Abstract:

In this study, the nattokinase-producing capacity of Bacillus subtilis subsp. subtilis BS21076 was improved by
sequential mutagenization with ultrasonic followed by UV light at the appropriate doses determined according to death
and mutation rates. The optimal mutation conditions of strain BS21076 were established as 60 min ultrasonic treatment at
45 kHz and 280 W followed by 120 s UV irradiation at a distance of 30 cm. A high nattokinase-producing mutant strain
named BSCZ-4, producing 1.96 times higher nattokinase than BS21076, was obtained after several rounds of primary and
secondary screening. After 20 consecutive passages, the diameter of the zone of dissolution produced by BSCZ-4 remained
stable in the range of 18–19 mm, suggesting good genetic stability. Using Box-Behnken response surface methodology,
the optimal culture conditions for the enhanced production of nattokinase by BSCZ-4 in solid-state fermentation were
determined as addition of 5% konjac power to the culture medium, an inoculum size of 8%, and 24 h culture at 34 ℃,
resulting in a nattokinase activity of (4 087.83 ± 93.75) U/g.

Key words: mutation, Bacillus subtilis, response surface methodology, process