strain r22, possesses at least seven genes, each of which encoded an extracellular protease orthologous to its counterpart in B. subtilis 168, aprE, bpr, epr, mpr, nprE, vpr, and wprA, but it was found to lack nprB. Inactivating the aprE ortholog alone resulted in a severe decrease in gamma-PGA production and in the total extracellular protease activity. The defect in gamma-PGA production of the mutant lacking the aprE ortholog was complemented when the medium was supplemented with sufficient glutamate. These results suggest that the alkaline senile protease encoded by aprE plays an indispensable role in supplying materials to produce gamma-PGA. On the other hand, simultaneous inactivation of all the protease genes except for aprE did not significantly affect either gamma-PGA production or total protease activity.