The Fundamental Study Of A Novel Fibrinolytic Enzyme: Nattokinase

Nattokinase is a novel fibrinolytic enzyme with strong and specific thrombolytic activity. In this paper, one strain high yielding nattokinase (NK) was obtained, then the liquid fermentation in shaking flask and fed-batch fermentation in 3.7L fermentator of this strain was optimized, at last isolation and preliminary purification of NK was finished.A wild strain of Bacillus subtil is producing NK named Bacillus subtilis HL-1 was obtained from natto through two-step screening strategy of casein plate selecting producing protease stain first and shaking culture determining secreting NK bacteria finally. The NK activity of B. subtilis HL-1 in liquid culture was 970 IU ?ml-1. Response surface methodology was used to optimize the fermentation condition of NK production of B. subtilis HL-1. In the fist optimization step, a Plackett-Burman design was used to evaluate the influence of related factors. Tryptone influenced NK production positively while NaiHPCU, temperature and age of seed negatively. The others had no significant influence on NK production. The path of steepest ascent was used to approach the optimal region of the fermentation condition subsequently. In the third step the concentrations of tryptone and NaaHPC^ and the age of seed were further optimized using central composite designs and response surface analysis. The optimized condition allowed the NK production to be increased from 970 IU ?ml"1 to 1314 IU ?ml'1.Large-scale liquid fermentation was conducted in Bioengineering KLF 3.7L fermentor. The effect of seed medium on NK production was invested and the result showed that the NK activity in the fermentation culture could be increased 30% (1698 IU ?ml"1) by adding up 1 g ?L"1 xylose into the seed medium. The experiments of batch and fed-batch fermentation revealed that fed-batch benefited NK production and feeding fresh culture medium into fermentator when cell density reduced was preferable, which led to NK activity of 2348 IU ?ml"1 in 2 L culture."7Zhejiang University master paper______________ABSTRACTPurification of NK mainly included four steps: removing cell by centrifugation, 20-60% saturation ammonium sulfate precipitation, Sephadex G-75 gel filtration and Sepharose CM Fast Flow ion-exchange chromatography and purified enzyme appeared single band on SDS-PAGE. The purification factor and NK activity recovery rate of SEC and AIE was 4.75 ^ 74.3 % and 1,32 > 77 %, respectively.