Mutation Breeding Of High-yield Strain Of Bacitracin And The Optimization Of Fermentation Conditions

Using Bacillus licheniformisrmis FUN-BL as an aboriginal strains, the positive mutants were screened by cup-plate method and HPLC after ultraviolet and diethyl sulfate.The aboriginal strains Fo mutated through ultraviolet (15W, the irradiation distance of 30 cm and the irradiation time of 120 sec) were screened in repeat to obtain the high-yield strain of bacitracin F167, whose titer was improved by 11.76%. Then F167 mutated through diethyl sulfate (dealt with 2% diethyl sulfate for 90 min)were screened in repeat to obtain the high-yield strain of bacitracin F2141, whose titer was improved by 32.19%. Making the high-yield strains F2141 do the genetic stability test for five successive generations, the titer followed 1003.23U/mL,989.34U/mL,976.35U/mL, 960.32U/mL,949.72U/mL. High-yield strains of bacitracin can be bred by ultraviolet and diethyl sulfate.Choosing 6 high yield strains of F210, F272, F294, F2133, F2141, F2156 which have been mutated by ultraviolet and diethyl sulfate as aboriginal strains of protoplast fusion. In order to obtain the protoplast, added 5% glycine into seed medium as pretreatment and adjust the bacteria concentration for 0.2(OD600). Adopting normal design of three factors three levels, the concentration of lysozyme 10 mg/mL, enzymolysis time 40 min, enzymolysis temperature 30℃ are studied. Using the conditions of preparation of protoplast do the protoplast fusion. And then 7 high-yield strains which titer were increased more than 10% were screened by cup-plate method and HPLC. Among them, the highest titer of Ff215 is 927.10 U/mL. It was increased by 15.73%than the starting strains.In this chapter, the components of the medium and the condition of culture were optimized. Using single-factor experiments to study five culture conditions and the research shows that pH 7.5, inoculation 2%, temperature 37℃, shaking rate 230r/min, liquid volume 20mL/mL are good. The results show that the Lys, Glu, Asp, tween-20, Ile, Thr, CuCl2 and CoCl2 were have an impact on the titer of bacitracin. Glu, Gu2+ and tween-20 were the three most important factors to the titer of bacitracin, and Glu on the impact of the titer of bacitracin was the largest. According to the Box-Benhnken, it proved that the response surface model exhibit a maximum, and the maximum was 955.79U/mL. After five parallel experiment, the titer of the mean value was 938.59U/mL. It was increased by 9.3% than the starting strains(856.15 U/mL).At last, the temperature, pH and protease on the effect of the activity of bacitracin and storability of bacitracin were studied after 50% saturated ammonium sulfate. The results show that the influence of temperature on the bacitracin activity is not obvious, and activity was reduced by 50% after 121℃,30min. Neutral protease almost had no effect on bacitracin, acid protease and alkaline protease can make bacitracin partially inactive. Placing 180d at room temperature, the decline of activity of bacitracin was not obvious.