Purification Of Antibacterial Active Substances From Paenibacillus Polymyxa Cp-S316 And Mutation Screening Of High-Yield Strains

A new antagonistic bacterium Paenibacillus polymyxa Cp-S316 isolated from soil of Mountain Tai and further identified with registration number AY292989 on Genbank. It showed broad inhibitory spectrum and great inhibition against animal and plant pathogenic bacteria and fungi. The shake-flask fermentation properties of Cp-S316 were excellent, and It can produced antibacterial and antifungal active substances, but the production of both active substances fluctuated greatly with different medium. Paenibacillus polymyxa Cp-S316 could produce polymyxin E and polymyxin E1 and at least three novel antibiotics against pathogenic bacteria. Meanwhile it could produce at lease one kind of antifungal active substances. As an original strain, Paenibacillus polymyxa Cp-S316 was treated by ultraviolet mutagenesis. The mutated strains were obtained, and several antibacterial active substances were isolated, purified, and identified, and the effect of antibacterial active substances from Paenibacillus polymyxa Cp-S316 on the integrity of Escherichia coli K12 membrane was studied by determining the variation of OD26o values of bacterial suspensions. The main results were as follows:1 Paenibacillus polymyxa Cp-S316 was treated by ultraviolet mutagenesis. The mutated strains of A17, E13, E18, E33 and F28 were obtained by prescreening of antibacterial active substances, screening and re-screening of shake flask culture. Compared with Cp-S316, the yield of antibacterial substances obtained from mutant A17 was increased by 91%. The subculture experiments indicated that the hereditary characteristic of high productivity of Paenibacillus polymyxa A17 and E18 were stable.2 Isolation and purification of antimicrobial active substances were operated. antimicrobial active substances were absorpted with cation exchange resin and desorbed with the 0.5 mol/L H2SO4 in dynamic. The desorption liquid was concentrated by vaporization and vacuum freezy-desiccation. antimicrobial active substances were further isolated gradually by methanol extraction, Sephadex G25 column chromatography, CM-Sepharose Fast Flow ion exchange column chromatography with 0-0.3mol/L NaCl as grads desorption solution. The antimicrobial active substances contained less impurity were got by Sephadex G25 column chromatography.Finally antimicrobial 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 antimicrobial active substances A. The conditions of RP-HPLC were suggested as Venusil XBP C18 column(250mm×20.0mm,5μm), detection wavelength 210nm, the mobile phase gradient elution of acetonitrile-buffer(23:77, v/v) at 30℃, with the buffer containing 0.1% TFA, flow rate 5.0mL/min, the volume of injection 5.0mL. Two antimicrobial active substances component was separated under the condition. The conditions of RP-HPLC for preparation of the antimicrobial active substances E were suggested as Venusil XBP C18 column (250 mm×20.0mm,5μm), detection wavelength 210nm, the mobile phase gradient elution of acetonitrile-buffer(17:83, v/v) at 30℃, with the buffer containing 0.1% TFA, flow rate 5.0mL/min, the volume of injection 5.0mL. Two antimicrobial active substances components were separated under the condition. At last antimicrobial active substances components E-2 and A-2 were showed single peak in UPLC chromatography.3 Inhibition of antibacterial active substances from Paenibacillus polymixa Cp-S316 on Escherichia coli were explored. The effect of antibacterial active substances from Paenibacillus polymyxa Cp-S316 on the integrity of Escherichia coli K12 membrane was studied by determining the variation of OD260 values of bacterial suspensions. The results showed that the antibacterial substances could damage the Escherichia coli K12 membrane which leaded to the leakage of RNA, DNA and other cytoplasmic macromolecules.