The Induced Mutation Of A Paenibacillus Strain And Isolation, Purification Of Its Antifungal Active Substances

Paenibacillus polymyxa Cp-S316, an antimicrobial bacterium, was isolated from soil of Mountaintai and further identified with registration number AY292989 on Genbank. From the research of the antimicrobial bacterium, it showed broad inhibitory spectrum and great inhibition against animal and plant pathogenic bacteria and fungi. The shake-flask fermentation properties of Cp-S316 was also excellent, and antibacterial and antifungal active substances were produced in the same medium, but the production of both active substances fluctuated greatly with different medium. The fermentation conditions and medium for antifungal active substances were optimized. The Cp-S316 was induced by ultraviolet ray and mutant strains with higher antifungal activity were obtained, which provide theoretical support for its industrial application. The route of isolation and purification for active substances was established, meanwhile,the active substances was be preparation. The main results were as follows.1. The Mutagenesis of Paenibacillus polymyxa Cp-S316 by UV radiationThe research indicated that Cp-S316 had high sensitivity to UV, when the irradiation time was 20s, the lethal rate could reach 95.3%. So the optimal irradiation was selected as 20s. After prescreening, first and re-screening, six high-yield mutant strains were obtained, which were named as c16, d39, d56, e19, h41 and h44. The titers were increased 123%,130%,142%,136%,141% and 125%, respectively. In order to investigate the fermentation stability, the mutant strains were tested after continuous culture generation by generation. The result showed that the mutant had a good genetic stability.2. Isolation purification and preparation of antifungal active substancesThe macroporous adsorptive resin X-5 was chosen to adsorb active substance, the optimal proportion between resin and active substance was 1:20 (m/v), and the optimal time of static adsorption was 4h. The optimum desorption solution was 0.01 mol/L HCl-70% ethanol desorbing in static state. When the proportion between desorption solution and resin was 2:1(v/m) in dynamic desorption, the desorption rate was 70%. Then the antifungal active substances were extracted by carbinol. The extracted active substances were further purified by CM-Sepharose Fast Flow ion exchange column chromatography with 0-0.5mol/LNaCl as grads desorption solution. The active substances contained less impurity were got by Sephadex G50 column chromatography.Then antifungal active substances were refined by reversed-phase high performance liquid chromatography (RP-HPLC). The method of RP-HPLC was set up for preparation of the antifungal active substances. The conditions of RP-HPLC were suggested as SinoChrom ODS-BP C18column(300×10.0mm,10μm), detection wavelength 210nm, the mobile phase gradient elution of acetonitrile-buffer(22:78,v/v) at 30℃, with the buffer containing 0.1% TFA, flow rate 0.5mL/min, the volume of injection 20μL. Finally antifungal active substances were preparation with preparative chromatography, The conditions of it were suggested as Venusil XBP C18柱(20x250mm),flow rate 0.5mL/min, the volume of injection 20μL,detection wavelength 210nm,two kinds of active substances were obtained,the Rt were 14.32min and18.12min. Made preparation for repetitious about these two kinds of active substances.The purity of the active substances was tested by UPLC, they both showed a single peak in UPLC chromatography.