| With the growing problem of antibiotic resistance, there is an urgent need for new antimicrobial agents. Ketolide antibiotics are prominent role in the flight against drug-resistant, and are the most hopeful antibiotic. Using the method of homologous recombination has constructed Saccharopolyspora erythraea M, which could synthesize a new ketolide compound,3-deoxy-3-oxo-5-desosamine-erythronolide B.But the yield was very low, and the production had no antibacterial activity. Saccharopolyspora erythraea 963 and 967 were mutant strains which obtained by 60Co-y rays irradiation on Saccharopolyspora erythraea M. Although their production processed antibacterial activity, the yield needed to be improved.Compound mutation usually has good effect. In order to obtain high-yield mutant, Saccharopolyspora erythraea 963 and 967 are repeated radiated by 60Co-y rays. Spores are diluted with normal saline and 5%,10%,20%,40%,80%glycerol properly, radiated with the different dose of 60Co-γrays(non-radiated spores as CK).The number of colony is counted and the lethality is calculated to choose appropriate spore dilute and the best 60Co-y ray dose. Select mutant from streptomycin-resistant plate of filter R3M, observe morphological, choose mutants with higher antibacterial activity after fermentation cultivation, and then calculate the mutation rate to study the repeated mutagenic effect of 6 Co-y rays on Saccharopolyspora erythraea 963 and 967.So as to discuss the streptomycin-resistance screening method, the mutant strains are screened from the R3M plate of increased streptomycin concentration using Saccharopolyspora erythraea 963 as the original strain at the same time.The experiment of the minimum inhibitory concentration of Streptomycin indicates that with the increased concentration of streptomycin, the inhibitory effect on Saccharopolyspora erythraea 963 and 967 also enhance.140μg/mL and 120μg/mL are their minimum inhibitory concentration of Streptomycin respectively because there are no olive colonies on the plate. In the streptomycin resistance screening of fixed concentration,130μg/mL and 110μg/mL are choosed separately; in the resistance screening of increased streptomycin concentration, concentration gradually increased from 130μg/mL to 140μg/mL,150μg/mL and 160μg/mL.By comparing normal saline and 5%,10%,20%,40%,80% glycerol on the growth effect of spores after irradiated and the experiments operability, normal saline is good for spore dilutent. It is found that the higher radiation dose, the higher lethality rate of spores. When the dose is 500Gy, the lethality rates of Saccharopolyspora erythraea 963 and 967 are about 90%.So 500Gy is optimal mutagen dosage.Using Saccharopolyspora erythraea 963 as original strain to screen, one orthomutation is obtained, whose antibacterial activity elevates significantly and genetic stability is good. The rate of orthomutation mutation of is 1.67‰.There are 12 physiological mutants, whose sporulation time, spore color change largely. The rate of physiologic mutation is 24.1‰. Compared with radiation once, it is helpful in improving screening efficiency of mutation breeding.The results of resistance screening of increased streptomycin concentration showed that although the rate of physiologic mutation is higher, it is not conducive to screen efficiently because of higher negative mutation rate comparing to the resistance screening of fixed streptomycin concentration.Using repeated mutagenesis with 60Co-γray supplemented by resistance screening of fixed streptomycin concentration is able to improve the rate of morphological mutation and positive mutation. The selected mutant has a good genetic stability. It overcomes uncertainty of random selection and the blindness, and increased the efficiency of strain breeding. The results laid the foundation of industrial application of Saccharopolyspora erythraea for the future study.
|
PDF Full Text Request