Abstract:Using Bacillus subtilis MX-6 isolated from natto as research subject，the effects of temperature，initial pH，inoculation amount，liquid medium volume and shaker speed on nattokinase activity were determined by single factor test.Box-Behnken design of response surface methodology was used to optimize the fermentation conditions of nattokinase.The maximal dissolution diameter of nattokinase in actual experiments was 20.71 mm with 36.69 ℃，initial pH 6.94，inoculation amount 3.03%，liquid medium volume 48.77 mL and shaker speed 170.05 r/min，and the dissolution diameter had been increased by 35.54%as compared to that of un-optimized conditions.It was demonstrated that the use of response surface methodology could effectively and successfully optimize the fermentation conditions of nattokinase production.