Construction Of Recombinant Bacillus Subtilis Strains For High-level Secretory Expression Of Nattokinase

Intravascular thrombosis, one of the main factors of cardia-cerebrovascular disease leading to the myocardial infarction and stroke in the elderly, has greatly threatened the health of human beings. Hence, developing safe and reliable thrombolytic drugs without side effects is of great significance. Nattokinase(NK, EC 3.4.21.62) is considered to be the most potential thrombolytic agents in the future due to its specific and strong activity to thrombolysis. However, the low level production of NK limits its application. Therefore, this study is focused on the enhancement of NK production.This thesis studied the effect of six different promoters on the expression of NK in Bacillus subtilis firstly. And for the improvement of NK production in B. subtilis WB800, a suite of high efficient expression vectors with tandem promoters in different forms were constructed. After preliminarily optimizing the culture medium components, we carried out the experiment of the best recombinant strain in 5 L fermenter. Natto solid-state fermentation experiments were also conducted, and NK production was compared with commercially available natto dried powder. The results of the research are as follows:(1) The effects of six different promoters on NK expression level were compared. According to the fibrinolytic activity analysis, we found that the recombinant strain B. subtilis WB800/pSG102(PP43) achieved the highest enzyme activity among the six strains. And at 36 h, the enzyme activity reached to 140.5 FU·mL-1, followed by recombinant strain B. subtilis WB800/pSG101(PHpaII) and B. subtilis WB800/pSG103(PBcaprE).(2) Two forms of promoters in tandem were studied:(a) make promoters in tandem entirely, and strains B. subtilis WB800/pBG107-B. subtilis WB800/pBG128 were achieved. B. subtilis WB800/pSG121(PHpa II-PHpaII-PP43) displayed the highest expression level among all these strains, and enzyme activity was up to 264.2 FU·m L-1 at 36 h;(b) perform the operation of making the core area sequences of promoters in tandem, and plasmids pSG131-pSG137 were constructed successfully. The analyses of fibrinolytic activity and SDS-PAGE indicated that the recombinant strain B. subtilis WB800/136(5CPP43) had the highest expression level of NK. The maximum value 258.0 FU·m L-1 was gained at 36 h.(3) We optimized the fermentation conditions of the strain B. subtilis WB800/pSG121(PHpaII-PHpaII-PP43) in the level of shake flask including inoculum age, carbon source, nitrogen source and carbon-nitrogen ratio. The optimal fermentation conditions were determined as follows: the inoculating age was 8-15 h; the carbon source and nitrogen source were glycerol and soya peptone; and the best C:N was 1:1. As a result, the activity of NK reached to 820.0 FU·m L-1 in 5 L fermenter after optimization.(4) The amount of supplementation of recombinant strain dried powder was studied, and the optimal adding amount was 5.7 mg per 100 g soybean. When compared with the natto dried powders which are commercially available in the same conditions, the strain B. subtilis WB800/pSG121(PHpaII-PHpaII-PP43) has more advantages.