Abstract: Nattokinase (NK), encoded by the aprN gene of Bacillus subtilis natto, has strong fibrinolytic activity both in vitro and in vivo. In this research, the aprN gene from B. subtilis was cloned and the codons which encode the first 30 amino acids were optimized on the basis of the codon preference of B. subtilis. Recombinant vector pHT01-aprN was constructed. Through restriction enzyme digestion analysis, PCR amplification and sequencing, the subcloned gene was confirmed to be aprN. The pHT01-aprN was transformed into B. subtilis 168 by electroporation, and the engineered bacterium (B.s 168/pHT01-aprN) was isolated on LB plates containing chloramphenicol. NK expression was induced by IPTG, and the highest enzyme activity in shaking flask culture was up to (289.00 ± 3.42) U/mL with good stability, which was 3.9 times as high as that of wild-type B. subtilis natto.

Key words: nattokinase, aprN gene, Bacillus subtilis, enzyme activity, engineered bacteria