Study Of Breeding High Spinosad-producing Strains By Genome Shuffling And Developing The New Preparation Of Stored-grain

Spinosad was a new microbial pesticides, which was secondary metabolite produced byaerobic fermentation of saccharopolyspora spinosa with high-effective insecticidal activityand safe for nontarget animal，lead to high benefit of economy and society. In this study, weenhanced production of spinosad with the strains of Saccharopolyspora spinosa by genomeshuffling which could satisfy the requirement of industrial production. Extraction andpurification of spinosad from fermentation broth was studied by using macroporous resinadsorption combined with silica gel chromatography technology and filtrating the prescriptionof spinosad by testing the conditions of thermal stability, low temperature stability, roomtemperature storage stability, particle size and dispersibility.1. We explored the conditions of preparation, regeneration, parents inactivated and fusionof protoplast of original strains. The results showed that the strain grown for65h was treatedby4.00mg/mL lysozyme at39℃for20min, the number of preparation and regenerationrate were92.30%and7.66%, respectively. The protoplasts were inactivated by heat60℃formore than90min and UV treatment more than200s, respectively. The inactive protoplastswere fused and regenerated of polyethylene glycol (PEG6000,50%) for15min, the fusionrate was about1.18%. After three rounds of genome shuffling, a high yielding strain,designated as S.spinosa3-652, was successfully isolated. It was increased about36.07%incomparison with that of the original strain. The subculture experiments indicated that the highproducer was stable.2. Extraction and purification of spinosad from fermentation broth was studied by usingmacroporous resin adsorption combined with silica gel chromatography technology. Firstly,the influences of the type of extraction solvents，extraction solvents volume and pH inspinosad extraction process were investigated. Secondly, the static and dynamic adsorptionbehavior of seven kinds of macroporous resins for spinosad extraction was studied and thenthe optimum macroporous resin was selected in the present work. Finally, the silica gelchromatography technology was explored for spinosad purification. The results showed thatethanol could extract the spinosad efficiently when the pH of fermentation broth was8.0andthe ratio of material and solvent was1:3(g/v). DM11was selected as the optimummacroporous resin to extract and separate spinosad under the experimental condition, and theadsorption and desorption ratio of DM11macroporous resin for spinosad was12508.00μg/g(wet resin) and93.47%, respectively. The maximum adsorption capacity of DM11macroporous resin for spinosad was reached at pH9.0. The optimal ratio of spinosad and macroporous resin for adsorption was2.5:1(mg:mL). When70.00~95.00%ethanol was usedas eluant, the desorption capacity reached97.50%. Then the silica gel columnchromatography was used for further purification of spinosad by gradient elution using amixture of petroleum ether: Ethyl acetate: Methanol (2:1:0.2and1:1:0.25, v/v/v), and thehighest purity of90.58%was obtained under these conditions.3. The results showed that xylene was selected as the cosolvent. Emulsifier was amixture of polyoxyethylene fatty acid ester: polyglycerol fatty acid ester (5:1, v/v), and theaddition was7.00%. Then the addition of glycerol, sodium hexametaphosphate and sodiumcarboxymethyl cellulose (CMC) was6.00%,0.80%,0.30%, respectively, distilled watersupplement.