Subtilisin NAT, a subtilisin-like serine protease present in fermented soybean “natto” extract, inhibits <i>Streptococcus mutans</i> biofilm formation

Abstract

Our previous study indicated that the water-soluble extract obtained from commercially available natto products inhibited the sucrose-dependent cariogenic biofilm formation of Streptococcus mutans. This inhibitory effect correlated with the protease activity levels of the extract. Subtilisin NAT (nattokinase), a subtilisin-like serine protease, is a dominant protease found in natto products. In this study, we investigated the effect of natto products produced by a subtilisin NAT-deficient mutant (aprN − ) of the Bacillus subtilis strain NBRC16449 on S. mutans biofilm formation. The mutation decreased the γ-poly glutamic acid (γ-PGA) levels and reduced protease activity in natto products. A significant reduction of the inhibitory effect was observed in the extract from natto products prepared with the aprN − strain. The γ-PGA obtained from the natto products did not affect biofilm formation. We propose that subtilisin NAT plays a major role in biofilm inhibition induced by natto extract.

Nattokinase, a Subtilisin-like Alkaline-Serine Protease, Reduces Mutacin Activity by Inactivating the Competence-Stimulating Peptide in Streptococcus mutans

“…Fermented soybean natto produced using this strain as a starter contains abundant proteases, among which nattokinase is the major one [ 25 ]. The protease activity at the concentration of nattokinase used in this study (final concentration 1 mg/mL) was equivalent to that of 10 g wet weight of natto product [ 8 ]. Nattokinase is essentially safe when administered at the recommended dose (200 mg/d); histopathological examination of the organs and tissues have shown no evidence of toxicity [ 9 ].…”

“…We previously found that natto, an unsalted fermented soybean food produced using Bacillus subtilis var. natto as a starter, inhibited S. mutans biofilm formation, and the inhibitory factor was nattokinase (subtilisin; EC 3.4.21.62) [ 7 , 8 ], which is a major extracellular enzyme produced by B. subtilis var. natto [ 9 ] and a subtilisin-like alkaline serine protease that exhibits potent thrombolytic activity and substrate specificity.…”

“…natto [ 9 ] and a subtilisin-like alkaline serine protease that exhibits potent thrombolytic activity and substrate specificity. Nattokinase did not show growth-inhibiting or bactericidal activity against S. mutans or other commensal bacteria in the oral cavity and inhibited the synthesis of insoluble glucan, the major component of the extracellular matrix of S. mutans [ 7 , 8 ]. Owing to its natural occurrence, safety, efficiency, and cost-effectiveness, this enzyme is considered a potential caries-preventive agent.…”