Screening And The Separation And Purification Of Antibacterial Protein Of Paenibacillus Kribbensis CX-7 Strain

74% of our country’s farmland soil lack phosphorus, and more than 95% of the phosphorus in the soil is ineffective phosphorus (apatite) that are very difficult to use by plants; about 60% of the arable land lack potassium, and arable land available K content is decreasing at a rate of 2×10-6-3×10-6 per year, causing 3 elements in the soil, nitrogen, phosphorus, potassium to be out of proportion, affecting the development of agriculture.The study of that how to supply available phosphorus and potassium to crops from widely distributed soil minerals, is of great scientific and practical significance. The study of crop pathogen antagonistic action exhibited by bacteria capable of efficiently solubilizing phosphorus and potassium is seldom reported. In this study, screening antagonistic bacteria strains of common pathogens with high activity of solubilizing potassium, aimed to obtain multi functional bacteria, which not only have efficient solubilization of phosphorus and potassium capabilities, but also exhibit the effect of biological control. And through laboratory experiments we developed a type of highly efficient microbial agents for the services of our country’s agricultural production.297 bacillus stains were isolated from different crops soil samples in the country’s different regions and 34 strains with the function of solubilizing or dissolving potassium were obtained using plate screening methods. Soluble phosphorus and potassium content in the fermentation liquid was determined by Mo - Sb antispetrophotography method and sodium tetraphenylboron method, and the selected strains have higher phosphorus and potassium solubilizing activity than the K-1 strain (AC10012). 3 strains have efficient solubilizing activity of organic phosphorus, which are NX-42、CX-14and BY-4. 15 strains have efficient solubilizing activity of inorganic phosphorus bacteria, which are BY-3, CX-9, SS-14, SS-9, BY-4, CC-14, CX-7, CX-24, SS-8, NX-43, NX-42, CX-8, CX-21, SS-12 and CC-34. 12 strains were be found to be high potassium dissolving bacteria, which are NX-11, NX-43, CX-9, CX-8, CX-7, BY-4, SS-12, NX-23, CX-14, BY-3, NX-12 and NX-42. From the above 34 strains we screened 5 strains showed antagonistic behauior against pathogenic bacteria antagonistic. The CX-7 strain, separated from wheat soil in Chang’an County, Xi’an, had high solubilizing capacity of phosphorus and potassium, and high antibacterial activity, so physiological and biochemical reactions and sequence analysis of 16S rDNA gene were done. The physiological and biochemical reactions showed the features of CX-7 strain were similar to Paenibacillus kribbensis. 16S rDNA sequence homology comparison was done on the NCBI website, and the phylogenetic tree was drawn by the Neigllbor-Joining method. The results showed that 16S rDNA sequence homology of CX-7 strain and Paenibacillus kribbensis at 98.7%. Therefore, combining physiological and biochemical reactions and 16S rDNA gene sequence analysis, CX-7 strain was identified as Paenibacillus kribbensis.The phosphate and potassium solubilizing activity of Paenibacillus kribbensis CX-7 strain were found to be organic phosphorus 3.18 mg/L, inorganic phosphorus 71.60 mg/L and potassium feldspar 3.44 mg/L. The results of the corn plant test inoculated with CX-7 strain showed that: leaf area increased by 5.6%, plant height increased by 8%, wet and dry weight increased by 21.6% and 68.3%, available potassium and available phosphorus content in the soil was higher than that of the control. CX-7 strain has good application effect of solubilizing phosphorus and potassium, and has the very good development prospects. Liquid fermentation conditions of antimicrobial substances produced by the CX-7 strain were optimized, the optimal culture medium of obtaining antibacterial proteins: sucrose 0.5%, soybean flour 0.5%, MgSO4 0.02%, K2HPO4 0.02%, yeast extract 0.07%, (NH4)2SO4 0.02%, initial pH value was 7, the capacity was 50 mL /250 mL shaking bottle, the best inoculation amount was 7%, and fermentation period was 36 h. The protein antibacterial activity showed obvious differences from the original and terminal optimization. The original antibacterial circle area was 379.94mm2, and the terminal was 509.25 mm2, and the activity increased by 34.03%.Antimicrobial protein secreted by CX-7 strain was separated and purified by DEAE-SePhamse Fast Flow anion exchange chromatography, and a single antimicrobial peptides was obtained, the molecular weight was detected to be about 50 kDa by SDS-PAGE. The antibacterial peptide was thermal stabile; the pH range in which the peptide was active was wide, and in the range of pH6-8 antibacterial activity was strong; under alkaline conditions it was stable, and in acidic conditions bacteriostatic activity disappeared.