Screening Og Enramycin Producing Strain And Optimization Of Fermemtatipon Conditions

Enramycin was one of polypeptide antibiotic produced by Streptomyces fungicidious, combined by not saturated fatty acid and ten kinds of amino acid. It mainly contained enramycin A and enramycin B, with a little C and D.It was active to Gram-positive bacteria especially to harmful Clostridium, as to inhibit the synthesis of material in the cell wall. In favour of animal growth, enramycin was widely used in the production of animal husbandry. This paper was to study enramycin production by the strain Sireptomyces fungicidious KS010.It was reviewed that establishing the method to detect the content of enramycin, breeding the high-productive strain of enramycin, optimizing fermentation conditions and so on.Firstly, the content of enramycin was detected by the method of Microbiological assay and HPLC. For Microbiological assay, the determination conditions were optimized through the investigations of extraction solution composition, extraction time and the concentratios of bacillus subtilis. The optimum deternmination conditions were acidic acetone solution B (acetone:2mol/LHCl:H2O=20:1:21, pH3.0) and80min for extraction. The calibration curve was linear within the range from0.56to35.79U/mL between the logarithm of concentration and the diameter of inhibition zone with the106spores/mL of test microorganism constration. The regression equation was Y=0.2436X-3.47436, the correlation coefficient was0.9960.The determination conditions for HPLC were optimized through the investigations of mobile phase composition, mobile phase flow and extraction time. The optimum deternmination conditions were acetonitrile:50mM KH2PO4(30:70), flow0.8mL/min in the detection wavelength of267nm. The calibration curve was linear within the range from0.26to275.72U/mL between the peak area and Titer concentration.The regression equation was Y=1.83099E-6X-0.52846,and the correlation coefficient was0.99947. Comparing the same sample in the two methods for detecting the content, both are of high repeatability and accuracy.So they could be used for content detection.After mutation by NTG treatment and agar screening method, the mutant M-19was screened out with a high enramycin yield up to32.62%.Then the strain M-19as the object, it was found that the60Co-γmutagenesis was better than the ultraviolet mutagenesis. After mutation by60Co-γtreatment, the mutant Streptomyces fungicidious C0250-42was obtained with a high enramycin yield at1853U/mL, which was up to68.00%from the original1041U/mL and also of good stability.From the one-way analysis and the response surface methodology, factors of production enramycin were studied.The best liguid fermentation condition was optimized: the yeast leaching powder1.29%,KH2PO410.84mmol/L,ZnCl28.74mmol/L, glucose5.93%, pH8.0,33℃.After fermentation for7days, the titer of enramycin was the highest at2065.82U/mL, which was up to98.30%from the original1041U/mL.The mutant Streplomyces fungicidious CO2.50-12was verified by250mL-triangular flask fermentation and used in the5L fermentor.After11days fermentation in the5L fermentor at the air volume and200r/min. the titer of enramycin was up to2476.48U/mL...