Construction of a genetically engineered strain of nattokinase and assessment of its fibrinolytic activity

A strain of Bacillus natto was isolated from natto and found to have a high yield of nattokinase. The aprN and pro-aprN gene fragments, encoding nattokinase from Bacillus natto, were amplified using two pair of primers and were expressed in Bacillus subtilis WB800N host cells using the pHT43 plasmid as a vector. In this system, the effect of leader peptides on nattokinase activity, revealing that these leader peptides mediate the folding function of nattokinase was explored. After optimizing the expression conditions (1 mmol/L IPTG inducer at 37°C, pH 7.5, cell culture OD600 of 0.6), maximal nattokinase enzymatic activity of  848.52 IU/mL after induction of fermentation for 4 h, was achieved, at which time maximal extracellular protein had been produced. The fermentation medium of the engineering strain was optimized, and purified nattokinase via salt precipitation and ultrafiltration was isolated. Relative to fermentation supernatants, the purification ratio of nattokinase reached 6.63, with a total recovery of 80%, and a specific enzyme activity of 11507.92 IU/mg. These results indicate that the nattokinase overexpression using the pHT43 vector in WB800N cells is an effective means of achieving efficient nattokinase production, and the engineered strains constructed herein have great promise as potential industrial strains for nattokinase production.   Key words: Nattokinase, aprN gene, Bacillus subtilis WB800N, Fibrinolytic activity, engineered strain, fermentation.