Fermentation Optimization Of Catalase From Bacillus Subtilis Based On Endogenous And Exogenous Stresses Induction And The Production Scale Up

Catalase (CAT) can decompose H₂O₂ into H2O and O2 effectively. Some strains have been used in industry to produce CAT. The problems of CAT fermentation in mainland China recently are low CAT production and low CAT content in fermentation broth, which was not good for downstream extraction.In this paper, oxidative stresses including endogenous and exogenous stresses on Bacillus subtilis WSHDZ-01 were investigated so as to increase CAT production in culture broth. The main research of this thesis follows:(1)By comparison of the effect of different endogenous stresses on CAT production, the superior addition was adding 2.0%(v/v) ethanol to both at 0 h. The total CAT activity and intracellular CAT activity were 126% and 150% higher than those of the control. In addition, different adding strategies had different influence on CAT production and biomass. For the over-expression of CAT, several strategies was investigated. The results show that multiple batch feeding can make Bacillus sp.WSHDZ-01 accumulate more intracellular CAT. By adding 2.0%(v/v) ethanol to both at 0 h,12 h,36 h in one fermentation cycle, the CAT activity can reach 15000 U/mL that is 132.6% higher than the control(6450 U/mL). Furthermore based on the consumption rate of ethanol under batch-adding, three-step constant speed fed-batch was researched. Total CAT activity reached highest-28990 U/mL .(2) The most important thing was to enhance the extracellular CAT production in the fermentation both when high intracellular CAT activity had achieved. H₂O₂ stress had a good effect on the secretion of CAT from intracellular to extracellular, while surfactants did little effect on CAT production. CAT activity was 43.5% higher than the control after adding 0.3% (v/v) H₂O₂ to the culture at 12 h. Within the tolerance of Bacillus sp.WSHDZ-01 to H₂O₂ CAT activity reached to 9744 U/mL with adding H₂O₂ twice at 12 h and 24 h, which was 51% higher than that of the control. Based on these results, a novel oxidative stress strategy combining the ethanol feeding and the pulse addition of H₂O₂ was proposed. With this strategy, the ratio of extracellular CAT to the total CAT reached 82.5%, increased by 18.6% than that of the control (without ethanol and H₂O₂ addition).(3) The scale up of CAT production in factory was investigated with Bacillus sp.WSHDZ-01 based on the previous results in flask and 3 L fermentor. The aeration rate and the agitation speed were scaled up by the related principles.By combining theory calculation with practical situation, CAT activity in 30L fermentor was 34700 U/mL at 54 h under fed-ethanol. In 1 m3 fermentor CAT activity reached 18240 U/mL at 64 h. In 5 m³ fermentor CAT activity reached 15000 U/mL at 64 h.These figures show that it is possible to apply Bacillus sp.WSHDZ-01 to CAT industrial production. Research in the future can do some works on temperature control and oxidative stresses.