Breeding Of Gentamicin High-Producing Strain And Studies On Optimization Of Its Fermentation Condition

By perfecting the operating condition of TLC, the optimum detecting system was obtained, and the detection limit and reproducibility were studied. Based on the optimum system, we successfully separated the main components (C1,C1a,C2) of GM and effectively detected 10 components of GM fermentation broth which was simply concentrated. In addition, the system was used to detect the component ratio combining with titer test, which showed good consistency with the results of HPLC. This method is simple, rapid and reliable. About HPLC system, contrast study of mobile phase showed that methanol: water:formic acid = 74:21:5 solution (sodium heptanesulfonate 4.0 g / L) used as the mobile phase was better. Studies on separation and purification showed that the weak acid DK110 macroporous resin was better (eluting peaks were more focused) than 732 resin.To get high-producing gentamiacin (GM) strains, which was resistant to the material produced by itself, Me07 was taken as the initial strain and was treated by UV, DES and microwave mutagenesis. The experimental results showed that DES treatment was the best way. Then, the GM production mutants was screened by two breeding ways of resistance plates and dynamic flasks combining with TLC analysis. The high-yield strain Me07-1D15 was obtained and compared with Me07, the yield of gentamicin in fermention broth was increased by 2.0 fold from 680 U/ml to 1400 U/ml,and C1 content was as high as 47.59%.The fermentation medium of gentamicin was optimized,and the results showed that starch was the best carbon source, the bean powder 1# was the most suitable nitrogen source. The results of Plackett-Burman test indicated that starch, soybean powder CoCl2 and CaCO3 were significant factors influencing the fermentation level. Then the single factor experiments and orthogonal combination experiments were did, and the results showed that the optimal concentration of significant factors were: starch 60 g/L, soybean powder 30 g/L, CoCl2 0.008 g/L, CaCO3 4 g/L.The optimization of fermentation conditions were studied, and the best fermentation conditions were obtained:seed age was 2 days, liquid volume in 500 mL flask was 40 mL, culture temperature was 34℃, the shaking speed was 250 r/min, the fermentation time was 8 days. Research on the fermentation process showed that rationally reducing the nutritional content of the fermentation medium could greatly shorten the fermentation cycle and maintain a high production level of GM. Besides, Application of different stress conditions like heat shock, feeding high ethanol and high NaCl concentrations during fermentation has found to be effective for the increased production of gentamicin. Production of gentamicin was increased to 1715 U/mL in medium supplemented with 0.6% NaCl to 144 hrs old culture. Production of gentamicin was increased to 1696 U/mL in medium supplemented with 4% ethanol to 96 hrs old culture, while, when the ethanol concentration was 5～6%, the synthesis of gentamicin was inhibited.