Research On Paenibacillus Polymyxa SIIA-1408 Producing Anti-bacterial Bioactive Product

Paenibacillus polymyxa is a plant growth promoting rhizobacteria and bio-control bacteria which possesses no pathogenicity to plant,can promote the growth of plant and prevent pest caused damage to plant,is a strain of promising applying potentiality.Paenibacillus polymyxa can produce many kinds of different antibacterial activity product.With the resistance state of gram negative bacteria becoming incresingly serious,more and more attention are put to Paenibacillus polymyxa,especially those who could produce polymyxin.Based on the above statement,the importance of research on the antibacterial activity product is self-evident.In this paper,bacillus SIIA-1408 was classified and determined,and its metabolism was studied,two components of polymyxin E and a product with wide range of antibacterial activity was isolated and purified,and the chemical structure of them was studied.The major research content including:（1）To classify and determine the strain bacillus SIIA-1408,the morphological characteristics,cultural characteristics,some conventional physiological and biochemical characteristics,and phylogenetic tree constructed by analysis of 16 s RNA gene sequence was studied,a conclusion was drawn that SIIA-1408 belongs to Paenibacillus genera and Paenibacillus polymyxa spieces.So SIIA-1408 was renamed Paenibacillus polymyxa SIIA-1408.（2）Combined Paenibacillus polymyxa SIIA-1408 fermentation liquor antibacterial activity with related articles’ s report and its analytical method on possible product,we preliminarily ascertain the main antibacterial bioactive product as polymyxin E.Based on this presumption,a proper pretreatment method was chosen for Paenibacillus polymyxa SIIA-1408 fermentation liquor and the preliminary separation process and reverse silica gel column chromatography was studied.Combined with preparative HPLC,two major component in SIIA-1408-28-22-30 was isolated and purified,and above 92% purity of both component was obtained.Through high resolution mass spectrum analysis,the chemical fomular of the above two component was elucidated,that were,C₅₃H₁₀₂N₁₆O₁₃,C₅₂H₁₀₀N₁₆O₁₃,identical with that of polymyxin E1,E2 respectively.Based on the above result,the Paenibacillus polymyxa SIIA-1408 axenic isolation was got and one strain named SIIA-1408-28 with a relative high polymyxin yield and good hereditary stability was chosen as a starting strain.Then,atmospheric and room temperature plasma（ARTP）mutation and NTG mutation was employed to SIIA-1408-28.Based on the polymyxin yield in the fermentation liquor,a strain named SIIA-1408-28-22-30 with a relative high yield（polymyxin yield was 16341.2 μg/ mL）and good hereditary stability was selected.Next,the fermentation conditions of SIIA-1408-28-22-30 was studied in shake flask.The impact that seven factors,namely,the inoculation time of shake flask seed;inoculum size;loading sample volume;initial pH of fermentation broth;shaker’s rotary speed;temperature;fermentation cycle have on the yeild was studied.The optimum fermentation medium constituent（composed of glucose,corn starch,black soybean meal,（NH₄）2SO₄,NH₄H2PO₄,MgSO₄,CaCO₃ and antifoaming agents）was ascertained by single factor experiment.Based on the result of single factor experiments,orthogonal experiment was applied in the optimization of fermentation medium.The polymyxin E yield under the optimized fermentation medium（made up of glucose 20.0 g/L,corn starch 70.0 g/L,black soybean meal 30.0 g/L,（NH₄）2SO₄ 20.0 g/L,NH₄H2PO₄ 0.6 g/L,MgSO₄0.05 g/L,CaCO₃ 1.0 g/L and antifoaming agents 0.9 g/L）was about 20352.7μg/ mL and about 1.587 times of that of initial strain SIIA-1408-28 under the primary fermentation conditions.（3）In the process of polymyxin E producing fermentation medium optimization,an interesting phenomenon was observed,namely,without adding（NH₄）2SO₄ to the fermentation medium,the yield of polymyxin was sharply decreased,while the yield of a product（temporarily named SIIA-1408-X）with a wide range of antibacterial activity was relative high.Further study,phenylalanine added to the fermentation medium was found to greatly pomote the yield of SIIA-1408-X,while（NH₄）2SO₄ and other ammonium salt found to obviously inhibited its yield.To testify the chemical structure of SIIA-1408-X,the preliminary separation process and reverse silica gel column chromatography was studied,and combined with preparative HPLC and crystallization process,the crystalline of SIIA-1408-X was obtained.Then,through negative and positive mass spectrum and single crystal diffraction analysis,SIIA-1408-X was turned out to be（S）-2-hydroxyl-3-phenyl-propionic acid,namely,phenyllactic acid.