Optimization Of Fermentation Conditions For High Yield Of Cry1 And Cry2 Proteins Of Bacillus Thuringiensis Strain 4.0718

In order to increase the production of insecticidal crystal proteins(ICPs)of Bacillus thuringiensis 4.0718,firstly, Plackett-Burman design was applied to evaluate the effectiveness of the related nutrition factors;it was found that the soybean powder and MnSO₄·H₂O were significant factors for Cryl and total ICPs production,but the yield of Cry2 wasn't affected remarkably in such medium.Then the steepest ascent experiment was adopted to approach the optimal region of the medium composition.Lastly,the optimized formula of the culture medium was as follow:starch 12,glucose 10,soybean powder 11.5,peptone 2.5,K₂HPO₄ 2.5,FeSO₄·7H₂O 0.02,MnSO₄·H₂O 0.02,MgSO₄·7H₂O 0.25 g/L,obtained by response surface methodology(RSM).The final yields of Cryl and Cry2 was 0.32 and 0.11mg/mL,increasing twice more than that in the medium optimized before.The median lethal concentration(LC₅₀)of optimal medium was 1.09μL/mL.The toxicity to Helicoverpa armigera was significantly enhanced than the control(LC₅₀ 3.10μL/mL).By the one-factor-at-a-time experiment,it was found that the optimal initial pH of the optimal culture medium was 8.0 and the level of anti foam soybean oil should be controlled to the range of 0 to 1(%,v/v).In order to increase cell density, the intial concentration of the optimized medium was geminated. The results showed that the high substrate concentration restrained the sporulation and ICPs production.But it was found that the ICPs yields were improved by increasing parts of carbon and nitrogen resourse with adjusting C/N ratio.The optimized feeding medium and technology were addition of total 12g/L glucose and beef powder(composed of 2:1)three times in sequence at 10,12,14h after inculated.It provided a good foundation for exploring fed-batch process in fermentor.The carbon and source of the optimized medium was adjusted。The optimal value was 18g/L glucose and 14.5g/L peptone.