High and Economical Nattokinase Production with Acetoin as a Useful Byproduct from Soybean Milk and Glucose

Nattokinase (NK) is a potent fibrinolytic enzyme with wide pharmaceutical and nutraceutical applications. Safe and high NK-yielding strains are urgently needed. In this study, the best strain NDF was isolated from one of the 11 natto samples and then identified as Bacillus subtilis. The effects of carbon and nitrogen sources on NK production were investigated, and glucose and soybean milk were finally selected as the optimal carbon and nitrogen sources, respectively. Acetoin, a valuable compound with versatile usages, was detected as the main byproduct of carbon overflow. In a 6-L fermenter, NK and acetoin reached their peak concentrations simultaneously (10,220 IU/mL and 25.9 g/L, respectively) at 25 h in a culture medium containing 180 g/L of soybean milk and 105 g/L of glucose. The NK product was verified by sequencing of the aprN gene and SDS-PAGE analysis. Only very limited kinds of proteins were found in the supernatant of the fermentation broth, and NK was one of the main bands. This study has developed an economical and high NK production method with acetoin as a useful byproduct.