Fermentation and Recovery Technique of Synergic Zwittermicin for Engineered Bacillus Thuringiensis G033A
|School||Chinese Academy of Agricultural Sciences|
|Keywords||Engineered Bt G033A Surface Response Methodology fermentation metabolism research recovery technology of Zwittermicin A|
Bacillus thuringiensis(Bt)plays an important role in pest controlling as microbial insecticide. Study of Bt have been developed with its widely used. There are some drawbacks of all traditional Bt products, such as narrow host range, low toxicity to target insect, resistance from insects. With the analysis of Bt gene and clone, Engineered Bt with high toxicity and broad spectum have been got. Recombinant strain G033A shows strong insecticidal activity to lepidopteran pests, including beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella), and cotton bollworm (Helicoverpa amigera).G033A fermentation recipe is optimized by Surface Response Methodology. By using Fractional Factors, Path of Steepest ascent and Central Composite Design and anysis of variance in predictive model was made to get an optimized recipe. Main components of optimized recipe is : 4 % glucose ,4.388 % peanut powder;0.391 % yeast extract;0.315 % corm steep powder and minerals。Potency of fermentation beer against Helicoverpa amigera is 5783IU/μl. RSM provides a new method for Bt recipe optimiziation.Research of batch and fed-batch fermentation process of G033A is done in 30 L automatic stainless fermentor. Parameters including reduction sugar, NH2-N, DO, pH and cell count and factors which influencing fermentation results are analyzed respectively. feed concentration and feed time are determined. In fed-batch process, by controlling pH 6.8-7.2, adjusting stir speed to keep DO above critical DO, maintaining reduction sugar lever about 0.5-1%, total glucose usage is 6.8%, feeding hydrosate plant aminoacid is 1%, high fermentation potency is got that parasporal crystal content of 130 kD/65kD is 0.58 % / 0.82 %, synergier folds is 5.5, cell count is 62E+8/ml, potency against Helicoverpa amigera is 7012 IU/μl. Research results show that fermentation technique is a critical process for engineered Bt production.Dissolved oxygen (DO) is a main restricted factor of Bt high- Density production. Agitation form, temperature and pressure can affect DO. By using proper agitation form, reducing fermentation temperature in log phase and increasing pressure, DO in log phase will be improved. During fermentation period, DO must be aboved than critical DO.Ion exchange technology is a way to recovery Zwittermicin A produced by G033A. By comparing different technology feasibility and characteristics of powder, ion-exchange technology is considered a practicable technology to recovery Zwittermicin A to get high potency and good characteristics Bt products.