The Study On Selection Of High-yield Bleomycin Producing Strain

Bleomycin (BLM) is a family of broad-spectrum antibiotics with special structures and functions. BLM consists of 5 molecule of amino acids, 2 molecule of hexoses and 1 molecule of amino-side chain. Structure of the family members is only different in substituted side chain. BLM is effective in treatment of squama cancer of head and cervix, and malignant lymphoma without decreasing leucocytes, inhibiting immune functions and damaging hemopoietic system. The anti-cancer mechanism of BLM is the inhibition of DNA synthesis by compound BLM formed from oxygen and iron.At present the titre of BLM is lower, such as the titre of parent strain U-43 is only 25μg/mL.In order to obtain higher BLM-producing strain, 5 different breeding methods were used to treat the parent strain U-43 separately. At last, high-production strain, L-20 was screened by the method of N~+ Ion implantation. Furthermore, the original fermentation medium was optimized .The concentration and The feeding time of the precursor of Spermine was selected which increase the production of BLM evidently. The followings are the results:1. The spore suspending-liquid of the starting strain U-43 was treated by the five methods of UV, Microwave, NTG, Electron beam, and N~+ Ion implantation. The titres of all strains obtained from the treatments of the first four methods were decreasing to some degree when the strains were selected in the second time by shaking flask fermentation. In the same time, the titres of these high-yield strains are unstable and decreasing dramatically when Genetic Stability Experiment of High-yield Strains were done for 5 generations. But the titre of the high-yield strain L-20 which was obtained by the method of N~+ Ion implantation was 42.25μg/mL. Its titre was 1.68 times as higher as that of the starting strain. When genetic stability experiments were done for 5 generations, the titre is stable and about 42.25μg/mL2. In order to improve the titre of high-yield strain L-20, Beef extract was added into the fermentation liquid. In order to screen an optimum medium which was fit for the fermentation of high-yield strain L-20, the best concentration of Inorganic Chemicals, Carbon resource and Nitrogen resource was selected by the orthogonal design method. The titre of fermentation liquid of L-20 is 45.35μg/mL with the optimum medium. In the end, the concentration and feeding time were selected in order to improve the production of BLM in the fermentation liquid. The result is that the titre of BLM can be improved from 45.35μg/mL to 60.84μg/mL when the concentration of the precursor Spermine is 0.02mg/ml and the feeding time is 36h.