Artial Properties And Purification Of Antifungal Substance From Bacillus Subtilis B25Strain

Banana wilt disease,caused by Fusarium oxysporum f.sp cubense. It is a soil-borne facultative parasite present in soil. It is very difficult to control Fusarium wilt because the fungus survives in the soil as chlamydospores during extended periods even in the absence of the host roots.Wilting symptoms and plant death caused by can be devastating, with losses as high as100%.It was a fatal wilt disease, result in serious threaten to banana plantations.Bacillus subtilis B25is found to produce antimicrobial substance, which has exhibited significant inhibitory effect on Fusarium oxysporum. The result of bioactivity tests showed B25prominently showed growth inhibition to some common pathogenic fungi, such as Colletotrichum gloeosporioides of mango and Colletotrichum gloeosporioides of rubber trees on potato dextrose agar (PDA) plates. In the paper, in detail describes the physiological and biochemical identification of B25;Fermentation medium and culture conditions of the strain were optimized;Subsequently, partial character and purification of antifungal protein were studied. All this lay a foundation for the development and application of antifungal protein and also for future genetic modification of the strain. What this paper work done is as follows:API50CHB bacillus biochemical identification system on the physiological and biochemical identification of strain B25, using API identification software to read code identification, the result show that B25is bacillus subtilis.The result found that Bacillus subtilis B25have obvious difference in antifungal bioactivity and growth rate in LB and NB mediums.Antifungal bioactivity of supernate in NB broth is more stronger than LB broth,however in LB broth B25grow faster, Antifungal substance was found when logarithmic growth was nearly finished, and increase to the highest point in stable phase.In order to improve the production of antifungal substances, the optimal culture meidium was determined by single factor and orthogonal design test in shake-flask. The result of single factor experiment is that:Glucose and sucrose were suitable external carbon source,Yeast Extract is an ideal nitrogen source, KH2PO4is suitable mineral salt;the optimum medium composition:Glucose1.5%, Bacterial peptone1.5%, beef extract0.5%, KH2PO41.0%.The optimums fermentation conditions of antifungal substance Production were original PH7.0, fermentation temperature30℃,amount of load60ml(in250ml flask).Ethanol and ethyl acetate and ammonium sulfate were used to extract antagonistic substance; The stability of it on temperature,pH,two proteinases (trypsin, proteinaseK);And estimated the molecular weight of antifungal protein with lOkDa ultrafiltration tube filtering; With special medium(protein, cellulose, chitin) test the activity situation of decomposition of antifungal protein.The results showed that The antagonistic substance can be extracted from cell-free culture broth supernatants by75%(w/v)(NH4)2SO4saturation. The antimicrobial substance not changed significantly even in100℃boil1hour;It tolerated acidic, neutral and slightly alkaline but was not stable in strong alkalie;With regard to the two proteinases,the activity decreased obviously when deal with trypsin and proteinaseK; It failed to pass lOkDa filter membrane, indicate that antifungal active substances is not small molecular weight of bacteriocin, but larger molecular weight of antifungal protein. The degrading test showed that the supernatant had protease activity and cellulase activity but didn’t have chitinase activity.The crude proteins were precipitated from the supernatant by ammonium sulfate.The dialysates were condensed to yield crude proteins The isolation procedure comprised ion exchange chromatography on DEAE-Sephadex Fast Flow and gel filtration chromatography on SephadexG-100. The purified antifungal fraction showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The active fraction was identified by LC-MS/MS. The analysis of the antifungal protein suggested that it might be a kind of hypothetical protein gi|154685475, account for82.7%of the total protein content, and other protein content between0.3%and6.2%, hypothetical protein gi|154685475from Bacillus amyloliquefaciens FZB42with a relative molecular mass of38708Da and it is a unknown protein.